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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.

We used an in vitro opsonophagocytic killing assay to measure the functional activity of antibody directed at the mucoid exopolysaccharide (MEP) antigen expressed by Pseudomonas aeruginosa strains isolated from cystic fibrosis patients. Rabbit antibodies raised to purified MEP were able to mediate phagocytic killing in the presence of human peripheral blood leukocytes and a low level (final concentration, 0.3%) of fresh normal human serum as a complement source. No bacterial killing was observed when peripheral blood leukocytes, antiserum, or complement was omitted. Specificity of the antibody for the MEP antigen was shown by adsorption and inhibition assays. Affinity-purified antibody to MEP also mediate phagocytic killing. These data indicate that antiphagocytic properties attributable to MEP can be overcome by specific antibody.

The mechanism of killing of 12 serum-sensitive strains of mucoid Pseudomonas aeruginosa isolated from patients with cystic fibrosis was investigated. A quantitative assay indicated that more than 90% of cells were killed in 50°7o normal human serum (NHS). All strains failed to grow in NHS concentrations of >10070. Killing was unaffected by adsorption of NHS with the mucoid bacteria or chelation with MgCl2-ethyleneglycol bis(j3-aminoethyl ether)N,N'-tetraacetate (MgC12-EGTA) but was abolished in serum heated to 50 C for 20 min. Incubation of NHS with mucoid P aeruginosa reduced the hemolytic capability of MgCl2-EGTA-chelated NHS against rabbit red blood cells by 56%-99070. Killing of the serum-sensitive mucoid strains was also seen in hypogammaglobulinemic serum. These data suggest that killing of such strains by NHS can occur via antibody-independent activation of the alternative pathway of complement. The importance of this finding lies in the implication that complement levels in the lungs of patients with cystic fibrosis who are colonized by these organisms are inadequate to deal with this chronic, progressive infection. The inability of patients with cystic fibrosis (CF) to deal effectively with chronic colonization by mucoid exopolysaccharide-producing strains of Pseudomonas aeruginosa suggests a potential im

A high-molecular-weight alkali-labile polysaccharide (PS) isolated from the slime of immunotype 1 Pseudomonas aeruginosa was tested for its ability to protect mice from lethal challenge with the live, homologous organism. Intraperitoneal (i.p.) injection of 10 to 25 microgram of the PS protected 60 to 70% of the mice against challenge with up to 50 50% lethal dose units. Although single immunization of mice with up to 250 microgram of PS effected protective levels of only 70%, two successive immunizations provided 100% protection. Subcutaneous and intravenous immunization with PS also provided protection to i.p. challenges with immunotype 1 P. aeruginosa, but not to i.p. challenge with immunotype 4 P. aeruginosa. Although lipopolysaccharide (LPS) was found to be more immunogenic than PS in out studies, contamination of the alkali-labile PS with LPS did not account for the protection seen. Alkali treatment (0.1 N NaOH, 37 degrees C, 2 h) of the PS destroyed its protective effectiveness, while similarly treated LPS retained its capacity for inducing immunity in mice. Adsorption and passive protection studies with sera raised to either PS or a mixture of PS and LPS indicated that antibody directed to the alkali-labile PS antigen was capable of contributing to the protection of mice against challenge with P. aeruginosa.

Evidence is presented for the recognition of a new species of Streptococcus isolated from abscess foci in an Amazon freshwater dolphin, Inia geoffrensis. The organism appears to be immunologically distinct from members of the recognized Lancefield groups of streptococci. Antigens prepared by five different extraction procedures do not react with antisera to Streptococcus groups A to U, whereas antisera prepared against the new isolate react well with the extracted homologous antigens but not with antigens from groups A to U. Based on cultural, morphological, biochemical, and serological results, it is suggested that this isolate belongs to a new species for which we propose the name Streptococcus iniae. The type strain of this new species is strain PW (=ATCC 29178).

The serotype distribution of bacteremic and nonbacteremic clinical isolates of Pseudomonas aeruginosa in relation to the Fisher immunotyping scheme, the International Antigenic Typing System (IATS), and high-molecular-weight polysaccharide determinants was investigated. Of 281 bacteremic isolates, 273 (97.2%) were serotyped by one of the seven IATS specificities that correspond to a Fisher lipopolysaccharide/high-molecular-weight polysaccharide specificity. In contrast, these seven serotypes accounted for only 68.5% of the 124 nonbacteremic clinical isolates. Review of the reported serotype distribution of P. aeruginosa isolates in Europe further supported the finding of a limited serotype distribution among bacteremic clinical isolates. Fifteen of the 17 IATS serotypes were found among all of the strains of P. aeruginosa serotyped, an indication that most of the IATS serotypes are present in the United States. Thus, only certain lipopolysaccharide immunotypes of P. aeruginosa occur as clinical bacteremic isolates, and a multivalent, high-molecular-weight polysaccharide vaccine directed at the lipopolysaccharide type determinants of P. aeruginosa has potential usefulness.

A neutral small molecular mass (approximately 6.5 kDa) polysaccharide comprising a pentasaccharide repeat unit was isolated from culture supernatants of Pseudomonas aeruginosa immunotype 4. The polysaccharide had a pentasaccharide repeating unit as follows (formula; see text) where Rha is rhamnose. The structure was determined using acid hydrolysis, solvolysis with anhydrous hydrogen fluoride, methylation analysis, and 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments. The polysaccharide bound antibody raised to the lipopolysaccharide of the seven P. aeruginosa Fisher-Devlin immunotype strains. Inhibition assays demonstrated the presence of a serologically similar polysaccharide in supernatants of these strains. Affinity-purified antibody to the polysaccharide bound to lipopolysaccharide and whole cells of the immunotype strains of P. aeruginosa in a Western immunoblot and colony blot assay, respectively. This polysaccharide seems to contain an antigenic determinant present in the core of the P. aeruginosa lipopolysaccharide or may represent another minor polysaccharide substituent on the lipopolysaccharide in addition to the O side chain.

"... perhaps the worst thing about cystic fibrosis is that it attracts a bacteria known as pseudomonas. The excessive, filthy mucus overwhelms the defense mechanisms in the lungs, so the pseudomonas bacteria colonize there. Pseudomonas is most common with cystic fibrosis, but it is also a threat to patients why have been severely burned, and it is found in the bloodstreams of certain types of cancer patients. Still, for all the research that has been done, there is as yet no antibiotic to deal with pseudomonas, and once it begins to march through the lungs, it multiplies with impunity and sweeps everything in its path. For any cystic fibrosis patient, pseudomonas is the harbinger of death." [1]

SUMMARY: The immunological basis for resistance of certain strains of Neisseria gonorrhoeae to the bactericidal action of normal human serum was studied by investigating the potential role of factors which are known to interfere with each of the sequential steps that result in immune lysis of Gram-negative bacteria. Strains of N. gonorrhoeae were characterized as serum-sensitive (sers) or serum-resistant (serr) on the basis of their sensitivity to lysis by the sera of six normal individuals. Neither intrinsic resistance to the lytic action of activated human complement nor inaccessibility of the cell membrane to C5b accounted for serr. Outer membrane lipopolysaccharide (LPS) was the target antigen for lytic antibody in normal human sera. The gross chemical composition and molecular size of the LPS of the strains were heterogeneous and no consistent patterns of differences between those extracted from serr and from sers strains were found. Neither IgA nor IgG ‘blocking’ antibody in normal human serum was responsible for serr. We conclude that serr results from the absence from the LPS of the strains of antigenic loci for the lytic antibody in most normal human sera, or, expressed as a function of the host, the absence from the sera of most normal humans of lytic antibody directed against LPS antigenic loci for immune lysis.

Alginic acid-like mucoid exopolysaccharide was isolated from three strains of Pseudomonas aeruginosa obtained from the sputa of patients with cystic fibrosis. Purified mucoid antigens were >99% uronic acid. With a hemagglutination assay, antibody responses to the mucoid exopolysaccharide were documented after immunization of rabbits with either whole mucoid organisms or purified mucoid exopolysaccharide. The mucoid antigen from one strain (no. 2192) was composed predominantly of a single serologic epitope shared among 40 alginate exopolysaccharides from different clinical isolates. The mucoid exopolysaccharide from the other two strains (nos. I and 258) had a serotype-specific determinant in addition to the common epitope. Analyses of antibody in sera from normal adults, children, and patients with cystic fibrosis culturepositive and culture-negative for mucoid P. aeruginosa showed a highly significant (P < 0.001) association between increased hemagglutination titers and positive cultures for mucoid P. aeruginosa. The characteristic alginic acid-like exopolysaccharide produced by isolates of Pseudomonas aeruginosa from the sputa of patients with cystic fibrosis (CF) has prompted much interest in a potential pathogenic role for this material. Studies on the in vitro effects of this exopolysaccharide in

An einem Meerschweinchenmodell der experimentellenPseudomonas aeruginosa-Pneumonie wurden die Faktoren untersucht, die die Wirksamkeit einer passiven Immunisierung mit Psomaglobin®N*, einem i.v.Pseudomonas-Immunglobulin, beeinflussen. Tiere, die 2 Stunden nach Infektion mit einer einmaligen intravenosen Infusion von Psomaglobin®N in einer Dosis von 500 mg/kg behandelt wurden, wiesen eine Uberlebensrate von 33% auf. Geringere Dosen waren weniger wirksam. Keines der mit Albumin behandelten Kontrolltiere uberlebte. Die Therapie mit Psomaglobin®N war wirksam, wenn sie 2 Stunden oder 8 Stunden nach der Infektion einsetzte. Begann sie erst 24 Stunden nach der Infektion, so zeigte sie dagegen keine Wirkung mehr. Neutropenische Tiere (vorangegangene Cyclophosphamid-Behandlung) uberlebten nach alleiniger Behandlung mit Psomaglobin®N nicht. Bei kombinierter Therapie mit Psomaglobin®N und Tobramycin stieg die Uberlebensrate im Vergleich zu einer alleinigen Tobramycin-Behandlung jedoch signifikant auf 86% gegenuber 43% (p < 0,05). Diese Befunde erlauben den Schlus, das die Gabe von Psomaglobin®N eine wirksame Therapie derP. aeruginosa-Pneumonie sein konnte.

A high-molecular-weight polysaccharide (PS) was isolated from the culture supernatant of a Fisher immunotype 3 (IT-3) strain of Pseudomonas aeruginosa. Consistent with previously reported findings for IT-1 and IT-2 PS, the preparation of IT-3 PS was found to be an immunogenic, nontoxic form of the O polysaccharide side chain on the lipopolysaccharide (LPS). The IT-3 PS was mainly carbohydrate in composition. It was serologically and chemically identical to LPS O side chain, but distinct from that structure in molecular size and immunogenicity. The IT-3 PS was nontoxic in mice and guinea pigs, nonpyrogenic in rabbits, and greater than 1,000-fold less reactive than IT-3 LPS in gelation of the Limulus amoebocyte lysate. Preliminary analyses by gas-liquid chromatography and 13C nuclear magnetic resonance have established the structural identity of IT-3 high-molecular-weight PS and the IT-3 O side chain. IT-3 PS was immunogenic in rabbits and mice. After active immunization, mice were protected against P. aeruginosa IT-3 intraperitoneal infection and burn wound sepsis. IT-3 PS also elicited protection against challenge with an IT-5 strain of P. aeruginosa, indicating that low-level contamination of the IT-3 PS with IT-3 LPS was not responsible for the immunogenic activity. These findings demonstrate the feasibility of preparing nontoxic immunogenic IT-3 PS capable of eliciting serotype-specific protective antibodies, employing methods similar to those previously described for the isolation of PS from other P. aeruginosa immunotypes.

Previous work has described small molecular weight neutral polysaccharides from isolates of Pseudomonas aeruginosa that appear to be associated with the lipopolysaccharide (LPS) and distributed across serologic barriers defined by antibody to the O side chain. We have isolated and characterized another of these structures obtained from culture supernatants of an immunotype 3 strain of P. aeruginosa. The isolated neutral polysaccharide has a tetrasaccharide repeat unit: (formula; see text) where Rha is rhamnose. The structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments, acid hydrolysis, methylation analysis, Smith degradation, and optical rotation determinations. Polyclonal antibodies raised to intact and alkali-treated (0.1 N NaOH, 56 degrees C, 2 h) LPS from the seven Fisher immunotype strains of P. aeruginosa bound well to the neutral polysaccharide. Antibodies affinity purified from these sera using immobilized neutral polysaccharide as well as a neutral polysaccharide-specific monoclonal antibody, E87, reacted with an antigenically similar structure found among many isolates of different LPS serotypes in a colony blot and with LPS from the seven Fisher immunotypes in an immunoblot. In an immunoblot assay, the neutral polysaccharide inhibited binding of the monoclonal antibody, E87, to material present in LPS preparations from a variety of serotypes. This structure may represent another P. aeruginosa neutral polysaccharide variant found associated with the LPS.

Three cell wall-derived Pseudomonas aeruginosa vaccines were evaluated for their capacity to provide protection from experimental pseudomonas pneumonia in guinea pigs. Two vaccines, Pseudogen, a heptavalent lipopolysaccharide vaccine, and PEV-01, a polyvalent glycine-EDTA cell wall extract, each produced hemagglutinating, opsonic, and serum bactericidal antibodies in guinea pigs. Significant protection from fatal hemorrhagic pneumonia was conferred by vaccination with these products. Purified, high-molecular weight polysaccharide vaccine was a less potent immunogen in guinea pigs and provided less protection from pneumonia. Protection from fatal pneumonia in vaccinees correlated with more rapid clearance of viable P. aeruginosa from lung tissue during the first 6 hr of infection. It appears that active immunization with certain cell wall-derived pseudomonas antigens can provide protection against experimental pseudomonas pneumonia.

A second strain of Streptococcus iniae has been recovered from an Amazon freshwater dolphin (Inia geoffrensis). This isolate differs from the first-described isolate in its ability to produce acid from lactose but not salicin and its inability to hydrolyze esculin. The two isolates share a common cell wall antigen that appears to represent the C polysaccharide grouping antigen of this species. In addition, there are strain-specific antigens associated with each isolate. The second strain has been designated strain BU (= ATCC 29177).

We studied the induction of human interleukin-1 (IL-1) production in strains of Staphylococcus aureus isolated from patients with nonmenstrual toxic shock syndrome (TSS). Of the 20 TSS-associated strains studied, 11 produced and nine did not produce TSS toxin-1 (TSST-1). Human monocytes were incubated with dilute staphylococcal supernatants, and IL-1 production was measured in a lymphocyte-activating factor assay. All 20 TSS-associated strains were potent inducers of IL-1, in comparison with none of 10 vaginal isolates of S. aureus from healthy women. TSST-1-positive strains were more potent than TSST-1-negative strains. Nine TSST-1-negative TSS-associated strains were compared with 14 strains of S. aureus from other clinical settings and were found to be significantly more potent inducers of IL-1 (P less than .01). Eight of these nine TSS-associated strains produced at least one staphylococcal enterotoxin. Stimulation of monocytes by products of S. aureus may play a role in the pathogenesis of TSS.

BALB/c mice immunized with Pseudomonas aeruginosa immunotype 1 polysaccharide develop protective T cell immunity to bacterial challenge. In vitro, T cells from immunized mice kill P. aeruginosa by production of a bactericidal lymphokine. The present study demonstrates that adoptive transfer of T cells from immunized BALB/c mice to granulocytopenic mice resulted in 97% survival on challenge with P. aeruginosa, compared with 17% survival with adoptive transfer of T cells from nonimmune BALB/c mice. This protection is specifically elicited by reexposure to the original immunizing antigen; adoptive recipients cannot withstand challenge with immunotype 3 P. aeruginosa. However, the adoptive recipients do survive simultaneous infection with both P. aeruginosa immunotypes 1 and 3. Adoptive transfer of T cells from the congenic CB.20 mice, which are unable to kill P. aeruginosa in vitro, provides only 20% protection to granulocytopenic mice. These studies indicate that transfer of specific immune T lymphocytes can significantly enhance the resistance to P. aeruginosa infection in granulocytopenic mice.

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.

A murine monoclonal antibody (MAb) was prepared against Pseudomonas aeruginosa immunotype-1 (It-1) lipopolysaccharide (LPS). The MAb bound It-1 LPS in the enzyme-linked immunosorbent assay and in the immunodiffusion and immunoblotting assays, agglutinated and opsonized It-1 bacteria, and protected against challenge with live It-1 organisms in a murine burn infection model. All of these activities were immunotype specific. Correlation of the opsonic and protective properties of the MAb with its recognition site on the LPS O side chain confirmed that such immunotype-specific determinants are important targets for protective antibodies in Pseudomonas disease. The functional equivalence of this MAb and polyclonal antibodies from hyperimmune plasma underscores the therapeutic potential of single MAbs which recognize critical determinants in the LPS O side chain.

Sera from humans vaccinated with a high-molecular-weight polysaccharide vaccine to Pseudomonas aeruginosa immunotype 1 (IT-1) were analyzed for duration of the immune response, specificity for the IT-1 determinant, and by assessing the immunoglobulin classes elicited. The ability of purified IgG, IgM, and IgA to interact with peripheral blood leukocytes, as well as purified polymorphonuclear neutrophils or mononuclear cells, was also examined in an opsonophagocytosis assay. Levels of antibody to IT-1 remained significantly (P < 0.001) elevated 21 months after immunization. Responses were generally specific to the IT-1 serotype determinant. Some vaccinees also responded to immunotype 2 and immunotype 5 determinants. IgG, IgM, and IgA serum antibodies were all elicited by vaccination. IgG and IgA were effective opsonins for P aeruginosa. IgM-mediated opsonophagocytosis required complement. Serum IgA was highly effective in conjunction with mononuclear cells in opsonophagocytosis of P aeruginosa, suggesting that these immune components may be capable of protecting neutropenic hosts. The role of antibody, complement, and phagocytic cells in human immunity to infection with Pseudomonas aeruginosa is well documented. Studies in animals [1] and patients [2-4] have suggested that antibodies to the lipopolysaccharide are associated with protection from death related to P aeruginosa sepsis. The requirement for phagocytic cells is indicated by the increased susceptibility of leukopenic patients to P aeruginosa infection [5] and by the resistance to bactericidal

Strains of Staphylococcus aureus isolated from patients with toxic shock syndrome (TSS) make a characteristic protein known as toxic-shock-syndrome toxin-1 (TSST-1), but the role of this protein in the pathogenesis of TSS is not certain. We have purified TSST-1 by using a combination of alcohol precipitation, isoelectric focusing, and gel chromatography. TSST-1 has an isoelectric point of 7.2 and a molecular weight of 23,100, in accordance with previously published determinations for this protein, and is serologically identical to pyrogenic exotoxin C and staphylococcal enterotoxin F. In highly purified form, TSST-1 is a potent inducer of interleukin-1 production by human monocytes, as quantitated in a thymocyte-proliferation assay. This capability is not attributable to contamination by other staphylococcal products or gram-negative endotoxin and can be blocked by hydrocortisone. Many features of TSS suggest that induction of interleukin-1 by TSST-1 in vivo may play a central role in the elaboration of this disease.

A human immunoglobulin G preparation, enriched in type-specific antibodies against Pseudomones aeruginosa lipopolysaccharide immunotypes 1, 2, 4, 6 (Fisher scheme), and suitable for intravenous infusion, has recently been developed. We evaluated the therapeutic efficacy of this preparation in a guinea pig model of P aeruginosa pneumonia. Intravenous infusion of 500 mg/kg of hyperimmune globulin into guinea pigs produced elevations in serum antibodies against P aeruginosa , which persisted for one week or longer. For study, animals were infected with type 1 or type 4 P aeruginosa challenge strains, then treated two hours after infection with a single intravenous infusion of 5% hyperimmune globulin (500 mg/kg), or 5% albumin. Cumulative survival rates for type 1 infection were 0/33 controls, and 12/36 globulin ( P P P aeruginosa immunoglobulin G preparation was more efficacious in treating pneumonia than was a conventional (nonhyperimmune) immunoglobulin G preparation. Finally, in experiments combining hyperimmune P aeruginosa globulin with tobramycin (1.7 mg/kg, six hours and 24 hours after infection), cumulative survivals from pneumonia were increased to 87%, as compared to survivals of 50% for tobramycin alone, 25% for globulin alone, and 0% in control groups. We conclude that passive immunization with a recently developed hyperimmune P aeruginosa immunoglobulin G preparation may be therapeutically useful in the management of life-threatening P aeruginosa pneumonia.

In this study we report the isolation and purification of the capsular polysaccharide elaborated by Staphylococcus aureus SA1 mucoid. The capsule was isolated from bacterial extracts and culture supernatants by a series of ethanol precipitations and enzyme digestions, followed by ion-exchange chromatography. Teichoic acid contamination was eliminated by oxidation with sodium metaperiodate, and the final product eluted in the void volume of a Sephacryl S-300 column. The purified capsular polysaccharide was analyzed by gas-liquid chromatography-mass spectroscopy, 13C and 1H nuclear magnetic resonance, amino acid analysis, immunelectrophoresis, and numerous biochemical assays. The major constituents of the capsule were 2-acetamido-2-deoxy-alpha-galacturonic acid (4-O linked), 2-acetamido-2-deoxy-alpha-fucose (3-O linked), and taurine. The polysaccharide also contained O-acetyl groups which were removed by mild alkaline hydrolysis. Serologically and biochemically, the capsule from strain SA1 mucoid appeared very similar to that produced by strain M. Purified capsular polysaccharide was immunogenic in both rabbits and mice. The optimal immunizing dose in mice was 0.1 microgram of purified capsular polysaccharide administered intraperitoneally. SA1 mucoid resisted opsonophagocytic killing by human leukocytes and complement. However, antibodies raised to the purified capsular polysaccharide neutralized the antiphagocytic effect of the capsule.

A comparison was made of the immunochemical properties of high-molecular-weight polysaccharide (PS) and lipopolysaccharides (LPS) from Pseudomonas aeruginosa immunotype 1 and 2 (IT-1 and IT-2) strains. High-molecular-weight PS was found to be an immunogenic, nontoxic form of the LPS serotype determinant found on the O side chain of the LPS. PS and O side chains were serologically identical. Immunization of animals with either whole bacterial cells or purified PS or LPS resulted in comparable levels of antibody directed at PS or O side chains. Chemical analysis of PS and LPS indicated the presence of monosaccharides in PS preparations that are not present in LPS, while PS and LPS share other monosaccharides. PS also lacks core-type sugars found in LPS such as 2-keto-3-deoxyoctulosonic acid, heptose, and glucosamine. PS from IT-1 P aeruginosa is immunogenic in humans, with essentially no toxicity noted. The lipopolysaccharide (LPS) obtained from Pseudomonas aeruginosa is of interest for the serologic classification of this organism and for the development of vaccines against infections due to P. aeruginosa [1-4]. Both the Fisher immunotyping scheme [5] and the International Antigenic Typing Scheme (IATS) base the serologic differences among strains of P. aeruginosa on antigenic determinants associated with the O polysaccharide side chain of the LPS [6]. Studies of the chemical composition of P. aeruginosa LPS have revealed the presence of such compounds as 2,6 dideoxyhexosamines, 2-keto-3-deoxyoctulsonic acid (KDO), and heptose as monosaccharide components [4, 5]. Some workers have suggested that the structure of P. aeruginosa LPS may be similar to that of the well-known enterobacterial LPS [6].

BackgroundA clinical study to investigate the leukotriene B4 (LTB4)-receptor antagonist BIIL 284 in cystic fibrosis (CF) patients was prematurely terminated due to a significantly increased risk of adverse pulmonary events. We aimed to establish the effect of BIIL284 in models of Pseudomonas aeruginosa lung infection, thereby contributing to a better understanding of what could have led to adverse pulmonary events in CF patients.MethodsP. aeruginosa DNA in the blood of CF patients during and after acute pulmonary exacerbations and in stable patients with non-CF bronchiectasis (NCFB) and healthy individuals was assessed by PCR. The effect of BIIL 284 treatment was tested in an agar bead murine model of P. aeruginosa lung infection. Bacterial count and inflammation were evaluated in lung and other organs.ResultsMost CF patients (98%) and all patients with NCFB and healthy individuals had negative P. aeruginosa DNA in their blood. Similarly, the P. aeruginosa-infected mice showed bacterial counts in the lung but not in the blood or spleen. BIIL 284 treatment decreased pulmonary neutrophils and increased P. aeruginosa numbers in mouse lungs leading to significantly higher bacteremia rates and lung inflammation compared to placebo treated animals.ConclusionsDecreased airway neutrophils induced lung proliferation and severe bacteremia in a murine model of P. aeruginosa lung infection. These data suggest that caution should be taken when administering anti-inflammatory compounds to patients with bacterial infections.

International audience; Cochlin, an extracellular matrix protein, shares homologies with the Factor C, a serine protease found in horseshoe crabs, which is critical for antibacterial responses. Mutations in the COCH gene are responsible for human DFNA9 syndrome, a disorder characterized by neurodegeneration of the inner ear that leads to hearing loss and vestibular impairments. The physiological function of cochlin, however, is unknown. Here, we report that cochlin is specifically expressed by follicular dendritic cells and selectively localized in the fine extracellular network of conduits in the spleen and lymph nodes. During inflammation, cochlin was cleaved by aggrecanases and secreted into blood circulation. In models of lung infection with Pseudomonas aeruginosa and Staphylococcus aureus, Coch(-/-) mice show reduced survival linked to defects in local cytokine production, recruitment of immune effector cells, and bacterial clearance. By producing cochlin, FDCs thus contribute to the innate immune response in defense against bacteria.

In this study, we determined that immunization with capsular polysaccharide from Staphylococcus aureus could protect mice against nonlethal infections induced by encapsulated staphylococci. We immunized mice with either formalin-killed bacteria or purified capsular polysaccharide (PCP) and challenged them with one of three related S. aureus strains that varied in capsule size. Quantitative cultures of blood and kidney from the animals were performed to evaluate protection. Immunization with whole bacteria protected mice against infection with the homologous strain. Mice immunized with PCP were protected when challenged intravenously with either a highly encapsulated S. aureus strain or a microencapsulated mutant but not with an unencapsulated mutant. Protection correlated with capsular antibody levels in the immunized animals. Immunity to staphylococcal infection could be passively transferred to naive animals by using immune serum. These experiments suggest that the S. aureus capsular polysaccharide merits further study as a potential vaccine candidate for preventing staphylococcal infection.

The safety and immunogenicity of a high molecular weight polysaccharide from immunotype 1 Pseudomonas aeruginosa were tested in a dose response fashion in adult volunteers. The vaccine lacked toxicity and pyrogenicity for experimental animals. Doses of 50, 75, 150, or 250 microgram were given to groups of individuals as a single dose subcutaneous injection. Doses of 150 and 250 microgram were associated with a significant rise in binding and opsonic antibody at 2 wk postimmunization. Titers remained unchanged for up to 6 mo. The vaccine was almost devoid of toxicity, eliciting no more than a slightly sore and tender arm at the site of injection. High molecular weight polysaccharide antigen appears to induce a good immune response following vaccination that is effective in mediating opsonophagocytic killing of live P. aeruginosa organisms.

Serogroup Y and W135 Neisseria meningitidis capsular polysaccharide vaccines were tested as monovalent and divalent preparations in groups of 10 adult human volunteers at a dose of 50 (monovalent) or 100 micrograms (divalent) injected subcutaneously. Reactogenicity was low for the group Y vaccine and the group Y-W135 combined vaccine; 3 of 10 volunteers developed systemic reactions after group W135 vaccination. All three vaccines induced significant homologous and heterologous binding and bactericidal antibody. Except for group W135 bactericidal antibody, homologous responses exceeded heterologous responses, and divalent and monovalent vaccines induced equivalent homologous responses. Homologous bactericidal antibody responses were maintained for 4 weeks in 85% of group W135 vaccinates and in 100% of group Y vaccinates. Bactericidal antibody was induced in 11 of 11 group Y and 12 of 15 group W135 volunteers without preexisting respective bactericidal activities, regardless of which vaccine they received. For all three vaccines, antibody levels declined only slightly over 6 months. Prevaccination antibody levels positively affected postvaccination binding antibody levels, but not bactericidal levels.

Pseudomonas aeruginosa is the principal pulmonary pathogen in patients with cystic fibrosis. All attempts to date to prevent or eradicate P. aeruginosa infections in these patients have been unsuccessful. Vaccination against P. aeruginosa has been proposed as a preventive strategy but it has not been adequately evaluated. The purpose of this study was to determine whether P. aeruginosa, present in the lungs of patients with cystic fibrosis, express surface antigens similar to those grown in vitro; this issue is of critical importance when choosing bacterial products as vaccine candidates. Lung sections from two patients who died of the pulmonary complications of cystic fibrosis were studied. Bacteria, both in lung sections and isolated from the lung sections and grown in vitro, reacted strongly with polyclonal and monoclonal antibodies against P. aeruginosa mucoid exopolysaccharide and outer membrane proteins F and H2; this suggested that these antigens are surface exposed in vivo. It was also found that bacteria in both lung sections were associated in situ with IgG, IgA, and C3 but not with IgM or C4.

We analyzed high molecular weight polysaccharide (PS) from the Fisher immunotype 2 (IT-2) strain of Pseudomonas aeruginosa for molecular composition and structure, then determined its immunogenicity in healthy adults. The PS was composed of 2-acetamido-2,6-dideoxygalactose (N-acetyl fucosamine) and glucose in a molar ratio of 2:1. Structural analysis by carbon-13 and proton nuclear magnetic resonance confirmed that the high molecular weight PS was structurally identical to that of the O-specific side chain of the lipopolysaccharide. PS differed from this material in molecular size. Immunization of 19 adult volunteers with doses of 50-100 micrograms of PS resulted in significant rises (P less than 0.04-P less than 0.0001) in binding antibody levels and killing antibody titers 2 and 4 wk postimmunization. The only reaction to the vaccine was localized tenderness at the immunization site. Analysis of the immunoglobulin isotype response to the vaccine showed a rise in specific serum IgG and IgA antibodies. Heterologous responses to other P. aeruginosa PS antigens were not seen. The antibody levels attained by vaccination were comparable with those in acute-phase serum samples of patients who survived sepsis with IT-2 P. aeruginosa and were significantly higher (P less than 0.03) than specific antibody levels in bacteremic patients who died. These results confirm that PS is a high molecular weight, immunogenic form of the P. aeruginosa IT-2 serotype antigen, eliciting levels of type-specific antibody comparable with those seen among patients surviving an episode of P. aeruginosa sepsis.

The mucoid exopolysaccharide of Pseudomonas aeruginosa is thought to confer antiphagocytic properties on mucoid strains of P. aeruginosa, thus allowing them to persist in the respiratory tract. It has also been speculated that the mucoid exopolysaccharide may be the adhesin for mucoid strains, but proof is lacking. We studied the role of the mucoid exopolysaccharide in adherence of mucoid strains in competitive experiments with purified mucoid exopolysaccharide, by measuring the binding of 14C-labeled mucoid exopolysaccharide to injured tracheas and testing whether an antibody against the major epitope of the mucoid exopolysaccharide inhibits adherence of these organisms. Our data show that the purified mucoid exopolysaccharide increased the adherence of four of the mucoid strains tested (by 50 to 300%; P less than 0.001) instead of inhibiting adherence. Radiolabeled mucoid exopolysaccharide bound much better to injured tracheal cells than to normal tracheal cells (P less than 0.001), and antibody against the antigen of strain 2192, the strain from which mucoid exopolysaccharide was prepared, inhibited the adherence of four of five mucoid strains but not the strain lacking this antigen. This antibody also failed to inhibit a nonmucoid revertant from strain 2192, which was previously shown to be inhibited by pili. These data strongly support the thesis that the mucoid exopolysaccharide is the adhesion for mucoid strains of P. aeruginosa.

(DBA/N[female] X CBA/2[male])F1 males have been reported to be deficient in producing antibodies against a number of antigens, including carbohydrates (I. Scher, Adv. Immunol. 35:1-71, 1982). We show that F1 male mice, in contrast to females, made less lipopolysaccharide (LPS)-specific antibodies after immunization with heat-inactivated Pseudomonas aeruginosa and had significantly less naturally occurring LPS-specific antibodies. Furthermore, neutropenic males were 50 to 1,000 times more sensitive to challenge with representative isolates belonging to the seven Fisher immunotypes. Administration to neutropenic F1 males of a human monoclonal antibody specific for the O carbohydrates of P. aeruginosa immunotype 2 LPS or administration of serum from rabbits immunized with heat-inactivated P. aeruginosa immunotype 1 raised the level of resistance to bacterial challenge close to that of females. The results show that the X-linked immunodeficient mouse is an excellent model with which to test the protective efficacy of P. aeruginosa-specific monoclonal antibodies.

Pseudomonas aeruginosa is a frequent respiratory tract colonizer in diseases in which mucociliary clearance is defective. The most striking of these is cystic fibrosis. The reasons for this organism's ability to colonize the respiratory tract and to persist there are not fully understood. Earlier studies showed that P. aeruginosa adheres preferentially to tracheobronchial mucin when compared with enterobacteria. We reasoned that if adherence to respiratory mucin protected P. aeruginosa from opsonophagocytic killing, then the ability of this organism to chronically colonize the respiratory tract could be partially explained. Using an opsonophagocytic killing assay with human polymorphonuclear leukocytes, we found that respiratory mucin protected six strains of P. aeruginosa from opsonophagocytic killing but did not protect poorly adhering strains of Escherichia coli, Staphylococcus aureus, or group B streptococci. Incubating P. aeruginosa with the mucin prior to addition to the opsonic assay inhibited phagocytic killing, whereas incubation of polymorphonuclear leukocytes with mucin did not, suggesting that inhibition was not due to an effect of mucin on leukocytes per se but was a consequence of bacterial adherence to mucin. Further studies indicated no decrease in the binding of either antibody or complement component C3 to the bacterial surface in the presence of mucin. This suggests that phagocytic inhibition may be due to a defect in uptake or destruction of mucin-coated bacteria by the leukocytes. Thus, the adherence of P. aeruginosa to respiratory mucin potentially contributes to its persistence in the respiratory tract by interfering with host immune responses.

Antibody responses were assayed in inbred strains of mice immunized with a high-molecular-weight polysaccharide (IT-1 PS) isolated from Fisher immunotype 1 Pseudomonas aeruginosa (P. aeruginosa 1). C3H/ANF mice generated the greatest antibody response after immunization with 1 microgram of antigen and could then survive challenge with 5 LD100 of P. aeruginosa 1 organisms. BALB/c mice produced lower levels of antibody and did so only after immunization with 50 micrograms of IT-1 PS. These mice could only withstand challenge with 1 LD100 of P. aeruginosa 1. The specificity of the antibody produced by these two strains differed. Antibody produced in C3H/ANF mice after primary immunization with either 1 microgram or 50 micrograms of IT-1 PS did not cross-react with other immunotypes of P. aeruginosa. Antibody produced in BALB/c mice after a primary immunization with 50 micrograms of IT-1 PS cross-reacted with polysaccharide isolated from Fisher immunotype 2 P. aeruginosa. These data indicate that, even within the same species, antibody responses to immunization with P. aeruginosa IT-1 PS may be qualitatively different. This kind of variation in immune response patterns will have to be considered in developing optimal regimens for immunization in human vaccine trials.

We used three related strains of Staphylococcus aureus to determine whether capsule size influenced bacterial virulence. Strain SA1 mucoid elaborated a large capsule demonstrable by transmission electron microscopy (TEM). Nonmucoid isolates were derived from strain SA1 mucoid by Tn551 insertional mutagenesis. By TEM, strain JL24 produced a "microcapsule," whereas strain JL25 was unencapsulated. Strain SA1 mucoid had a 50% lethal dose for mice greater than 3,000-fold lower than that of strains JL24 and JL25. Quantitative cultures of blood and kidney from animals challenged intravenously revealed that strain SA1 mucoid was cleared less readily from the bloodstream and kidneys than the nonmucoid mutants. In an in vitro assay, only strain SA1 mucoid demonstrated antibody-dependent, complement-mediated opsonophagocytosis by human leukocytes. Strains JL24 and JL25 were opsonized for phagocytosis by complement alone. Thus a highly encapsulated strain of S. aureus was more virulent in mice than two related nonmucoid strains. The microencapsulated mutant was not more virulent than the unencapsulated mutant.

We have developed a rabbit model of toxic shock syndrome that uses a subcutaneous infusion pump to administer toxic shock syndrome toxin 1 (TSST-1). A dose of 150 micrograms, infused at a constant rate over a period of 7 days, resulted in a characteristic illness highlighted by fever, conjunctival hyperemia, cachexia, and lethargy. The illness was uniformly fatal, with a mean interval until death of 3.2 +/- 0.4 days. Serial determinations of serum chemistries confirmed the multisystem nature of this illness. Rabbits developed profound hypocalcemia, with levels falling from 15.5 +/- 0.2 to 7.6 +/- 0.4 mg/dl under the influence of TSST-1. Blood urea nitrogen and creatinine rose dramatically, in the setting of oliguria or anuria. Serum glutamicpyruvic transaminase was the most reliable indicator of hepatic dysfunction, with the mean rising from 48 U/liter before administration of TSST-1 to 546 U/liter among rabbits surviving 2 days of the infusion. Creatine phosphokinase also rose dramatically in 10 of 16 rabbits. Rabbits demonstrated relative neutrophilia and lymphopenia as well as an increase in the partial thromboplastin time. Histopathologic examination demonstrated disease of multiple organs, particularly the liver, spleen, and lymph nodes, all of which demonstrated inflammation, thrombosis, hemorrhage, and erythrophagocytosis. The concurrent administration of prednisolone with TSST-1 prevented death in four of four rabbits and greatly lessened the morbidity. Rabbits were not protected from morbidity or mortality by the concurrent administration of polymyxin B. We believe that a constant, subcutaneous infusion of TSST-1 in rabbits provides a reproducible model for studying the pathogenesis of TSS.

We developed a competitive, enzyme-linked immunosorbent assay for the quantitation of toxic shock syndrome toxin 1 (TSST-1). Polyvalent immunoglobulin G from immunized rabbits was used as the capture antibody, and alkaline phosphatase conjugated to purified toxin served as the indicator enzyme. A standard curve was generated with each experiment, from which the concentration of toxin in culture supernatants was extrapolated. The assay was useful for determining toxin concentrations of 0.03 to 0.5 micrograms/ml, which is a substantial, practical improvement over immunodiffusion methods. Staphylococcal enterotoxins A through E were not significantly cross-reactive in the assay, and staphylococcal protein A did not interfere with quantitation of TSST-1. By testing a variety of staphylococcal strains, we found 100% concordance between toxin determinations made with our assay and those made by the investigators from whom the strains were obtained. The competitive, enzyme-linked immunosorbent assay is a highly reproducible, inexpensive means of determining TSST-1 concentrations and may have broad applicability in the field of toxic shock research.

Patients with cystic fibrosis commonly harbor in their lungs strains of Pseudomonas aeruginosa that have a mucoid coating considered virtually pathognomonic for the disease. We found that strains of Escherichia coli with a morphologically similar mucoid coating were present in the respiratory tracts of eight (11.8 per cent) of 68 patients with cystic fibrosis whose sputum cultures yielded Esch. coli, as compared with none of 89 patients without cystic fibrosis who had Esch. coli in sputum. Mucoid strains of Esch. coli were also recovered from the stools of five (11.1 per cent) of 45 patients with cystic fibrosis, as compared with one (0.7 per cent) of 150 patients without cystic fibrosis. The mucoid substances purified from Esch. coli were biochemically and antigenically distinct from those of P. aeruginosa. We conclude that the respiratory tract in cystic fibrosis offers an environment conducive to the production of a mucoid coating not only by P. aeruginosa but by other gram-negative bacilli as well.

We explored the genetic basis for the differing immune responses observed in inbred strains of mice to a high-molecular-weight polysaccharide (PS) from Pseudomonas aeruginosa immunotype 1 (IT-1). Previous studies have shown that C3H mice immunized with this antigen produce only immunotype-specific antibody. BALB/c mice immunized with IT-1 PS produce both anti-IT-1 PS antibody and antibody cross-reactive with PS from P. aeruginosa immunotype 2 (IT-2). In the current study, we observed that, in addition, these two strains differ in their ability to respond to low immunizing doses of IT-1 PS. C3H mice generated a protective antibody response after a 1-microgram immunization, whereas BALB/c mice failed to produce protective antibody after receiving 1 microgram of PS. Both strains generated protective levels of antibody after a 50-micrograms immunization. Genetic analysis of these response patterns indicates that the ability to produce cross-reactive antibody and the ability to respond to a 1-microgram immunization are independently inherited traits. In addition, the responsiveness of C3H mice to a 1-microgram immunization with the production of protective levels of antibody is not linked to the mouse major histocompatibility (H-2) complex, to sex-linked genes, or to a single gene outside the H-2 complex.

High-molecular-weight polysaccharide from Pseudomonas aeruginosa immunotypes 1 and 2 gave cross-protection in outbred CD-1 mice challenged with the heterologous immunotype organism. Both active immunization with 50 micrograms of polysaccharide, as well as passive transfer of immune serum were effective. The basis for this cross-protection is the ability of high doses of polysaccharide to induce antibody formation to both homologous and heterologous immunotype determinants.

A rabbit model of catheter-related bacteremia was developed to study immunity to the capsular polysaccharide/adhesin (PS/A) of coagulase-negative staphylococci. Catheters colonized by coagulase-negative staphylococci were inserted into the right jugular vein and attached to a subcutaneous osmotic pump, and blood cultures were obtained over 14 days. Nonimmune rabbits were bacteremic for 6-8 days after infection, hypoglycemic, and hyperlipidemic and had strong immune responses to teichoic acid but not to PS/A. PS/A immunization, but not teichoic acid immunization, reduced the number of bacteremic days by approximately 60%, diminished the hypoglycemia and hyperlipidemia, and ablated the immune responses to teichoic acid. Passive infusion of PS/A-specific polyclonal and monoclonal antibodies using a separate, noninfected catheter-pump combination implanted in the left jugular protected against both bacteremia and hematogenous colonization of this contralateral catheter.

A murine corneal scratch model has been used extensively to study various aspects of the pathogenesis of Pseudomonas aeruginosa, a common etiologic agent of corneal infections. This model uses mild inhalation anesthetics which keep the animals immobile for a relatively short time and promote the interaction between the infecting organisms and the corneal wound. Under these circumstances, only a small number of P. aeruginosa isolates delivered at inocula of > 10(7) CFU are infectious. We determined that this model is useful for studying other P. aeruginosa strains given at lower doses if injectable anesthetics are administered prior to infection to keep the animals immobile for 15 to 30 min. Under these conditions, eight clinical isolates of P. aeruginosa tested at doses of 10(8) CFU per eye induced corneal perforation and/or phthisis in C3H/HeN mice. The 50% infective doses of several strains were between 3 x 10(2) and 1 x 10(5) CFU per mouse eye. When this modified anesthetic procedure was used to evaluate the roles of different P. aeruginosa virulence factors in eye infections, pathology was not observed when eyes were inoculated with 10(8) CFU of strains deficient in production of a complete lipopolysaccharide or the RpoN sigma factor. A strain with a point mutation in the fur gene, involved in production of iron-regulated factors, showed decreased virulence, while a mutant deficient in both hemolytic and nonhemolytic phospholipase C was fully virulent. By modifying the anesthesia procedure, the corneal scratch model allows rapid evaluations of the roles of P. aeruginosa virulence factors in corneal infections.

Previously, we isolated a high-molecular-weight immunogenic polysaccharide (designated PS) from Pseudomonas aeruginosa immunotype 1 (IT-1). The method which we used was modified to permit the isolation of a similar PS from P. aeruginosa IT-2. This antigen was composed primarily of carbohydrate, had a complex monosaccharide composition, including sugars not found in the lipopolysaccharide, and was nonpyrogenic in rabbits and nontoxic in mice at high doses. This material protected mice from challenges with live homologous cells. P. aeruginosa IT-2 PS gave a line of identity with the O side chain of the lipopolysaccharide, but different from this polysaccharide in molecular weight, chemical composition, and ability to immunize mice actively. Lipopolysaccharide from P. aeruginosa IT-2 contained an immunological determinant not found on P. aeruginosa IT-2 PS, which was detected due to its stability during treatment with dilute alkali. Thus, we recovered a high-molecular-weight PS antigen from P. aeruginosa IT-2, which was serologically identical to the lipopolysaccharide O side chain but was chemically and physically distinct. Also, like P. aeruginosa IT-1 strains, P. aeruginosa IT-2 contains an alkali-stable immunodeterminant on the lipopolysaccharide that may represent a core-like antigen.