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Biomedical subjects

D E Woods

Publications and source records attributed to D E Woods.

At least 19 recordsLinked to original sources
PubMed1992

The effects of purified 25-kDa lipase from a clinical isolate of Pseudomonas cepacia in the lungs of rats.

Nanogram quantities of a 25-kDa lipase purified from culture supernatants of Pseudomonas cepacia 90ee, a sputum isolate from a cystic fibrosis (CF) patient, were placed in the lungs of healthy rats. The resulting pathological changes included large amounts of proteinaceous exudate, the accumulation of polymorphonuclear leukocytes and red blood cells, and disorganization of alveolar structure. Pseudomonas cepacia 90ee immobilized in agar beads was also placed in the lungs of rats in a model of chronic infection. This resulted in bronchopneumonia and a milder inflammatory response than that elicited by the purified enzyme.

Animals↗
PubMed1991

In vivo regulation of virulence in Pseudomonas aeruginosa associated with genetic rearrangement.

A chronic pulmonary infection model was used to induce conversion to the mucoid phenotype by Pseudomonas aeruginosa PAO. At 6 months after initial inoculation, organisms isolated from infected lungs demonstrated the mucoid phenotype. Significant decreases (P less than .01) were seen in the levels of exotoxin A, exoenzyme S, phospholipase C, and pyochelin produced by the mucoid P. aeruginosa PAO rat lung isolates that returned to parental levels after reversion to the nonmucoid phenotype. In addition, lipopolysaccharide of the mucoid PAO lung isolates failed to react with serotype B-specific antibody in contrast to the original PAO and the revertant PAO organisms. Digestion of chromosomal DNA and hybridization with P. aeruginosa virulence factor-specific probes demonstrated that conversion to the mucoid phenotype was associated with rearrangement of chromosomal DNA upstream of the exotoxin A gene. Analysis of DNA from revertant organisms revealed hybridization patterns identical to the original PAO organism.

Animals↗
PubMed1991

Hygromycin B inhibits synthesis of murine coronavirus RNA.

The aminoglycoside hygromycin B inhibits the infection of mouse hepatitis virus (MHV) A59 both in vitro and in vivo. In probing the mechanism by which hygromycin B exerts its antiviral effect, we describe here studies which point to inhibition of viral RNA synthesis as the key step in virus replication which is affected by the drug. Cells which are infected with MHV do not take up higher levels of hygromycin B than do uninfected ones. Comparative assays of MHV replication and MHV protein synthesis in the presence of hygromycin B and another aminoglycoside, neomycin, indicate that hygromycin B is the more-effective antiviral agent and that its antiviral activity likely does not involve phosphoinositide-mediated processes such as those inhibited by neomycin.

Animals↗
PubMed1991

Pseudomonas aeruginosa exoenzyme S is an adhesion.

Exoenzyme S from Pseudomonas aeruginosa has been studied as an adhesion for glycosphingolipids and buccal cells. Binding of exoenzyme S to gangliotriosylceramide (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer), gangliotetraosylceramide (Gal beta 1-3 GalNAcT beta 1-4 Gal beta 1-4Glc beta 1-1Cer), and lactosylceramide (Gal beta 1-4Glc beta 1-1Cer) separated on thin-layer chromatograms was observed. Binding curves for exoenzyme S with dilutions of gangliotetraosylceramide immobilized on plastic plates were similar to previously reported results for the intact bacteria. Binding of exoenzyme S to sialylated counterparts of these glycosphingolipids was not seen, indicating that the addition of a sialic acid residue interferes with binding. Exoenzyme S and monoclonal antibody to exoenzyme S inhibit the binding of P. aeruginosa to buccal cells. The presence of exoenzyme S on the surface of P. aeruginosa was detected by immunogold labeling of bacteria with antibodies to exoenzyme S. Results of these studies led us to conclude that exoenzyme S is an important adhesion of P. aeruginosa.

ADP Ribose Transferases↗
PubMed1990

Cloning and expression of the Pseudomonas aeruginosa exoenzyme S toxin gene.

The gene for exoenzyme S, an ADP-ribosyl transferase, was cloned from Pseudomonas aeruginosa strain DG1 using an oligonucleotide probe based on the partial N-terminal amino acid sequence to screen a library of DG1 SstI fragments inserted into pKT230 in Escherichia coli DH1. A positive clone, designated pPD3, hybridized with the oligonucleotide probe and contained a 15 kb SstI insert. In E. coli minicells pPD3 expressed a single protein of Mr 68,000. This protein was localized primarily in the periplasm in E. coli. A 3.6 kb HindIII-BamHI fragment was subcloned into the vector pT7-4 which contains the promoter from bacteriophage T7 to construct pT7-4HB. In E. coli strains expressing the T7 RNA polymerase on a second plasmid, the Mr 68,000 protein was expressed and shown to react with antibodies to exoenzyme S. No enzymatic activity was detected in cell sonicates or culture supernatants of E. coli (pPD3). Cell sonicates of E. coli (pT7-4HB) however were cytotoxic to HeLa cells and this cytotoxicity was neutralizable with anti-exoenzyme S antiserm. Thus, exoenzyme S expressed in E. coli is toxic but not enzymatically active. When plasmids carrying the exoenzyme S gene were introduced into P. aeruginosa, there was a significant increase in ADP-ribosyl transferase activity, indicating that the plasmid encoded protein is enzymatically active in P. aeruginosa.

ADP Ribose Transferases↗
PubMed1990

Subinhibitory concentrations of tetracycline inhibit surface expression of the Pseudomonas aeruginosa ferripyochelin binding protein in vivo.

The effects of subinhibitory concentrations of tetracycline on surface expression of Pseudomonas aeruginosa ferripyochelin binding protein (FBP) and P. aeruginosa virulence in a pulmonary infection model in rats were examined. Rats were inoculated intratracheally with P. aeruginosa strain DG1 embedded in agar beads. One half of the inoculated animals served as untreated controls while the other half received daily injections of 15 mg/kg tetracycline. FBP was shown to be surface exposed in bacteria isolated from control animals using indirect immunofluorescence and immunoelectron microscopy. No FBP was detectable, however, on the surface of bacteria isolated from the lungs of animals treated with tetracycline. The numbers of bacteria recovered from the lungs of infected animals did not differ between control and tetracycline treated groups, although the degree of pathology observed in tetracycline treated animals was significantly lower than in untreated controls (P = 0.002, one way ANOVA). Sub-inhibitory doses of tetracycline reduced proteolytic activity in vitro, but had no effect on the activities of exotoxin A or exoenzyme S. These studies suggest that exposure to subinhibitory concentrations of tetracycline can repress FBP surface expression as well as proteolytic activity in P. aeruginosa leading to a significant decrease in lung injury during infections due to this organism.

Animals↗
PubMed1990

3-deoxy-D-manno-2-octulosonic acid in the lipopolysaccharide of various strains of Pseudomonas cepacia.

Six clinical isolates of Pseudomonas cepacia (representing the five serotypes of the organism) were examined for the presence of 3-deoxy-D-manno-2-octulosonic acid (KDO) in their lipopolysaccharide (LPS). Purified LPS was examined for the presence of KDO by the thiobarbituric acid (TBA) assay and by gas chromatography. All strains possessed KDO. One strain possessed KDO that was detectable by the TBA assay after mild acid hydrolysis with 0.04 M H2SO4 at 100 degrees C for 20 min. The other strains also possessed KDO but it was only demonstrable by the TBA assay after strong acid hydrolysis (4 M HCl for 60 min at 100 degrees C). All six purified LPS preparations were shown to possess KDO by two separate gas chromatography procedures. LPS isolated from the six strains of P. cepacia was toxic for mice.

Animals↗
PubMed1989

Subinhibitory antibiotics reduce Pseudomonas aeruginosa tissue injury in the rat lung model.

Using the agar-bead rat lung model, we evaluated the effects of subinhibitory antibiotic treatment upon Pseudomonas aeruginosa exoenzyme expression and lung injury in vivo. One hundred and twenty-eight animals were separated into two groups of 64 animals. One group was inoculated with P. aeruginosa DG1, and the other with P. aeruginosa 3740. Each of these two groups was divided into four subgroups of 16 animals on the basis of ten-day antibiotic treatment with ciprofloxacin, tobramycin and ceftazidime or untreated controls. P. aeruginosa DG1 is non-mucoid and expresses significant yields of exoenzyme S and elastase. P. aeruginosa 3740 is a mucoid organism isolated from the sputum of a cystic fibrosis patient, and demonstrates modest elastase activity only (10% of DG1 levels). Lung bacterial counts were similar in treatment and control groups. Lungs from antibiotic-treated rats demonstrated fewer histological changes than those from untreated animals (P less than 0.001). DG1 lung isolates from antibiotic-treated animals yielded less elastase and exoenzyme S compared with isolates from untreated animals (P less than 0.001). No detectable decrease in elastase or mucoid phenotype was observed in 3740 lung isolates from antibiotic treated animals. Thus, antibiotic protection against lung injury by P. aeruginosa may involve modulation of virulence factors.

ADP Ribose Transferases↗
PubMed1989

Production of an extracellular toxic complex by various strains of Pseudomonas cepacia.

Six isolates of Pseudomonas cepacia, representing various serotypes of the organism and possessing similar degrees of virulence in mice, were examined for their production of an extracellular toxic complex (ETC) in vitro. This compound is lethal for mice and produces extensive lung pathology in rats; it is composed of a surface carbohydrate antigen, lipopolysaccharide and protein. All six isolates produced the ETC. The LD50 values for the six ETC preparations ranged from 395 micrograms for strain 61g to 1750 micrograms for strain 90ee. Only two of the six ETC preparations contained ketodeoxyoctonate detectable by the methods used, and these two were the most toxic. Rabbit antiserum to the ETC of a serotype D strain could significantly protect mice only against serotype D strains. Examination of the various phases of growth of P. cepacia showed that there was extracellular release of the ETC beginning in the early logarithmic phase and continuing through the late stationary phase. The presence of the ETC in the supernatant fluids was due to release of this material rather than to cell lysis. In addition, at least one strain of P. cepacia was shown to produce an alginic acid-like compound.

Animals↗
PubMed1989

Inhibition of Pseudomonas aeruginosa exoenzyme expression by subinhibitory antibiotic concentrations.

We examined the effects of subinhibitory concentrations of ciprofloxacin, tobramycin, and ceftazidime on Pseudomonas aeruginosa exoenzyme expression in vitro and in vivo. Exotoxin A, exoenzyme S, phospholipase C, elastase, and total protease activities were suppressed by antibiotics at concentrations as low as 1/20 of the MIC over a 24-h period in broth. Continuous 10-day exposure of P. aeruginosa DG1 broth cultures to antibiotic levels equal to 1/10 of the MIC reduced exoenzyme S activity in all treatment groups. Elastase activity was reduced only by ciprofloxacin and tobramycin treatment. This suppressive effect of the antibiotics persisted throughout the 10 days and was not influenced by the increase in MIC of ciprofloxacin detected during the course of the experiment. Rats chronically infected with P. aeruginosa were treated with subinhibitory doses of antibiotics and compared with untreated controls. Bacterial numbers in lung homogenates from each of the four study groups were identical. However, the lungs from antibiotic-treated rats had significantly less histological damage than those from control rats (P less than 0.001). The protective effect was greatest for ciprofloxacin and tobramycin. Further, P. aeruginosa isolates from ciprofloxacin- and tobramycin-treated rats demonstrated significantly less exoenzyme S and elastase activity than isolates from untreated rats (P less than 0.001). Isolates from ceftazidime-treated lungs expressed less exoenzyme S activity (P less than 0.001) but an equivalent amount of elastase activity as isolates from controls. The suppression of P. aeruginosa exoenzymes may arrest progressive lung injury during chronic P. aeruginosa lung infections.

ADP Ribose Transferases↗
PubMed1989

Purification and characterization of an extracellular protease from Pseudomonas cepacia.

An extracellular proteinase (PSCP) produced by Pseudomonas cepacia was purified from culture supernatants by ammonium sulfate precipitation, anion exchange chromatography on DEAE-Sephacel, and G200 gel filtration chromatography. The protease has an apparent Mr of 34,000 by electrophoresis. Substrates cleaved by the protease include gelatin, hide powder, and collagen but not human immunoglobulin G (IgG), IgM, secretory IgA, or IgA. The enzyme had the characteristics of a metalloprotease, a pH optimum of 6, and a temperature optimum of 45 degrees C. Intratracheal instillation of purified PSCP into rat lungs produced a bronchopneumonia characterized by polymorphonuclear cell infiltration and proteinaceous exudation into large airways. Rats responded immunologically to active immunization with PSCP, but this response was not protective against subsequent lung infection with P. cepacia. PSCP was shown to have antigenic similarity with Pseudomonas aeruginosa elastase by an immunoblotting technique. Sera from 10 cystic fibrosis patients, with and without a previous history of P. cepacia colonization, were shown to possess antibody reactive against PSCP.

Amino Acids↗
PubMed1989

Development of a rat model for respiratory infection with Bordetella pertussis.

Considerable effort directed toward designing a safe and effective vaccine for Bordetella pertussis in which the cellular and/or acellular antigens necessary to confer immunity are known has been hampered by lack of information on the pathogenesis of the natural infection. The study presented here describes an animal model of lung infection by B. pertussis encased in agar beads in adult (200- to 220-g) male Sprague-Dawley rats. At 3 and 7 days after inoculation with phase I B. pertussis, organisms could be recovered from the lungs of rats; however, organisms were not recoverable at days 10 and 14 but reappeared in lung homogenates at day 21. Histopathological examination revealed findings similar to those seen in human disease. At day 3, a mild lymphocytic infiltrate was present in the bronchi, with progressive lymphoid hyperplasia peribronchially. By day 7, a necrotizing inflammation of the tracheobronchial mucous membranes, characterized by both mononuclear and polymorphonuclear cells, was noted. Phase III B. pertussis organisms were not recoverable from the lungs of inoculated rats at day 3 after inoculation, nor were histological changes noted in these animals. Clinical findings in phase I B. pertussis-infected rats included hypoglycemia, circulating lymphocytosis, and paroxysms in which air was forcibly expelled from the mouth or nose.

Animals↗
PubMed1988

Culture-amplified immunological detection of Mycoplasma pneumoniae in clinical specimens.

We have employed a monoclonal antibody directed to a Mycoplasma pneumoniae membrane-associated protein antigen in a culture-amplified detection system. Eleven culture-positive sputum specimens were detected significantly more early by the immunoassay. Specimens culture-positive for other probable mycoplasma spp. were negative by this technique.

Antibodies, Monoclonal↗
PubMed1988

Effect of pyochelin on Pseudomonas cepacia respiratory infections.

Exogenously supplied pyochelin influenced the virulence of Pseudomonas cepacia pyochelin-negative strains in a chronic pulmonary infection model in rats. Groups of rats were inoculated transtracheally with agar beads containing P. cepacia or P. aeruginosa strains, saturated with either pyochelin or PBS. Supplementation of the inocula with pyochelin had no effect on the number of bacteria recovered from the lungs. The availability of pyochelin, however, increased the degree of pathology observed in lungs infected with pyochelin-negative strains of P. cepacia. The area of pathological involvement in the lung was about 2-fold larger, when pyochelin was present. Inclusion of pyochelin in the inoculum had no effect on the degree of pathology observed in lungs infected with a pyochelin-positive P. aeruginosa strain. Pyochelin was shown to stimulate in vitro growth of P. cepacia, but it had no effect on production of lipase or protease, factors which may be involved in P. cepacia virulence. These studies support our hypothesis that pyochelin may be important for dissemination in P. cepacia infections.

Animals↗
PubMed1988

Alteration of pulmonary structure by Pseudomonas aeruginosa exoenzyme S.

Intratracheal administration of purified Pseudomonas aeruginosa exoenzyme S elicited extensive, grossly observable damage in the rat lung within 2 h. Light and electronmicroscopy revealed injury and necrosis of bronchial epithelium, type I pneumocytes and capillary endothelial cells after 1 h; associated haemorrhage, fibrinous exudation and released type II cell lamellar bodies in alveolar lumina after 1-12 h; progressively increasing accumulations of polymorphonuclear leucocytes in the bronchi and alveoli and in alveolar septae (interstitial pneumonia) after 1-12 h; collapse of alveolar septal connective tissue and damage to pulmonary arterioles and venules. Treatment of monolayer cultures of bronchial fibroblasts with purified exoenzyme S elicited vacuolation of the cells with apparent membrane damage as revealed by light and electronmicroscopy. In-vivo production and activity of P. aeruginosa exoenzyme S may be an important pathogenicity determinant in the necrotising lung injury characteristic of P. aeruginosa pneumonia.

ADP Ribose Transferases↗