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G Döring

Publications and source records attributed to G Döring.

At least 37 records · Page 2Linked to original sources
PubMed2000

Direct quantitative transcript analysis of the agr regulon of Staphylococcus aureus during human infection in comparison to the expression profile in vitro.

Bacteria possess a repertoire of distinct regulatory systems promoting survival in disparate environments. Under in vitro conditions it was demonstrated for the human pathogen Staphylococcus aureus that the expression of most virulence factors is coordinated by the global regulator agr. To monitor bacterial gene regulation in the host, we developed a method for direct transcript analysis from clinical specimens. Quantification of specific transcripts was performed by competitive reverse transcription-PCR, and results were normalized against the constitutively expressed gene for gyrase (gyr). Using sputum from cystic fibrosis (CF) patients infected with S. aureus we examined the transcription of the effector molecule RNAIII of agr, of spa (protein A), generally repressed by agr, and of hla (alpha-toxin), generally activated by agr. In the CF lung RNAIII was expressed poorly, indicating an inactive agr in vivo. Despite the low level of RNAIII expression, spa was detectable only in minute amounts and an irregular transcription of hla was observed in all sputum samples. After subculturing of patient strains agr-deficient isolates and isolates with unusual expression profiles, i.e., not consistent with those obtained from prototypic strains, were observed. In conclusion, the agr activity seems to be nonessential in CF, and from the described expression pattern of spa and hla, other regulatory circuits aside from agr are postulated in vivo.

Bacterial Proteins↗
PubMed2000

Adherence of Staphylococcus aureus to endothelial cells: influence of capsular polysaccharide, global regulator agr, and bacterial growth phase.

The adherence of Staphylococcus aureus to human endothelial cells (EC) is probably an important step in the pathogenesis of systemic staphylococcal infections. We examined the influence of type 5 capsular polysaccharide (CP5) production, the global regulator agr, and the bacterial growth phase on S. aureus adherence to EC. Whereas S. aureus Newman showed maximal adherence to EC in the logarithmic phase of growth, an isogenic agr mutant showed maximal adherence in the stationary growth phase. S. aureus adherence to EC and CP5 expression were negatively correlated: a mutation in the agr locus diminished CP5 production and led to increased adherence. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence to EC. S. aureus Newman cells that adhered to EC did not express CP5. A Newman cap5O mutant was acapsular and showed significantly greater adherence to EC than the parental strain did (P<0.005). Complementation of the cap5O mutation in trans restored CP5 expression and reduced EC adherence to a level similar to that of the parental strain. The enhanced adherence shown by the cap5O mutant was similar in magnitude to that of the agr mutant or the cap5O agr double mutant. Cells of the cap5O mutant and cap5O agr double mutant harvested from stationary-phase cultures adhered significantly better than did cells harvested in the exponential growth phase. These data are consistent with the postexponential and agr-independent expression by S. aureus of at least one putative EC adhesin, whose binding domain may be masked by CP5.

Bacterial Adhesion↗
PubMed2000

Fractional analysis of bronchoalveolar lavage fluid cytology in cystic fibrosis patients with normal lung function. Bronchoalveolar lavage for the evaluation of anti-inflammatory treatment (BEAT) study group.

Cystic fibrosis (CF) is associated with a neutrophil dominated airway inflammation. So far bronchoalveolar lavage (BAL) studies in CF have used pooled BAL samples which may be more representative of the alveolar compartment rather than the airways. To assess whether the first sample of a BAL is more sensitive in the evaluation of airway inflammation, the authors have studied 105 stable CF patients aged 5-37 yrs with a mean forced expiratory volume in one second (FEV1) of 96+/-15% (mean+/-SD). BAL cytology of the first and pooled samples were compared to reference values obtained in children without respiratory disease. Absolute cell counts and the percentage of neutrophils were significantly increased in CF patients. If the 95% confidence interval was used as a cut-off point, 17/105 CF patients had a normal percentage of neutrophils in pooled BAL samples, but only three also had a normal percentage of neutrophils in the first BAL aliquot. Therefore, neutrophil dominated airway inflammation is more pronounced in the first, mainly bronchial, bronchoalveolar lavage sample suggesting that sequential analysis of bronchoalveolar lavage fluid may have a higher sensitivity to detect early inflammatory changes in CF patients.

Adolescent↗
PubMed1999

Simultaneous determination of Pseudomonas aeruginosa elastase, human leukocyte elastase and cathepsin G activities by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) is a new method for analysing proteolytic activities simultaneously present in incubation mixtures. Here we demonstrate that MEKC differentiates between the enzymatic activities of Pseudomonas aeruginosa elastase (PsE) and human leukocyte elastase (HLE) or cathepsin G (Cat G) in assays using the chromogenic peptide substrates Suc-Ala-Ala-Ala-NA or Suc-Ala-Ala-Pro-Phe-NA, respectively (where Suc = succinyl and NA = 4-nitroaniline/u-nitroanilide). When PsE and Cat G were incubated at equimolar ratio with Suc-Ala-Ala-Pro-Phe-NA, the PsE-specific cleavage products PheNA and Suc-Ala-Ala-Pro were detected whereas inhibition of the metalloproteinase PsE with EDTA resulted in detection of NA and Suc-Ala-Ala-Pro-Phe only. Similarly, when PsE and HLE were incubated at equimolar ratio with Suc-Ala-Ala-Ala-NA, the PsE-specific cleavage products Suc-Ala and Ala-Ala-NA were detected whereas at an PsE-HLE ratio 1:50, both the PsE-specific and the HLE-specific cleavage products NA and Suc-Ala-Ala-Ala were separated. MEKC also allowed determination of the kinetic constants for the interactions of PsE, Cat G and HLE with the substrates considered.

Amino Acid Sequence↗
PubMed1999

Broadly protective vaccine for Staphylococcus aureus based on an in vivo-expressed antigen.

Vaccines based on preferential expression of bacterial antigens during human infection have not been described. Staphylococcus aureus synthesized poly-N-succinyl beta-1-6 glucosamine (PNSG) as a surface polysaccharide during human and animal infection, but few strains expressed PNSG in vitro. All S. aureus strains examined carried genes for PNSG synthesis. Immunization protected mice against kidney infections and death from strains that produced little PNSG in vitro. Nonimmune infected animals made antibody to PNSG, but serial in vitro cultures of kidney isolates yielded mostly cells that did not produce PNSG. PNSG is a candidate for use in a vaccine to protect against S. aureus infection.

Animals↗
PubMed1999

Serine proteinase inhibitor therapy in alpha(1)-antitrypsin inhibitor deficiency and cystic fibrosis.

Proteinase-antiproteinase imbalances are recognized in several diseases including the two most common lethal hereditary disorders of white populations, alpha(1)-antitrypsin (alpha(1)-AT) deficiency and cystic fibrosis (CF). In alpha(1)-AT deficiency, the type Z variant of alpha(1)-AT forms polymers in the endoplasmic reticulum of hepatocytes resulting in liver disease in childhood. The block in alpha(1)-AT processing in hepatocytes significantly reduces levels of circulating alpha(1)-AT. This may lead in young adults to panacinar emphysema due to insufficient protection of the lower respiratory tract from neutrophil elastase, permitting progressive destruction of the alveoli. In CF, chronic bacterial lung infections due to impaired mucociliary clearance lead to a vigorous influx of neutrophils in the airways. Released levels of neutrophil serine proteinases, particularly elastase, exceed the antiproteinase capacity of endogenous serine proteinase inhibitors in the airways. Progressive proteolytic impairment of multiple defense pathways in addition to endobronchial obstruction and airway wall destruction are thought to be responsible for the reduced life expectancy in CF patients. Strategies to augment the antiproteinase defenses in the airways of patients with severe alpha(1)-AT deficiency or CF include the intravenous or aerosol administration of serine proteinase inhibitors. Studies in both patient groups using plasma-derived or transgenic alpha(1)-AT, recombinant secretory leukoprotease inhibitor or synthetic elastase inhibitors show promising results concerning drug safety and efficacy.

Adult↗
PubMed1999

Implementation of an interactive computer-assisted infection monitoring program at the bedside.

A new computer-assisted infection monitoring (CAI) software program has been developed for use in an intensive-care unit (ICU). By means of an interactive dialogue with physicians at the bedside, infection diagnoses and therapeutic decisions were recorded prospectively during a 3-month test period. By linking epidemiological data with information about therapeutic decisions, CAI could assess the quality of the therapeutic decisions. Antibiotics chosen empirically before the availability of any culture results, matched the antibiotic susceptibility patterns of the subsequently identified pathogens in 74% of the cases. Therapy chosen in collaboration with the computer after the pathogen was known, but before sensitivity results were available, corresponded with the eventual antibiograms of the microorganisms in 90% of the cases. Data analysis by CAI allowed us to assess critically the diagnostic and therapeutic habits in our ICU. Using the query-by-example method, CAI automatically calculated device-associated infection rates.

Adolescent↗
PubMed1999

PCR and blood culture for detection of Escherichia coli bacteremia in rats.

Critically ill patients often develop symptoms of sepsis and therefore require microbiological tests for bacteremia that use conventional blood culture (BC) techniques. However, since these patients frequently receive early empirical antibiotic therapy before diagnostic procedures are completed, examination by BC can return false-negative results. We therefore hypothesized that PCR could improve the rate of detection of microbial pathogens over that of BC. To test this hypothesis, male Wistar rats were challenged intravenously with 10(6) CFU of Escherichia coli. Blood was then taken at several time points for detection of E. coli by BC and by PCR with E. coli-specific primers derived from the uidA gene, encoding beta-glucuronidase. In further experiments, cefotaxime (100 or 50 mg/kg of body weight) was administered intravenously to rats 10 min after E. coli challenge. Without this chemotherapy, the E. coli detection rate decreased at 15 min and at 210 min after challenge from 100% to 62% of the animals with PCR and from 100% to 54% of the animals with BC (P, >0.05). Chemotherapy decreased the E. coli detection rate at 25 min and at 55 min after challenge from 100% to 50% with PCR and from 100% to 0% with BC (P, <0.05). Thus, at clinically relevant serum antibiotic levels, PCR affords a significantly higher detection rate than BC in this rat model. The results suggest that PCR could be a useful adjunct tool supplementing conventional BC techniques in diagnosing bacteremia.

Animals↗
PubMed1998

Placebo-controlled, double-blind, randomized study of aerosolized tobramycin for early treatment of Pseudomonas aeruginosa colonization in cystic fibrosis.

In chronic Pseudomonas aeruginosa pulmonary infection of patients with cystic fibrosis (CF), antibiotic therapy generally fails to eradicate the bacterial pathogen. The mucoid bacterial phenotype, high sputum production by the host, and low airway levels of antibiotics seem to be responsible for the observed decrease in antibiotic efficacy. We hypothesized that early antibiotic treatment by inhalation in CF patients may be able to prevent or at least delay airway infection. In a prospective placebo-controlled, double-blind, randomized multicenter study, 22 CF patients received either 80 mg b.i.d. of aerosolized tobramycin or placebo for a period of 12 months shortly after the onset of P. aeruginosa pulmonary colonization. Two patients in the tobramycin and six patients in the placebo group stopped inhalation before the 12 month treatment period. Using life table analysis, the time to conversion from a P. aeruginosa-positive to a P. aeruginosa-negative respiratory culture was significantly shorter in the tobramycin-treated group than in the placebo group (P < 0.05, log rank test). Lung function parameters and markers of inflammation did not change in either group during treatment. The results of this study suggest that early tobramycin inhalation may prevent and/or delay P. aeruginosa pulmonary infection in CF patients.

Administration, Inhalation↗
PubMed1998

Localization of Staphylococcus aureus in infected airways of patients with cystic fibrosis and in a cell culture model of S. aureus adherence.

Staphylococcus aureus causes chronic respiratory tract infections in patients with cystic fibrosis (CF). Using immunofluorescence and scanning and transmission electron microscopy we located S. aureus in lung specimens of three infected CF patients, in a nasal polyp of one CF patient, and in a suspension cell culture system of primary nasal epithelial cells in vitro. Very little of S. aureus was attached to the lung epithelium, whereas abundant S. aureus was detectable in the mucus of obstructed airways. Similarly, S. aureus adhered to components of secreted mucus on primary nasal epithelial cells of CF patients and healthy control subjects, grown as cell balls in vitro (bacteria/cell +/- SD: CF: 21.9 +/- 1.5; controls: 22. 0 +/- 5.8). Mucus depletion of cell balls prior to incubation with S. aureus resulted in a significantly reduced binding (bacteria/cell +/- SD: CF: 4.2 +/- 0.3; P < 0.001; controls: 5.0 +/- 1.3; P < 0. 007). Binding of S. aureus to cell balls from CF patients or control subjects did not differ significantly. When cell balls were treated with human neutrophil elastase, hypersecretion caused removal of S. aureus from cell-associated mucus. The results suggest that S. aureus adheres primarily to mucus components of the respiratory epithelium and that significant differences do not exist in binding of S. aureus to CF or non-CF cells.

Adolescent↗
PubMed1998

Catalase, myeloperoxidase and hydrogen peroxide in cystic fibrosis.

An oxidant-antioxidant imbalance with damaging consequences for the bronchial epithelium has been hypothesized in the airways of patients with cystic fibrosis (CF). It is based on the assumption that neutrophils entering the lumen of the infected airways undergo activation and release toxic oxygen metabolites and myeloperoxidase (MPO), an enzyme which transforms hydrogen peroxide (H2O2) into highly toxic oxygen metabolites. Our aims were to substantiate this hypothesis. H2O2 levels were measured in breath condensates of 63 CF patients and 51 normal subjects. In CF sputum samples, activities and concentrations of MPO and catalase (CAT) were determined. MPO/H2O2-mediated cytotoxicity of CF sputum was measured in cell culture assays. H2O2 levels were similar in CF patients and normal subjects (mean +/-SD) 0.97 +/- 0.69 versus 1.11+/-0.78 microM; p=0.427). Concentrations and activities of CAT (0.31+/-0.18 microM; 105+/-69 units) and MPO (5.93+/-4.8 microM; 87.8+/-75 units) were detectable in 38 CF sputa. Addition of H2O2 to in vitro cells preincubated with CF sputum did not induce cytotoxicity even when CAT was removed from sputum. Sputum MPO together with H2O2 did not inactivate alpha-proteinase inhibitor. Preincubation of MPO with sulphated glycoconjugates or deoxyribonucleic acid (DNA) totally inhibited its cytotoxic effect. In conclusion, catalase, sulphated glycoconjugates and deoxyribonucleic acid may prevent myeolperoxidase-mediated oxygen radical generation in cystic fibrosis sputum.

Adolescent↗
PubMed1998

Ulcer bed infection. Report of a case of enlarging venous leg ulcer colonized by Pseudomonas aeruginosa.

We report a case of ulcer bed infection in an enlarging venous leg ulcer without clinical signs of cellulitis in the surrounding tissues. Signs of infection in the leg ulcer were: 1) cocci-like structures and bacteria-like rods around vessel walls in the viable ulcer bed, 2) vasculitis-like inflammation of deeply situated vessels of the viable tissue, 3) Pseudomonas aeruginosa-specific antibodies in the serum (other than against exotoxin A), 4) extensive epidermolysis of normal human skin by the wound exudate in vitro, and 5) P. aeruginosa exotoxin A in the wound exudate (23 ng/ml). In an in vitro cell assay, the wound exudate was cytotoxic and rabbit antibodies to exotoxin A, but not a serine proteinase inhibitor, inhibited this cytotoxicity. P. aeruginosa exotoxin A might contribute to the pathogenesis of the ulcer enlargement. The ulcer improved after the third skin graft, probably mainly due to effective treatment with a long-stretch compression bandage.

ADP Ribose Transferases↗
PubMed1997

Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial.

Ad CFTR, a replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator (CFTR), was administered by aerosolization in a single escalating dose to three pairs (cohorts) of cystic fibrosis (CF) patients. Buffer only was administered to the nose and lungs 9-14 days before nasal instillation of virus followed the day after by aerosolization of Ad CFTR to the lung. Nasal doses (defined in terms of viral plaque forming units, pfu) were 10(5), 10(7), and 4 x 10(8), whereas aerosolized doses were 10(7), 10(8), 5.4 x 10(8) for each cohort, respectively. No acute toxic effects were observed in the first 4 weeks after virus treatment. Shedding of infectious Ad CFTR was never detected, whereas detection of vector DNA sequences and CFTR expression demonstrated DNA transfer to the nose and airways of patients. No significant deviations in immunological and inflammatory parameters were observed in serum and in bronchoalveolar lavage (BAL). Importantly, for all patients, the serum anti-adenovirus antibody levels did not change significantly from baseline and no antibodies against adenovirus were found in BAL.

Adenoviridae↗
PubMed1997

Regulation of Staphylococcus aureus capsular polysaccharide type 5: CO2 inhibition in vitro and in vivo.

Staphylococcus aureus capsular polysaccharide type 5 (CP5) expression was investigated in lung tissue and nasal polyps of two cystic fibrosis (CF) patients, in rats, and in vitro using ELISA and IFA. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1%-5% of cells expressed CP5. When rats were challenged with CP5-positive S. aureus in the granuloma pouch model, only 1%-5% of CP5-positive cells were detectable in pouch exudates. CF and pouch isolates, however, reexpressed CP5 (70%-90% of cells) when grown in vitro with air. Addition of > or = 1% CO2 to air or to O2/N2 gas mixtures reduced CP5 expression significantly (P < .001) in a dose-dependent manner (6%-1% CP5-positive cells). The results show that S. aureus does not produce CP5 in CF airways and in rat granuloma pouches and that CO2 is an environmental signal that regulates CP5 expression.

Animals↗
PubMed1997

A multicenter vaccine trial using the Pseudomonas aeruginosa flagella vaccine IMMUNO in patients with cystic fibrosis.

Pseudomonas aeruginosa is a major pathogen in patients with cystic fibrosis (CF). In CF patients the opportunistic pathogen causes chronic pulmonary infections which are difficult to treat with antibiotics. Loss of lung function is the major cause of death in CF. Vaccination against P. aeruginosa is a possible way to prevent these infections and flagella antigens of P. aeruginosa seem to be promising vaccine candidates. In vitro and animal studies showed that flagella antigens were protective both as actively administered immunogens and in passive studies in compromised animals. Phase I studies using IMMUNO's flagella vaccines in healthy individuals revealed that, intramuscularly administered, these vaccine preparations were well tolerated, showed no adverse side effects and gave rise to high and longlasting antibody titers in the circulation of the individuals. Furthermore, immunisation with a flagella vaccine elicited specific anti-flagella antibodies not only systemically, but also in the secretory immune system of the airways. Consequently, a phase III multicenter vaccine trial using the Pseudomonas aeruginosa 5142/1210-Flagella Vaccine IMMUNO was initiated. The study design is placebo-controlled, randomized and double-blind, involving 400 CF patients without P. aeruginosa lung infection m 16 CF centers in Germany, France and Italy. The study will start in the fall of 1996 and will be carried out for 2 years.

Adult↗
PubMed1997

Cystic fibrosis respiratory infections: interactions between bacteria and host defence.

In cystic fibrosis, impaired mucociliary clearance leads to chronic endobronchial bacterial infection, mostly caused by Pseudomonas aeruginosa. In the early stage of infection, the pathogen produces several extracellular protein toxins which may contribute to its multifactorial virulence before specific antibodies are produced. P. aeruginosa successfully resists phagocytosis by neutrophils, which dominate the local inflammatory response, by switching from a nonmucoid variant to a mucoid phenotype. Chronic infection and inflammation are characterized by neutrophil-released proteinases which may provide favourable growth conditions for the bacterial opportunist. Aerosol therapy with serine proteinase inhibitors is being investigated in cystic fibrosis.

Adult↗