Antigenic complexes of Pseudomonas aeruginosa slime: their isolation and biological properties

The possibility of using antigenic complexes contained in the extracellular slime of P. aeruginosa clinical strains belonging to different serological groups as the components of a chemical vaccine has been revealed. Animal experiments have demonstrated a high immunogenicity of these preparations, as well as their low toxicity. The use of slime antigens stimulates the production of specific antibodies exerting a protective action against infection with homologous P. aeruginosa strains.     

Involvement of skeletal muscle gene regulatory network in susceptibility to wound infection following trauma

Despite recent advances in our understanding the pathophysiology of trauma, the basis of the predisposition of trauma patients to infection remains unclear. A Drosophila melanogaster/Pseudomonas aeruginosa injury and infection model was used to identify host genetic components that contribute to the hyper-susceptibility to infection that follows severe trauma. We show that P. aeruginosa compromises skeletal muscle gene (SMG) expression at the injury site to promote infection. We demonstrate that activation of SMG structural components is under the control of cJun-N-terminal Kinase (JNK) Kinase, Hemipterous (Hep), and activation of this pathway promotes local resistance to P. aeruginosa in flies and mice. Our study links SMG expression and function to increased susceptibility to infection, and suggests that P. aeruginosa affects SMG homeostasis locally by restricting SMG expression in injured skeletal muscle tissue. Local potentiation of these host responses, and/or inhibition of their suppression by virulent P. aeruginosa cells, could lead to novel therapies that prevent or treat deleterious and potentially fatal infections in severely injured individuals.     

The modelling of a Pseudomonas aeruginosa wound process

P. aeruginosa wound infection was induced in white mice to test new preparations against P. aeruginosa. This model ensures the nearest approximation to the course of P. aeruginosa chronic infection, i.e. it reproduces the focus of inflammation and the prolonged course of the disease (the positive decision on application No. 4, 324, 555 of November 2, 1987, has been obtained). The essence of the method consists in obtaining the model of P. aeruginosa wound infection by a combined trauma of the skin (burn and incision): P. aeruginosa is introduced in a dose of 6 X 10(9)-8 X 10(9) microbial cells into the burn blister through the incision made 3 hours after the burn and then 20-24 hours later in a dose of 10(9)-2 X 10(9) microbial cells, introduced under the crust formed by that time.     

The protective activity of 2 normal immunoglobulin preparations for intravenous administration in experimental Pseudomonas aeruginosa infection

The antibody levels in 18 batches of the preparations of human immunoglobulin, Immunovenin and Immunovenin-Intact, for intravenous injection were determined in the enzyme immunoassay with the use of the mixture of P. aeruginosa lipopolysaccharide antigens of seven immunotypes. The average antibody titers in these preparations were identical. The preparations were found to have protective action against P. aeruginosa experimental infection in mice.     

Immunotyping of freshly isolated strains of Pseudomonas aeruginosa

During 1972-1982 the bacteriological study of 1391 patients with thermal burns was carried out. As the result of clinico-bacteriological studies, the occurrence of P. aeruginosa was found to increase from 39.3% to 70.5% during this period. The immunotyping of P. aeruginosa cultures isolated in 3 burn-treatment centers showed that strains belonging to immunotypes 2, 3, 7 and 3/7 were most frequently isolated from burn wounds. These strains were found to be the cause of hospital infections in burn-treatment hospitals. In connection with the data thus obtained immunological preparations intended for the prophylaxis and treatment of P. aeruginosa infection should include P. aeruginosa strains, immunotypes 2, 3, 7 and 3/7.     

Immunological study of the cellular components of Pseudomonas aeruginosa. VI. The toxicity and protective properties of its mucus and its fractions]

Capsule-like mucus was obtained from two newly isolated mucoid strains of P. aeruginosa and then treated with ethanol. The mucus was fractionated by the method of differential centrifugation (at 15, 000 g for 1 h, at 105, 000 or 170, 000 g for 3 h) and gel chromatography in columns packed with Sepharose 4B. The sediment fractions contained 30--80% of high molecular polysaccharide protein (peptide) mucus components which were toxic for mice and protected 25--77% of rats against experimental P. aeruginosa infection. The supernatant fluid fractions contained 60--80% of predominantly protein components with molecular mass between 20,000 and 60,000 daltons. These mucus components were slightly toxic for mice and rats and protected 80--100% of rats against P. aeruginosa infection.     

Immunologic study of the cellular components of bacillus pyocyaneus. V. Antigenic analysis of slime and its fractions]

Various slime fractions were obtained from newly isolated mucoid strains of P. aeruginosa by the method of ultrafiltration or differential centrifugation with subsequent gel chromatography. Purified slime was found to react with a broader spectrum of typing O sera than the corresponding cell wall lipopolysaccharides. Erythrocytic diagnostic preparations produced on the basis of slime antigens allowed to reveale the presence of circulating antibodies against P. aeruginosa. The slime components with molecular weight of 30,000--100,000 daltons and greater contained common antigenic determinants, and the slime components with molecular weight of 10,000--30,000 daltons contained both specific antigenic determinants and those also common to the high molecular components.     

Protective effects of affinity-purified antibody and truncated vaccines against Pseudomonas aeruginosa V-antigen in neutropenic mice

Virulent P. aeruginosa strains express PcrV, one of the translocational components of the type III secretion system. PcrV has been reported to be a protective antigen against lethal P. aeruginosa infection. The PcrV region, which contributes to protective immunity against P. aeruginosa infection, was investigated by using genetically engineered, truncated PcrV proteins and affinity-purified anti-PcrV antibodies against the truncated PcrV proteins. The efficacy of active and passive immunization against PcrV was tested in mice with cyclophosphamide-induced immunosuppression by intraabdominal challenge of P. aeruginosa . Active immunization with either full-length PcrV1-294 or PcrV139-294 significantly improved the survival of mice infected with P. aeruginosa , while PcrV139-258, PcrV139-234, PcrV197-294, and PcrV261-294 were not protective. These results suggest that an effective PcrV vaccine needs to contain not only the Mab166 epitope (PcrV144-257) but also the carboxyl terminal tail of PcrV. In the case of passive immunization, administration of affinity-purified anti-PcrV IgG against either PcrV1-294 or PcrV139-258 showed significantly higher efficacy against lethal P. aeruginosa infection than did original anti-PcrV IgG and Mab166. The increased efficacy of affinity-purified anti-PcrV IgG implies that more potent anti-PcrV strategies are possible. The results of this study are crucial to the development of an effective PcrV vaccine for active immunization and to an appropriate blocking anti-PcrV antibody against P. aeruginosa infection in humans.     

Protective cross activity of the extracellular mucus of Pseudomonas aeruginosa

Extracellular slime was isolated from 15 P. aeruginosa typing strains of different O-serotypes (immunotypes). The isolated slime, partially purified by ethanol precipitation, was later referred to as crude slime. Glycolipoprotein was obtained from crude slime and lipopolysaccharide (LPS) was obtained from acetone-dried microbial cells by the method of aqueous-phenol extraction. All these antigenic preparations were studied in the active mouse cross-protection tests: immunized mice were challenged with 7 strains of different immunotypes, strain No. 170 019 or toxigenic strain PA-103. In experiments on mice the slime of different P. aeruginosa serotypes (immunotypes) was found to stimulate immunity to intraperitoneal infection with P. aeruginosa, both homologous or heterologous in respect to their immunotype, including toxigenic strains. Slime glycoprotein also stimulated active cross-immunity in mice, but the level of this immunity was higher than that of immunity stimulated by crude slime. LPS showed mostly weak protective activity in experiments on mice.     

铜绿假单胞菌及L型诱导巨噬细胞凋亡的实验研究

目的研究铜绿假单胞菌（PA）及L型诱导巨噬细胞凋亡的能力,比较二者的差异。方法用生物素断端标记（TUNEL）法检测PA及L型感染巨噬细胞2、4、8、12、16和20h后各时间段的细胞凋亡率,Giemsa染色观察细胞凋亡情况,硝酸还原酶法检测培养液中一氧化氮（NO）的浓度变化。结果 PA及L型能诱导巨噬细胞发生凋亡,与对照组比较差异有统计学意义（P〈0.05）;L型诱导细胞的凋亡率弱于原菌（P〈0.05）;PA及L型感染组培养液NO浓度较对照组明显升高（P〈0.05）。结论 PA及L型可诱导巨噬细胞发生凋亡,L型较其原型诱导细胞凋亡的能力弱,NO可能在巨噬细胞凋亡中发挥一定作用。PA及L型可通过诱导巨噬细胞凋亡,发挥致病作用。     

Experimental immunological effectiveness and safety of pyoimmunogen vaccine against Pseudomonas aeruginosa infection

Pyoimmunogen, a polycomponent vaccine against P. aeruginosa infection, has been obtained in laboratory and semi-industrial conditions. The microbial biomass obtained from the strains belonging to O-serotypes (immunotypes) most frequently occurring in clinical practice has been used for producing protective antigens. The preparations have been found to contain proteins (peptides) and carbohydrates in the ratio 6 : 1 to 8 : 1, as well as traces of 2-keto-3-desoxyoctanate, which is indicative of the low content of endotoxin. The immunogenicity of the preparations has been studied experimentally by the active immunization of mice. In these experiments the animals vaccinated in a single injection were found to be protected from challenge with both homologous and heterologous P. aeruginosa strains. The high level of protection from infection caused by toxigenic strain PA-103 was registered. The preparations have low toxicity: LD50 for mice exceeds 2 mg (in protein content): after the multiple administration (7-10 times) of the preparation to mice and rats the weight of the experimental animals was not significantly different from the weight of the control animals.     

Use of an immunoenzyme method for detecting specific antibodies to Pseudomonas aeruginosa in patients with acute and chronic lung diseases

ELISA in which P. aeruginosa slime preparations are used as antigens permits the detection of antibodies to this microorganism in the blood sera of patients with acute and chronic pulmonary diseases induced by P. aeruginosa. This assay makes it possible to find out the etiology of infection even before the results of bacteriological investigation are obtained.     

Results of using polyvalent Pseudomonas aeruginosa vaccine in children with burns by various medical centers

Polyvalent Pseudomonas aeruginosa vaccine, prepared at the Institute of Hematology from 10 hospital strains isolated from burn wounds, was administered to 32 children with extensive and deep burns. The vaccine was well tolerated. The vaccine produced a high degree of the immunity against Pseudomonas aeruginosa infection. Agglutinin serum titre increased significantly. Vaccination either prevented or inhibited the infection of burn wounds with Pseudomonas aeruginosa in all immunized children. The symptoms of Pseudomonas aeruginosa infection usually disappeared following one or two vaccinations. Bacteriemia caused by P. aeruginosa was not observed in 31 out of 32 children. In the remaining child transient bacteriemia was noted. No septicemia caused by P. aeruginosa was seen. Due to the high efficiency of the polyvalent P. aeruginosa vaccine all burned children with burns exceeding 10% of the total body surface should by vaccinated to prevent the life-threatening infections with Pseudomonas aeruginosa.     

Principle of constructing polyvalent Pseudomonas aeruginosa vaccines]

Dependence of the range of protective action of P. aeruginosa vaccine on the number of its composites was studied. A principle of the selection of strains who vaccines differed in vivo by immunological specificity was applied to construction of the experimental preparations and modelling a polyvalent vaccine. Increase of the number of components in the vaccine was accompanied by increase of its protective action range. However, with the increase of the number of polyvaccine components in the polyvaccine the accretion of the protective effect expressed in the mean protective index per component displayed a gradual reduction. It was calculated theoretically that a 6--7-component vaccine should provide protection from 94--96% of the P. aeruginosa strains; as to further increase of the number of components--it would induce overloading of the vaccine with a possible absence of any effect.     

Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa , suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real-time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function.     

The protective effect of preparations made from plant seedlings in experimental Pseudomonas aeruginosa infection

The influence of preparations obtained from oat and wheat seedlings (immunostimulating factors IF-1 and IF-2, respectively) on the natural resistance of mice to P. aeruginosa infection was studied. IF-1 and IF-2 were introduced intraperitoneally in a single injection in doses of 100 micrograms and 1000 micrograms per mouse 2 and 7 days prior to the inoculation of P. aeruginosa strain 8 in doses of 1 and 10 LD50. The presence of substances capable of stimulating the immunobiological reserves of the body in actively growing plants (seedlings) was shown.     

Control of Pseudomonas aeruginosa in the lung requires the recognition of either lipopolysaccharide or flagellin

Acute lung infection due to Pseudomonas aeruginosa is an increasingly serious problem that results in high mortality especially in the compromised host. In this study, we set out to ascertain what components of the TLR system are most important for innate immunity to this microorganism. We previously demonstrated that TLR2,4-/- mice were not hypersusceptible to infection by a wild-type P. aeruginosa strain. However, we now find that mice lacking both TLR2 and TLR4 (TLR2,4-/- mice) are hypersusceptible to infection following challenge with a P. aeruginosa mutant devoid of flagellin production. We demonstrate that this hypersusceptibility is largely due to a lack of innate defense by the host that fails to control bacterial replication in the lung. Further evidence that a response to flagellin is a key factor in the failure of TLR2,4-/- mice to control the infection with the mutant strain was obtained by demonstrating that the intrapulmonary administration of flagellin over a 18 h period following infection, saved 100% of TLR2,4-/- mice from death. We conclude that the interactions of either TLR4 with LPS or TLR5 with flagellin can effectively defend the lung from P. aeruginosa infection and the absence of a response by both results in hypersusceptibility to this infection.     

Endotoxin of Pseudomonas pseudomallei detected by the body weight-decreasing reaction in mice and comparison of it with those of P. cepacia and P. aeruginosa.

The heat-treated whole cells, culture supernatants, and extracted endotoxin preparations of Pseudomonas pseudomallei were examined for endotoxin by the mouse body weight-decreasing (BWD) test. The experiments were conducted also with those of P. cepacia and P aeruginosa. Endotoxin was detected in all the samples of P. pseudomallei. Endotoxin of P. cepacia was detected in whole cells, but not in culture supernatant. The BWD activity of P. aeruginosa was 30 times as high as that of P. pseudomallei. This result was confirmed by the experiments with endotoxin preparations. In the limulus amebocyte lysate gelation (LAL) test, however, the endotoxin preparations of the two species showed the same level of activity.