Pseudomonas aeruginosa exotoxin A antibodies in rapidly deteriorating chronic leg ulcers.
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ADP Ribose Transferases↗
Biomedical subjects
G Döring
Publications and source records attributed to G Döring.
Cleavage of lymphocyte surface antigens CD2, CD4, and CD8 by polymorphonuclear leukocyte elastase and cathepsin G in patients with cystic fibrosis.
Polymorphonuclear leukocytes (PMN) accumulating in airways of patients with cystic fibrosis (CF) as a response to chronic endobronchial bacterial lung infection, release lysosomal serine proteinases such as PMN-elastase at concentrations of approximately 0.5 microM to 5 microM into the airway lumen. Immunohistology of CF lung material and fluorescence activated cell sorter analysis of sequential CF bronchoalveolar lavages demonstrated loss of the CD4 and CD8 Ag on CD3+ T lymphocytes in sputum-filled airways. In 10 CF sputum samples 1.0%, 19.1%, and 15.7% of all CD3+ T lymphocytes expressed CD4, CD8, and CD2, respectively. Incubation of CF sputum supernatant fluids with peripheral blood T lymphocytes resulted in total reduction of CD4 and CD8 but not CD2. Addition of alpha 1-proteinase inhibitor abolished surface Ag cleavage completely. Purified PMN-elastase and cathepsin G cleaved CD2, CD4, and CD8 on peripheral blood T lymphocytes at proteinase concentrations of 0.83 to 8.3 microM in a dose-dependent manner. Cleaved CD4 and CD8 were reexpressed on the surface of T lymphocytes after 24 h in the absence of PMN-elastase. Incubation of a CD4+ T cell clone with PMN-elastase lead to a significant reduction of cytotoxicity toward target cells and significantly reduced IL-2 and IL-4 production. The results suggest a temporary functional impairment of T lymphocytes in foci of high inflammation characterized by stimulated PMN, which may lower tissue destruction.
Adult↗
Double-blind comparison of the acute effects of two relevant doses of oral nicorandil on central hemodynamics, left ventricular function, and myocardial contractility.
Nicorandil is a nicotinamide derivative with potent vasodilator properties. Oral and sublingual administration of this compound in patients with coronary artery disease resulted in a predominant reduction of afterload and a small decrease in left ventricular preload. The effects of nicorandil in different doses on contractile left ventricular (LV) function, however, are not well defined. The aim of the present study was to assess the effects of nicorandil on hemodynamics, left ventricular volume and function, as well as the LV contractility index dP/dt measured by Millar-tip manometers. A total of 16 patients with coronary artery disease were included in this study. After diagnostic coronary angiography and ventriculography, the patients randomly received either 10 or 20 mg of oral nicorandil. The hemodynamic parameters, cardiac output, and LV end-diastolic and systolic pressures were determined after 15, 30, and 60 minutes, and a second angiogram was performed 60 minutes after administration. The predominant effect was a decrease in the mean aortic pressure with a concomitant decrease in the peripheral arterial resistance. Clinically relevant changes in aortic pressure, systemic vascular resistance, and rate-pressure product, however, were documented only after oral administration of 20 mg nicorandil, as opposed to minimal changes in the 10 mg group. LV volume and cardiac output were not changed significantly. LV contractility remained unchanged during the observation period. The hemodynamic profile of this compound is dose-dependent afterload reduction without a change in contractility. Because there was no concomitant increase in heart rate, calculated oxygen consumption was reduced, especially in the higher dose group.
Adult↗
Parenteral application of a Pseudomonas aeruginosa flagella vaccine elicits specific anti-flagella antibodies in the airways of healthy individuals.
To test the hypothesis that a Pseudomonas aeruginosa flagella (FLA) vaccine, intramuscularly administered, elicits specific antibodies in the respiratory tract, antibody titers against FLA were determined in sera and bronchoalveolar lavage (BAL) fluids of 10 healthy human adults before and after immunization. Immunization produced significantly increased anti-FLA antibody titers in all sera (mean reciprocal titers: IgG: 26,711; IgA: 2,767; IgM: 421) and on respiratory epithelial lining fluids (mean reciprocal titers: IgG: 112; IgA: 76; sIgA: 26). Significant differences in class-specific serum and BAL anti-FLA titers suggested airway antibody production. The study shows that parenteral immunization provides not only high systemic antibody titers but also local antibody production in the human respiratory tract.
Adult↗
[Reactive oxygen species and arachidonic acid metabolites of polymorphonuclear leukocytes].
The activation of polymorphonuclear leukocytes by stimulants from host or parasites leads to various metabolic processes and to enhanced oxygen uptake. Oxygen is enzymatically processed to from reactive oxygen compounds and metabolites of arachidonic acid which are involved in killing of microbial pathogens and influence many processes in inflammation. Their activities are regulated intracellularly by antioxidants. A lack or exhaustion of these systems may lead to cell and organ damage in which DNA, proteins, lipids and sugars may be involved. Antioxidants and inhibitors of the arachidonic acid metabolism are applied to reduce these pathological changes. An essential aspect of microbial pathogenicity is the multifold modification of the formation of reactive oxygen compounds and arachidonic acid metabolites.
Animals↗
Allotypes of alpha 1-antitrypsin in patients with cystic fibrosis, homozygous and heterozygous for deltaF508.
In cystic fibrosis (CF) neutrophil released serine proteinase activity may facilitate Pseudomonas aeruginosa lung colonization, leading to chronic infection. Since such activity is mostly controlled by alpha 1-antitrypsin (alpha 1-AT), we postulated that with CF carrying deficient alpha 1-AT variants might be at higher risk for P. aeruginosa acquisition and might reveal other phenomena, specific for serine proteinase activity. In 215 Danish patients with CF, homozygous (80%) or heterozygous (20%) for the major CF mutation deltaF508, alpha 1-AT variants were determined. Carriage of deficient alpha 1-AT variants was correlated to an earlier onset of P. aeruginosa lung infection (P < 0.0001), higher total IgG (P < 0.0001), and P. aeruginosa-specific serum antibodies (P < 0.0001). The two groups did not differ in lung function, probably due to intensive antimicrobial treatment.
Adolescent↗
A new extracellular protein of Pseudomonas aeruginosa PA103 regulated by regA.
The expression of exotoxin A (ExoA) from Pseudomonas aeruginosa is influenced by iron and is under the control of the regulatory gene regA. To test whether regA plays a role in the expression of other iron-regulated proteins a RegA- mutant was constructed by insertional mutagenesis. The polypeptide pattern of this mutant (PA103R) was compared with the parental strain (PA103) and a trans-complemented strain PA103R(pREX18) after growth of the strains in conditions containing low or high concentrations of iron. An iron-regulated 42 kDa protein (RRP) was identified and purified from the culture supernatant of PA103 and PA103R(pREX18) which was missing in PA103R. Database analysis of the N-terminal sequence of this regA-regulated protein (RRP) revealed no similarity to other proteins. Preliminary investigations into the function of RRP revealed that it has no proteolytic or cytotoxic activity. Using two-dimensional electrophoretic analysis of whole cells, a technique which allowed separation of over 600 polypeptides, we were unable to identify any other iron-regulated protein whose expression was regulated by regA.
ADP Ribose Transferases↗
Iron release from transferrin by pyoverdin and elastase from Pseudomonas aeruginosa.
Pseudomonas aeruginosa produces the siderophores pyoverdin and pyochelin as well as receptors for siderophores in response to iron deprivation. Previously, it has been shown in vitro that at neutral pH purified pyoverdin acquires iron from transferrin only in the presence of P. aeruginosa elastase (LasB), which proteolytically degrades transferrin. We constructed a LasB-negative mutant, PAO1E, by insertional mutagenesis to investigate whether this mutant differs in growth from the parental strain PAO1 in an iron-depleted medium supplemented with transferrin or human serum. PAO1 and PAO1E did not differ in growth with 1.25 microM Fe2-transferrin as the only iron source. Urea gel electrophoresis indicated iron release from intact transferrin during the logarithmic growth phase of PAO1 and PAO1E. A total of 333 microM LasB was synthesized from PAO1 after onset of stationary-phase growth. Quantification of pyoverdin by spectroscopy revealed that up to 900 microM pyroverdin was produced during growth of the strains in medium supplemented with Fe2-transferrin or 10% human serum. Incubation of Fe2-transferrin and purified pyoverdin in concentrations similar to those found in the culture supernatant resulted in release iron from transferrin after 10 h at 37 degrees C. However, LasB significantly enhanced the rate constant for iron acquisition of pyoverdin from transferrin. We conclude that P. aeruginosa can use transferrin as an iron source without further need of LasB or pH changes. This is further supported by experiments with P. aeruginosa K437, which has a defective iron uptake system, and its LasB-negative mutant, K437E. Though K437 and K437E did not differ in growth with Fe2-transferrin as the only iron source, their growth was significantly reduced relative to that of PAO1 and PAO1E.
Bacterial Proteins↗
The role of neutrophil elastase in chronic inflammation.
Passively released or actively secreted elastase from neutrophils has been linked to the pathologic processes of a variety of inflammatory diseases, including idiopathic pulmonary fibrosis, rheumatoid arthritis, adult respiratory distress syndrome, and cystic fibrosis. The serine proteinase has a broad substrate specificity and may attack a number of host proteins outside of the neutrophil, including lung elastin and fibronectin. Such a proteolysis may change the normal surrounding tissue and the protein pattern of an inflammatory focus. Additionally, it acts as a potent secretagogue in minute amounts. The reason that neutrophil elastase is present in considerable concentrations outside of the neutrophil during chronic inflammation and that the major endogenous serine proteinase inhibitor for neutrophil elastase, alpha 1-proteinase inhibitor, is easily inactivated by proteolytic and oxidative attack is unclear. Released neutrophil elastase may also be involved in regulating chronic inflammation. In a feedback mechanism, neutrophil elastase inhibits neutrophil stimulation and concomitant elastase release by cleavage of immunoglobulins, complement components, and complement receptor type 1 on neutrophils. Besides a number of harmful effects of neutrophil elastase in inflammation, the latter mechanism, although considerably impairing phagocytosis, may be beneficial particularly in the light of persistent bacterial pathogens in the human lung affected by cystic fibrosis.
Animals↗
Molecular epidemiology of Pseudomonas aeruginosa in an intensive care unit.
Genotyping was used to analyse Pseudomonas aeruginosa isolates from sink drains and 15 intubated patients as part of a 3-month prospective study of strain transmission in a medical-surgical intensive care unit. Ninety percent of all washbasin drains were persistently contaminated with several P. aeruginosa genotypes. In 60% (9/15) of the patients, P. aeruginosa colonization or infection was hospital-acquired: P. aeruginosa strains isolated from these patients were present in hospital sinks or in other patients before their admission. Since all patients were immobile, personnel were the probable route of transmission of P. aeruginosa in the hospital. The mechanism of strain transmission from sinks to hands during hand washing was investigated in a children's hospital. When P. aeruginosa was present at densities of > 10(5)/c.f.u. per ml in sink drains, hand washing resulted in hand contamination with P. aeruginosa via aerosol generation in the majority of experiments or P. aeruginosa was detected using an air sampler above the washing basin. High P. aeruginosa cfu were present at 4.30 h in the eight sinks (5.4 x 10(5)-7.0 x 10(10) c.f.u./ml), whereas at 13.00 h P. aeruginosa c.f.u. were significantly lower (3.1 x 10(2)-8.0 x 10(5) c.f.u./ml). These data reveal that the danger of bacterial contamination of hands during hand washing is highest in the morning. The identified transmission routes demand more effective hygienic measures in hospital settings particularly concerning personnel hands and sink drains.
Adolescent↗
Typing of Staphylococcus aureus by pulsed-field gel electrophoresis, zymotyping, capsular typing, and phage typing: resolution of clonal relationships.
Sixty-nine Staphylococcus aureus isolates from two epidemiologically unrelated sources were typed by pulsed-field gel electrophoresis after SmaI digestion of chromosomal DNA (genome typing), and the results were compared with those obtained by other typing methods: phage typing with the international set of phages, capsular serotyping with monoclonal antibodies against capsular polysaccharides type 5 and 8, and zymotyping by polyacrylamide agarose electrophoresis for esterase polymorphism. A good correlation of S. aureus types was found by these four typing methods. Differentiation increased in the order capsular typing < zymotyping < phage typing < genome typing, yielding 2, 10, 20, and 26 different S. aureus types, respectively. Five of the 26 genome types were further divided into several subtypes revealing clonal relationships. When 36 French S. aureus isolates were compared with 33 German S. aureus isolates, 3 strains representing clonal populations were identical in both groups. S. aureus isolates from patients with cystic fibrosis were also typed at the beginning and the end of a 4-week summer camp for these patients. The results suggested a possible strain transmission during the summer camp. We conclude that genome typing by pulsed-field gel electrophoresis is a powerful tool not only for strain identification but also for the resolution of the clonal relationships of S. aureus strains.
Bacterial Typing Techniques↗
Age-dependent pulmonary clearance of Pseudomonas aeruginosa in a mouse model: diminished migration of polymorphonuclear leukocytes to N-formyl-methionyl-leucyl-phenylalanine.
Ten-, 20-, and 35-day-old mice were subjected to an aerosol containing Pseudomonas aeruginosa. The lung clearance of the organism was decreased in mice under 20 days of age. This deficiency was accompanied by decreased migration of polymorphonuclear leukocytes (PMN) to the airways in response to the P. aeruginosa challenge. Similar results were obtained in both outbred, C5-sufficient Swiss mice and inbred, C5-deficient DBA/2 mice. The diminished clearance of P. aeruginosa was related to a transient, age-related decrease in PMN chemotaxis to formyl-methionyl oligopeptides. PMN chemotaxis levels similar to those seen in adults were regained by day 35 after birth.
Age Factors↗
Comparison of four procedures for measuring elastase production by Pseudomonas aeruginosa strains from cystic fibrosis patients.
Forty-five Pseudomonas aeruginosa strains were isolated from the sputa of cystic fibrosis patients. The elastase production of each strain was assayed in the culture supernatant using four different procedures, i.e. two immunological assays (RIA and ELISA), and two enzymatic assays, the latter employing either elastin or tetraalanine as substrate, with conductometric measurement of substrate hydrolysis. Elastase concentrations were determined from standard curves prepared with the same purified elastase, and expressed in mg of elastase per litre of supernatant. The resulting values were in the range reported in the literature, and differed greatly from one strain to another (0-230 mg/l). Linear relationships were found when assays were compared in pairs. Significant correlation coefficients were obtained (r greater than 0.76, p less than 0.001) but the values were quite different for different assays. Thus, ELISA measurements were always from three to five times higher, and RIA results were from two to five times lower, than those from the other assays. Enzymatic assays with elastin gave higher values than those using tetraalanine. Most P. aeruginosa strains produce two other proteinases, alkaline proteinase and Las A protein. Both enzymes have limited elastolytic and peptidasic activities. The presence of alkaline proteinase does not result in falsely elevated elastase values, but an increase of elastase activity was observed when Las A was preincubated with elastin. Since this increase was not observed when tetraalanine was used as the substrate, the presence of Las A in the supernatants could explain the differences observed between the enzymatic assays. The assay with the synthetic substrate is therefore preferred.
Cystic Fibrosis↗
Antianginal and anti-ischemic efficacy of nicorandil in comparison with isosorbide-5-mononitrate and isosorbide dinitrate: results from two multicenter, double-blind, randomized studies with stable coronary heart disease patients.
The purpose of the two double-blind studies summarized in this article was to compare the antianginal and anti-ischemic effects of nicorandil with those of two different nitrate preparations. A total of 129 patients with stable New York Heart Association functional class II or III coronary heart disease were enrolled in the studies. Ninety-five patients received nicorandil, 34 received isosorbide dinitrate (ISDN), and 63 received isosorbide-5-mononitrate (MN). In study 1, nicorandil was compared with MN in a crossover design with 54 protocols eligible for efficacy assessment of MN and 52 eligible for nicorandil, respectively. Twenty milligrams of nicorandil and 20 mg MN administered b.i.d. for 4 weeks were equally effective in the treatment of stress-induced angina. Both drugs prolonged bicycle exercise tolerance and reduced weekly anginal attack rates. In study 2, nicorandil and ISDN were administered to two parallel groups of patients at a dose of 10 mg t.i.d. for 2 weeks and then 20 mg t.i.d. for 4 weeks. Under the assumption that the repetitive administration of nitrates with short dosing intervals might induce the development of tolerance to the nitrate mechanism of action, the t.i.d.-dosing regimen had been chosen in this study. Thirty-two protocols from those receiving nicorandil and 34 protocols from those receiving ISDN were eligible for efficacy assessment. Both drugs increased exercise capacity and reduced ST-segment depression at identical work loads with no significant difference between groups (p > 0.05). For both drugs, the higher doses were more effective than the lower doses. tolerance to the nitrate mechanism of action did not develop with either drug.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Pseudomonas aeruginosa LasB mutant constructed by insertional mutagenesis reveals elastolytic activity due to alkaline proteinase and the LasA fragment.
The extracellularly secreted endopeptidase elastase (LasB) is regarded as an important virulence factor of Pseudomonas aeruginosa. It has also been implicated in the processing of LasA which enhances elastolytic activity of LasB. In order to investigate the role of LasB in virulence and LasA processing, a LasB-negative mutant, PAO1E, was constructed by insertional mutagenesis of the LasB structural gene, lasB, in P. aeruginosa PAO. An internal 636 bp lasB fragment of the plasmid pRB1803 was ligated into a derivative of the mobilization vector pSUP201-1. The resulting plasmid, pBRMOB-LasB, was transformed into Escherichia coli and transferred by filter matings to the LasB-positive P. aeruginosa strain, PAO1. Plasmid integration in the lasB site of the chromosome was confirmed by Southern blot analysis. Radioimmunoassay and immunoblotting of PAO1E supernatant fluids yielded no detectable LasB (less than 1 ng ml-1 LasB). The absence of LasB in PAO1E was further proven by the inability of its culture supernatant fluid to cleave transferrin or rabbit immunoglobulin G (IgG) after a 72 h incubation. The residual proteolytic activity of PAO1E culture supernatant fluid was attributed to alkaline proteinase (Apr), since it was totally inhibited by specific antibodies against Apr. Residual elastolytic activity in culture supernatant fluid of PAO1E was due to the LasA fragment and to the combined action of the LasA fragment with Apr on elastin. The sizes of purified LasA from PAO1 and PAO1E were identical (22 kDa). These results show that, besides LasB and the LasA fragment, Apr may also act on elastin in the presence of the LasA fragment and that the proteolytic processing of LasA in P. aeruginosa is independent of LasB.
Bacterial Proteins↗
Cross-colonization and routes of infection assessed with a DNA probe.
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Child↗
Immunology of the chronic lung infection.
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Chronic Disease↗
Generation of Pseudomonas aeruginosa aerosols during handwashing from contaminated sink drains, transmission to hands of hospital personnel, and its prevention by use of a new heating device.
Pseudomonas aeruginosa was isolated from sinks of washing basins, showers, toilets and bathtubs, from the personnel and patients of a mixed infectious disease ward in a German children's hospital during a prospective 4-week epidemiological study. 81% of all sinks were contaminated with P. aeruginosa strains. Upon entering the hospital, all personnel hand cultures were P. aeruginosa-negative. However, during duty, 42.5% of the personnel members carried different P. aeruginosa strains on their hands. Detection of P. aeruginosa strains in sinks preceding the isolation of identical genotypes from personnel hands suggested a transmission route from sinks to hands. Opening of water taps generated aerosols containing P. aeruginosa sink organisms which contaminated hands during hand washing. Survival times of various P. aeruginosa strains in aerosols was dependent on strain characteristics, light and humidity, and t 1/2 differed between 3-76 min. Heating of washing basin sinks to 70 degrees C with a new, safe and inexpensive device inhibited bacterial growth in sinks, generation of P. aeruginosa aerosols, and resulted in hand cultures negative for P. aeruginosa after washing.
Aerosols↗