[show abstract][hide abstract] ABSTRACT: The effects of rabbit-derived polyclonal Ab against PcrV, a protein involved in the translocation of type III secreted toxins of Pseudomonas aeruginosa, was investigated in two animal models of P. aeruginosa sepsis. In a mouse survival study, the i.v. administration of anti-PcrV IgG after the airspace instillation of a lethal dose of P. aeruginosa resulted in the complete survival of the animals. In a rabbit model of septic shock associated with Pseudomonas-induced lung injury, animals treated with anti-PcrV IgG intratracheally or i.v. had significant decreases in lung injury, bacteremia, and plasma TNF-alpha and significant improvement in the hemodynamic parameters associated with shock compared with animals treated in a similar manner with nonspecific control IgG. The administration of anti-PcrV F(ab')(2) showed protective effects comparable to those of whole anti-PcrV IgG. These results document that the therapeutic administration of anti-PcrV IgG blocks the type III secretion system-mediated virulence of P. aeruginosa and prevents septic shock and death, and that these protective effects are largely Fc independent. We conclude that Ab therapy neutralizing the type III secretion system has significant potential against lethal P. aeruginosa infections.
The Journal of Immunology 12/2001; 167(10):5880-6. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although many patients and physicians support the concept of advance care planning, only a small percentage of patients actually have the necessary discussion with health care providers. Hospital-based physicians other than primary care providers often are needed to increase physician, patient, and proxy communication about advanced directives. This study evaluated the effectiveness of a 5-10-min discussion designed to foster dialogue between patients and their proxies in a preoperative evaluation clinic. The discussions were lead by anesthesiologists.
A randomized controlled trial was conducted from September 1998 through May 1999 in a preoperative evaluation clinic at University of California, San Francisco, a tertiary care center. English-speaking patients aged 65 yr or older who were scheduled for elective surgery were randomized to receive a short information session stressing the importance of communication about end-of-life care between the patients and their proxies. Patients randomized to the control group received the standard preoperative anesthesia screening. An admitting counselor questioned all patients (control and intervention) about whether they have an advanced directive as part of the registration process before their arrival in clinic.
The intervention significantly increased discussions about end-of-life care between patients and their proxies. Eighty seven percent of patients reported having discussions with their proxies as compared with only 66% of control patients (P = 0.001). The intervention also increased durable power of attorney completion rate to 27% as compared with 10% completion rate by controls.
The preoperative evaluation period can be an opportunity to encourage patient and proxy communication about end-of-life care.
[show abstract][hide abstract] ABSTRACT: The ability of Pseudomonas aeruginosa to secrete specific toxins using the type III-mediated pathway has been reported. To determine the association of this phenotype with human illness, immunoblot analysis was used to detect expression of type III secretory proteins in P. aeruginosa isolates from respiratory tract or blood cultures of 108 consecutive patients. Relative risk of mortality was 6-fold greater with expression of the type III secretory proteins ExoS, ExoT, ExoU, or PcrV. Phenotype was independently correlated with toxicity in cellular and murine models. Prevalence of this phenotype was significantly higher in acutely infected patients than in chronically infected patients with cystic fibrosis. These results suggest that the type III protein secretion system is integral to increased P. aeruginosa virulence. A positive phenotype is a predictor of poor clinical outcome. In the future, such analyses may help distinguish potentially lethal infection from colonization and help determine appropriate therapy for critically ill patients.
The Journal of Infectious Diseases 07/2001; 183(12):1767-74. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: The effect of antibiotics on the acute lung injury induced by virulent Pseudomonas aeruginosa PA103 was quantitatively analyzed in a rat model. Lung injury was induced by the instillation of PA103 directly into the right lower lobes of the lungs of anesthetized rats. The alveolar epithelial injury, extravascular lung water, and total plasma equivalents were measured as separate, independent parameters of acute lung injury. Four hours after the instillation of PA103, all the parameters were increased linearly depending on the dose of P. aeruginosa. Next, we examined the effects of intravenously administered antibiotics on the parameters of acute lung injury in D-galactosamine-sensitized rats. One hour after the rats received 10(7) CFU of PA103, an intravenous bolus injection of aztreonam (60 mg/kg) or imipenem-cilastatin (30 mg/kg) was administered. Despite an MIC indicating resistance, imipenem-cilastatin improved all the measurements of lung injury; in contrast, aztreonam, which had an MIC indicating sensitivity, did not improve any of the lung injury parameters. The antibiotics did not generate different quantities of plasma endotoxin; therefore, endotoxin did not appear to explain the differences in lung injury. This in vivo model is useful to quantitatively compare the efficacies of parenteral antibiotic administration on Pseudomonas airspace infections.
Antimicrobial Agents and Chemotherapy 11/1999; 43(10):2389-94. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two antiinflammatory therapies that have been effective in preventing acid-induced lung injury were evaluated. Specifically, their effects on a subsequent bacterial-airspace challenge were compared. Bacteria were instilled 24 h after acid-induced lung injury. Pseudomonas aeruginosa PAO-1 was used as the bacteria, because its effects in healthy lungs was documented previously.
New Zealand white rabbits were anesthetized and three pretreatments were administered: (1) pentoxifylline pretreatment (a 20-mg/kg bolus dose and then 6 mg x kg(-1) x h(-1) given intravenously), (2) 1 ml anti-tumor necrosis factor alpha antiserum given intravenously, or (3) normal saline given intravenously. The pretreatment doses were shown previously to prevent acid-induced lung injury. Then 1.2 ml/kg hydrochloric acid (HCl), pH 1.25, was instilled into the rabbits' right lungs. All the animals underwent mechanical ventilation for 8 h. Twenty-four hours after the acid instillation, the rabbits were anesthetized again and 2 ml/kg (10(9) colony forming units/ml) PAO-1 was instilled into their left lungs. The rabbits' breathing was aided by mechanical ventilation for another 8 h, and then they were killed and exsanguinated.
Both pretreatments attenuated the acid-induced lung injury of the noninstilled left lungs. Arterial oxygen tension and the lung edema of pretreated, acid-exposed animals were significantly and almost equally improved (compared with no pretreatments) by either of the pretreatments. However, when the bacteria were instilled into the left lungs 24 h after the acid injury, the pentoxifylline pretreatment but not the anti-tumor necrosis factor alpha pretreatment prevented much of the bacteria-induced lung injury. Pentoxifylline pretreatment significantly improved the measurements of left lung edema and epithelial and endothelial permeability. There was also a trend for improved oxygenation in the pentoxifylline-pretreated and infected animals. In contrast, the anti-tumor necrosis factor alpha pretreatment did not prevent the bacteria-induced lung injury and increased some of the measurements of lung injury.
Two antiinflammatory therapies that prevented acid-induced lung injury to the noninstilled left lungs had significantly different effects on a subsequent bacteria-induced lung injury to the left lungs. The therapies differed in their mechanism of tumor necrosis factor alpha blockade, and this may have affected the bacteria-induced injury to the lungs.
[show abstract][hide abstract] ABSTRACT: To evaluate the role of alveolar macrophages (AMs) in acute Pseudomonas aeruginosa pneumonia in mice, AMs were depleted by aerosol inhalation of liposomes containing clodronate disodium. AM-depleted mice were then intratracheally infected with 5 x 10(5) CFU of P. aeruginosa. In addition to monitoring neutrophil recruitment and chemokine releases, lung injury was evaluated soon after infection (8 h) and at a later time (48 h). At 8 h, depletion of AMs reduced neutrophil recruitment, chemokine release, and lung injury. At 48 h, however, depletion of AMs decreased bacterial clearance and resulted in delayed movement of neutrophils from the site of inflammation with aggravated lung injury. With instillation of 5 x 10(7) CFU of bacteria, AM-depleted mice showed low mortality within 24 h of infection but high mortality at a later time, in contrast to non-AM-depleted mice. These results demonstrate that depletion of AMs has beneficial early effects but deleterious late effects on lung injury and survival in cases of P. aeruginosa pneumonia.
Infection and Immunity 08/1998; 66(7):3164-9. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gastric acid aspiration can result in acute lung injury. In this study, the authors determined whether alveolar macrophages express cyclooxygenase-2 as a source of inflammatory mediators after acid aspiration.
Seventy-five microliters of hydrochloric acid solution, pH 1.15, was instilled into one lung in mice. After exposure, alveolar macrophages were harvested, and competitive polymerase chain reaction and enzyme-linked immunosorbent assay were performed to measure expression of cyclooxygenase-1 and -2, interleukin-1beta and -6, tumor necrosis factor-alpha, and inducible nitric oxide synthase (iNOS). The authors used immunocytochemistry to demonstrate expression of cyclooxygenase-2 in alveolar macrophages. Selective cyclooxygenase-2 blockade using N-2(-cyclohexyloxy-4-nitrophenyl) methane-sulphonamide was done to characterize prostaglandin-cytokine interaction.
Acid aspiration induced upregulation of cyclooxygenase-2 and interleukin-6. Tumor necrosis factor-alpha and iNOS were not upregulated. Interleukin-1beta was upregulated even with saline instillation but could not be detected in the supernatant of the cell culture. Alveolar macrophages harvested from mice instilled with acid showed a trend toward more production of prostaglandin E2 and produced higher concentrations of interleukin-6 compared with alveolar macrophages from mice instilled with saline. Selective cyclooxygenase-2 blockade significantly decreased release of interleukin-6 from alveolar macrophages harvested from mice instilled with acid.
Acid aspiration induces strong expression of cyclooxygenase-2 and production of interleukin-6 in alveolar macrophages. Selective cyclooxygenase-2 blockade reduced production of interleukin-6 by acid-stimulated alveolar macrophages. These studies suggest that the induction of cyclooxygenase-2 plays an important role in the systemic inflammatory response induced by acid aspiration.
[show abstract][hide abstract] ABSTRACT: The alveolar epithelium is not injured by the apical application of moderate doses of Pseudomonas aeruginosa strains that produce protease. To determine the effect of Pseudomonas proteases on the basolateral surface of the alveolar epithelium, a series of experiments were done, in which P. aeruginosa strains that produce and do not produce proteases were administered intravenously. Subsequently, an innocuous dose of bacteria was instilled into the lungs of the rabbits. Although all the intravenous Pseudomonas strains increased the extravascular lung water to a similar degree, only the intravenous administration of the protease-producing P. aeruginosa strain increased the vulnerability of the alveolar epithelium to injury by the subsequent airspace bacteria. Bacteremia secondary to P. aeruginosa strains producing proteases could increase the chances of developing acute lung injury.
American Journal of Respiratory Cell and Molecular Biology 02/1998; 18(1):129-35. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cardiac risk assessment is a crucial aspect of perioperative evaluation because it is estimated that 1 million people, regardless of gender, will have perioperative cardiac complications at a cost of 20 billion dollars. Unfortunately, establishment of optimal guidelines for selected patient subgroups, particularly women, are lacking. More prospective studies are needed to help evaluate the most cost-effective, yet accurate way to noninvasively assess the presence of coronary artery disease in women.
[show abstract][hide abstract] ABSTRACT: We have previously shown that injury to lung epithelial type I cells can be detected biochemically by measuring the airway fluid content of a type I cell-specific protein, rTI40, in a model of severe acute lung injury [M. C. McElroy, J.-F. Pittet, S. Hashimoto, L. Allen, J. P. Wiener-Kronish, and L. G. Dobbs. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L181-L186, 1995]. The first objective of the present study was to evaluate the utility of rTI40 in the assessment of alveolar injury in a model of milder acute lung injury. Rats were exposed to 18 parts/ million NO2 for 12 h; control rats received filtered air for 12 h. In NO2-exposed rats, the total amount of rTI40 in bronchoalveolar fluid was elevated 2-fold compared with control values (P < 0.001); protein concentration was 8.5-fold of control values (P < 0.001). The increase in rTI40 was associated with morphological evidence of injury to type I cells limited to the proximal alveolar regions of the lung. The second objective was to correlate the severity of alveolar type I cell injury with functional measurements of lung epithelial barrier integrity. NO2 inhalation stimulated distal air space fluid clearance despite a significant increase in lung endothelial and epithelial permeability to protein. These data demonstrate that rTI40 is a useful biochemical marker for mild focal injury and that exposure to NO2 alters lung barrier function. Taken together with our earlier studies, these results suggest that the quantity of recoverable rTI40 can be used as an index of the severity of damage to the alveolar epithelium.
The American journal of physiology 12/1997; 273(6 Pt 1):L1228-34. · 3.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Increasingly, patients with advanced lung disease are being offered operative procedures. The assessment of the perioperative risk of these patients must include not only the assessment of their lung disease, but the assessment of the patient's cardiovascular disease, their age, and their other medical problems. Knowledge of the stress of particular surgical procedures is also of importance in risk assessment, and is addressed in this article.
Clinics in Chest Medicine 10/1997; 18(3):483-94. · 2.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pseudomonas aeruginosa is the most frequent Gram-negative pathogen causing nosocomial pneumonia. Four different strains of P. aeruginosa (including three isogenic transposon mutants) were utilized in experiments in mice to characterize the specific patterns of cytokine generation in response to bacterial products and cytotoxicity. Intratracheal instillation of any of the strains led to the up-regulation of IL-1beta, IL-6, and TNF-alpha mRNA. Instillation of the cytotoxic strains (PA103, PA103tox::omega) led to IL-10 mRNA up-regulation in the lungs and increased concentrations of IL-10 in the blood. In contrast, the instillation of the noncytotoxic strains (PA01, PA103exsA::omega) did not lead to an increase in IL-10 mRNA in the lungs or to an increase of IL-10 concentration in blood. IL-10 production appears to be a response to either cellular injury or to specific cytotoxic exoproducts produced by the bacteria. The systemic administration of rIL-10 significantly decreased the lung injury and the mortality in mice who had received the cytotoxic strains. The improvement in survival induced by administration of rIL-10 required the concomitant presence of IFN-gamma, as blockade of IFN-gamma with a neutralizing Ab led to 100% mortality, despite the administration of rIL-10. These results suggest that IL-10 is produced in response to specific bacterial products and that there is a potential role for IL-10 in the treatment of cytotoxic P. aeruginosa pneumonia.
The Journal of Immunology 10/1997; 159(6):2858-66. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Acid instillation leads to direct lung and to secondary systemic organ injury, probably via activated macrophages and neutrophils. This study investigated the effects of neutrophil elastase on organ injury after unilateral lung acid instillation by administrating a specific neutrophil elastase inhibitor, ONO-5046, before acid instillation.
Three groups of anesthetized rabbits (n = 12 in each group) underwent tracheostomies, and instillations were made into their right lower lobe airspaces with either phosphate buffered saline (pH, 7.4; volume, 1.2 ml/kg; n = 12) or HCl (pH, 1.25; volume, 1.2 ml/kg; n = 24). In half of the acid-instilled rabbits, ONO-5046, 10 mg/kg, was given intravenously 15 min before the HCl instillation, and then 10 mg x kg(-1) x h(-1) of the drug was continuously infused throughout the experiment. The other groups of animals received the vehicle intravenously. Anesthesia and mechanical ventilation was continued for 8 h, whereas arterial blood gases were sampled intermittently. Eight hours after saline or acid instillation, the animals were killed, and their lungs, heart, kidneys, liver, and small intestines were harvested. Wet-to-dry weight ratios (W/ D) and myeloperoxidase (MPO) assays of these organs were done, and elastase assays on the bronchoalveolar lavage fluids (BALF) obtained from each lung also were performed.
Pretreatment with ONO-5046 attenuated the physiologic changes seen in the vehicle-treated animals. Significant decreases in W/D of the noninstilled lungs and of the small intestine and normalization of the oxygenation of the experimental animals occurred. The ONO-5046 pretreatment did not affect the neutrophil sequestration in the lungs or in the other organs as determined by neutrophil counts in BALF and by the MPO assays.
A neutrophil elastase inhibitor, ONO-5046, administered immediately before acid instillation attenuated the physiologic changes seen in the vehicle-treated animals. The drug blocked neutrophil elastase but did not block neutrophil sequestration in the lungs, although the drug improved measurements of lung injury.
[show abstract][hide abstract] ABSTRACT: We compared the efficacy of gene transfer in vitro and in vivo using various formulations of DNA-lipid complexes based on the novel cationic lipid EDMPC (1,2-dimyristoylsn-glycero-3-ethylphosphocholine, chloride salt). In vitro studies analyzed delivery of marker genes to four established cell lines, including two of pulmonary origin. The in vivo analysis used intralobar delivery of marker genes and CFTR to mice and rats. We observed a lack of positive correlation between those DNA-EDMPC formulations that delivered DNA most efficiently in vitro and those that worked best in vivo. Intralobar DNA delivery to rodents mediated by EDMPC was efficient. The high level of gene delivery by DNA-EDMPC formulations demonstrates that efficient lipid-mediated gene transfer to the lung is possible.
[show abstract][hide abstract] ABSTRACT: The production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non-covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non-cytotoxic derivative PA103 exsA::omega and several cytotoxic and non-cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70-kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70-kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non-cytotoxic strains or PA103 exsA::omega. To clone the gene encoding this potential cytotoxin we used Tn5Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU:: Tn5Tc, which no longer expressed the 70-kDa extracellular protein but maintained expression of ExoT. PA103 exoU::Tn5Tc was non-cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU::Tn5Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co-ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.
[show abstract][hide abstract] ABSTRACT: We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa-induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon-tagged non-cytotoxic mutants of a cytotoxic and lung-virulent strain of P. aeruginosa (PA103). The transposon-insertion site was determined by using an inverse polymerase chain reaction followed by DNA-sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pill, based on their resistance to phage PO4 and/or loss of twitching motility (twt-). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III protein-secretion apparatus appears to be involved in this process.
[show abstract][hide abstract] ABSTRACT: Inhaled nitric oxide (NO) has been shown to prevent oxidant-induced lung injury in isolated-perfused lung models, whereas NO-derived oxidants may contribute to acute lung injury secondary to hyperoxia. Whether inhaled NO improves or contributes to oxidant-mediated lung injury may depend on the timing of NO administration or how lung injury is assessed. The objective of these studies was to determine whether inhaled NO (20 ppm) was protective or harmful to the different lung barriers when it was administered with 95% O2 for 60 h in Sprague-Dawley rats by measuring fluid transport and permeability to protein across the lung endothelium and the alveolar epithelium. Inhaled NO significantly attenuated the O2-mediated lung endothelial injury and abolished the increase in the bronchoalveolar lavage fluid content of rTI40, a specific and sensitive marker of alveolar epithelial type I cell injury, that occurs secondary to hyperoxia. In conclusion, inhaled NO administered with high concentrations of O2 may protect the lung endothelium and the alveolar epithelium against O2-mediated injury.
The American journal of physiology 05/1997; 272(4 Pt 1):L631-8. · 3.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pseudomonas aeruginosa, an opportunistic pathogen, is capable of establishing both chronic and acute infections in compromised hosts. Previous studies indicated that P. aeruginosa displays either a cytotoxic or an invasive phenotype in corneal epithelial cells. In this study, we used polarized MDCK cells for in vitro infection studies and confirmed that P. aeruginosa isolates can be broadly differentiated into two groups, expressing either a cytotoxic or an invasive phenotype. In vivo infection studies were performed to determine if cytotoxic and invasive strains displayed differential pathology. Invasion was assayed in vivo by in situ infection of mouse tracheal tissue followed by electron microscopy. Both cytotoxic and invasive strains entered mouse tracheal cells in situ; however, more necrosis was associated with the cytotoxic strain. In an acute lung infection model in rats, cytotoxic strains were found to damage lung epithelium more than invasive strains during the short infection period of this assay. The expression of cytotoxicity requires a functional exsA allele. In the strains tested, the ability to invade epithelial cells in vitro appears to be independent of exsA expression. Since ExsA is a transcriptional regulator of the exoenzyme S regulon, chromosomal preparations from invasive and cytotoxic strains were screened for their complement of exoenzyme S structural genes, exoS, encoding the 49-kDa ADP-ribosyltransferase (ExoS), and exoT, encoding the 53-kDa form of the enzyme (Exo53). Invasive strains possess both exoS and exoT, while cytotoxic strains appear to have lost exoS and retained exoT. These data indicate that the expression of cytotoxicity may be linked to the expression of Exo53, deletion of exoS and perhaps other linked loci, or expression of other ExsA-dependent virulence determinants. In the absence of a functional cytotoxicity pathway (exsA::omega strains), invasion of eukaryotic cells is detectable.
Infection and Immunity 03/1997; 65(2):579-86. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aerosolization of imipenem/cilastatin was compared with continuous intravenous infusions of the antibiotic for pharmacokinetic/pharmacodynamic analysis. The concentrations of imipenim/cilastatin in bronchoalveolar lavage fluids (BAL) obtained from rats exposed to the aerosolized antibiotic were significantly greater than the concentrations in BAL in the rats that had received intravenous infusions of imipenem/cilastatin. The two methods of antibiotic delivery were compared for their effects on bacterial-induced lung injury in rats that had Pseudomonas aeruginosa instilled into their airspaces. The aerosolization of antibiotic was associated with significantly decreased bacterial-induced lung injury. The high concentrations of antibiotic in the airspaces secondary to aerosolization appears to kill bacteria more quickly and preserve lung epithelial and endothelial integrity better than systemic delivery of the same antibiotic.
Journal of Antimicrobial Chemotherapy 12/1996; 38(5):809-18. · 5.34 Impact Factor