Animals in medical research. Official statement of the American Thoracic Society.
Explore the source record for details and available documents.
Animal Experimentation↗
Biomedical subjects
R B Fick
Publications and source records attributed to R B Fick.
Airway adherence of Pseudomonas aeruginosa: mucoexopolysaccharide binding to human and bovine airway proteins.
The ability of Pseudomonas aeruginosa (PA) to adhere to cells of the upper and lower airways is considered the initial step in its colonization and subsequent infection. Many potential adhesins exist, but few eukaryote receptors have been reported. This study evaluates the adherence of PA and the well-recognized adhesin mucoexopolysaccharide, of the mucoid variant, to tracheal epithelial cells and airway secretions. A number of tools were used to systematically examine this process, each in increasing detail. Scanning electron microscopy of infected tracheal cell monolayers showed that although nonmucoid PA adhered most often as individual organisms, mucoid strains frequently were adherent in the form of clusters of microcolonies. Partially purified mucoexopolysaccharide was itself adherent to the tracheal cell monolayers. More quantitative studies of carbon 14-labeled PA adherence to tracheal cell monolayers demonstrated that (1) bacterial adherence was temperature dependent; (2) mucoid PA was more adherent than nonmucoid PA (p less than 0.005); (3) proteinase treatment of the intact monolayers increased adherence of mucoid PA; and (4) adherence of nonmucoid PA was partially inhibited by pretreatment with N-acetylglucosamine, a normal constituent of airway mucus (control, 9.5 +/- 2.0 bacteria/cell; N-acetylglucosamine, 5.8 +/- 1.5 bacteria/cell; p = 0.05). These observations suggest that N-acetylglucosamine may play a role in the epithelial cell receptor for ligands on the surface of nonmucoid strains of PA. Pretreatment of mucoid PA with N-acetylglucosamine, D-arabinose, D-mannose, or N-acetylneuraminic acid did not affect adherence. Partially purified proteins isolated from the apical plasma membrane of bovine tracheal epithelial cells probed in a Western analysis with the iodine 125-labeled PA mucoexopolysaccharide revealed two binding proteins of 45,000 mol wt and 104,000 mol wt. Human airway secretions of patients with chronic PA infection were screened by the dot blot technique and demonstrated selective binding of mucoexopolysaccharide ligand. Western analysis of these biologically important secretions revealed that PA mucoexopolysaccharide bound to a 65,000 mol wt protein in airway secretions of infected individuals. This glycoprotein is similar to a previously described cell receptor for bacterial type 1 fimbriae.
Animals↗
The immunoglobulin G subclass composition of immune complexes in cystic fibrosis. Implications for the pathogenesis of the Pseudomonas lung lesion.
It has been shown that pulmonary macrophage (PM) phagocytosis of Pseudomonas aeruginosa (PA) is inhibited in the presence of serum from cystic fibrosis (CF) patients colonized by Pseudomonas, and that these sera contain high concentrations of IgG2 antibodies. The goal of these studies was to investigate the role that IgG2-containing immune complexes (IC) play in this inhibition of both PM and neutrophil phagocytosis. We found that serum IgG2 concentrations were elevated significantly in CF patients with chronic PA colonization and that in selected sera from CF patients with chronic PA colonization (CF + IC, n = 10), the mean IC level was significantly elevated (2.90 +/- 0.22 mg/dl [SEM]). IgG2 comprised 74.5% of IgG precipitated in IC from CF + IC sera. An invitro phagocytic assay of [14C]PA uptake using CF + IC whole-sera opsonins confirmed that endocytosis by normal PM and neutrophils was significantly depressed. Removal of IC from CF + IC sera resulted in significantly decreased serum IgG2 concentrations without a significant change in the other subclass concentrations, and enhanced [14C]PA uptake by PM (26.6% uptake increased to 47.3%) and neutrophils (16.9% increased to 52.6%). Return of the soluble IgG2 IC to the original CF sera supernatants and the positive control sera resulted in return of the inhibitory capacity of the CF + IC sera. We conclude that immune sera from patients with chronic Pseudomonas infections characterized by elevated IgG2 subclass level functions poorly as an opsonin. In these individuals, IgG2 contributes significantly to circulating IC and removal of IC, matched by a simultaneous fall in IgG2, improves bacterial uptake by neutrophil and mononuclear phagocytes. IgG2 antibodies exert antiphagocytic effects by both direct inhibition and the formation of IC.
Adult↗
Lung humoral response to Pseudomonas species.
Immunoglobulin G (IgG) is quantitatively the predominent immunoglobulin protein in the distal airway of the human host. IgG enters the distal lung from circulating pools and plasma cells located largely in the interstitium. Although all four IgG subclasses are present in human lung secretions, only subclasses G3 and, to a lesser degree, G1 attach to pulmonary macrophage membrane receptors. IgG opsonic antibodies are essential for the optimal clearance of a very troubling pathogen. Pseudomonas aeruginosa. Nosocomial pneumonia caused by this gram-negative bacillus responds poorly to potent antimicrobial agents and is associated with a 70% mortality. To a great extent the morbidity and mortality resulting from nosocomial pneumonia caused by Pseudomonas spp. are attributable to ineffective humoral immune response to this bacterium. IgG2 antibodies in response to pseudomonal lipopolysaccharide are poorly opsonic in the macrophage system, and derepression of the potent pseudomonal elastase, an enzyme with broad substrate specificity, contributes to the disruption of other IgG antibodies.
Antibodies, Bacterial↗
Controversies in the management of pulmonary disease due to cystic fibrosis.
Explore the source record for details and available documents.
Adrenal Cortex Hormones↗
Pathogenesis of the pseudomonas lung lesion in cystic fibrosis.
Explore the source record for details and available documents.
Antibody Formation↗
Functional importance of cystic fibrosis immunoglobulin G fragments generated by Pseudomonas aeruginosa elastase.
We examined the functional importance of immunoglobulin polypeptide fragments generated by Pseudomonas aeruginosa elastase (Pseudomonas elastase). The purpose of this study was to determine whether the elastase produced by Pseudomonas aeruginosa cleaves human IgG into immune fragments that functionally inhibit opsonophagocytosis. Our results confirm that IgG isolated from patients with cystic fibrosis (CF) incubated with purified pseudomonas elastase results in the generation of two major polypeptide fragments and that, furthermore, these fragments significantly inhibit bacterial uptake by human neutrophils. After 75 minutes bacterial uptake was six times greater when intact IgG was used as an opsonin (uptake 90.2% +/- 18.6% SEM) compared with a IgG was used as an opsonin (uptake 90.2% +/- 18.6% SEM) compared with a mixture of pseudomonas-lipopolysaccharide-reactive Fab and F(ab')2 fragments generated by pseudomonas elastase (uptake 15.4% +/- 0.8% SEM, p less than 0.001). Hydrolyzed CF IgG antibodies consistently resulted in a level of bacterial uptake less than that of normal saline negative controls (NS): (at 10 minutes, NS 26.6% vs CF 16.8%, p less than 0.05; at 75 minutes, NS 28.2% vs CF 15.4%, p less than 0.01. This suggests that the immune polypeptides are active inhibitors of the essential neutrophil phagocyte-bacterial cell interaction. Intact immune IgG reversed the defect in opsonophagocytosis. When intact IgG was mixed with IgG fragments the phagocytic rates increased directly with increasing amounts of intact IgG. We conclude that the elastase exoproduct secreted by Pseudomonas aeruginosa is capable of cleaving IgG into functionally important fragments that inhibit bacterial uptake. Furthermore, this inhibition can be overcome by increasing amounts of a commercially available preparation of intact immune IgG.(ABSTRACT TRUNCATED AT 250 WORDS)
Antibodies, Bacterial↗
Nontuberculous mycobacterial lung disease. Substantiation of a less aggressive approach.
A nonsurgical, less aggressive, less toxic chemotherapeutic protocol for the management of nontuberculous mycobacterial (NTB) pulmonary infections has been uniformly applied to patients in our institution between 1972 and 1985. Forty-three nonimmunocompromised patients with active lung disease caused by Mycobacterium avium-intracellulare (MAI) (n = 26), M kansasii (n = 16), and M xenopi (n = 1) were identified retrospectively. Eighteen MAI patients were treated with three or four antituberculosis agents resulting in sputum conversion and clinical improvement in 12 (67 percent). Additionally, 11 out of 16 (69 percent) patients completing therapy or still undergoing therapy for persistent MAI disease, achieved sputum conversion and clinical improvement after prolonged therapy (3.6 +/- 0.5 years [SEM]). When M kansasii was identified as the etiologic agent, all patients were treated with four or fewer antituberculosis agents and 14 out of 16 patients (88 percent) achieved sputum conversion and clinical improvement throughout the follow-up period. We conclude that the use of three or four chemotherapeutic agents in the treatment of NTM lung disease provides an excellent probability of successful outcome even in MAI infections.
Adult↗
Pseudomonas aeruginosa and the airways disease of cystic fibrosis.
The gram-negative organism Pseudomonas aeruginosa plays a major role in the morbidity and mortality of patients with cystic fibrosis (CF). Inherent properties of this organism, together with alterations in the CF airway, lead to colonization of the CF patient and, ultimately, contribute to the destructive lung lesion of CF. Innovative antibiotic therapies for the CF patient are discussed, including new potent oral and parenteral pseudomonicidal antibiotics, the re-emergence of nebulized delivery systems, and the validation of home intravenous therapy.
Anti-Bacterial Agents↗
Are lymphocyte beta-adrenoceptors altered in patients with cystic fibrosis?
1. Beta-adrenergic responsiveness may be decreased in cystic fibrosis. In order to determine whether this reflects an alteration in the human lymphocyte beta-receptor complex, we studied 12 subjects with cystic fibrosis (six were stable and ambulatory and six were decompensated, hospitalized) as compared with 12 normal controls. 2. Lymphocyte beta-receptor mediated adenylate cyclase activity (EC 4.6.1.1) was not decreased in the ambulatory cystic fibrosis patients as compared with controls. In contrast, decompensated hospitalized cystic fibrosis patients demonstrated a significant reduction in beta-receptor mediated lymphocyte adenylate cyclase activity expressed as the relative increase over basal levels stimulated by the beta-agonist isoprenaline compared with both normal controls and stable ambulatory cystic fibrosis patients (control 58 +/- 4%; ambulatory cystic fibrosis patients 51 +/- 7%; decompensated hospitalized cystic fibrosis patients 28 +/- 5%; P less than 0.05). 3. Our data suggest that defects in lymphocyte beta-receptor properties in cystic fibrosis patients may be better correlated with clinical status than with presence or absence of the disease state.
Adenylyl Cyclases↗
Penetration of cefotaxime into respiratory secretions.
The objective of this study was to quantitate cefotaxime and its active metabolite, desacetyl cefotaxime, in the distal airways and to compare these levels to concentrations in plasma. Respiratory secretions were obtained from the subsegmental level in 17 adult patients undergoing fiber-optic bronchoscopy within 2 h after receiving four doses of cefotaxime (2 g intravenously every 6 h). In 11 patients, cefotaxime levels measured by high-pressure liquid chromatography in bronchial secretions were below detectable limits (less than 0.5 mg/liter); however, levels of desacetyl cefotaxime exceeded 1.5 mg/liter in 9 of these 11 patients (range, 1.6 to 10 mg/liter). Concentrations of desacetyl cefotaxime in lung secretions (6.9 +/- 0.85 [standard error] mg/liter) was 77% of mean levels of desacetyl cefotaxime in plasma (8.9 +/- 1.26 mg/liter). In summary, concentrations of desacetyl cefotaxime in bronchial secretions are markedly higher than those of cefotaxime.
Adult↗
Candidacidal factors in murine bronchoalveolar lavage fluid.
Respiratory secretions provide an efficient method for protecting the large surface area of the lower respiratory tract. To determine whether lung secretions contribute to antifungal defenses, we tested bronchoalveolar lavage fluid for fungicidal activity. Candida albicans (blastoconidia) was incubated in unconcentrated cell-free lavage fluid from Swiss Webster mice and then cultured quantitatively to measure residual viability. In control buffer the residual fractions of viable fungi were 1.03 +/- 0.12 at 60 min and 0.84 +/- 0.05 at 120 min, whereas the residual fractions in lavage fluid were 0.64 +/- 0.07 and 0.23 +/- 0.05, respectively (P less than 0.05 by t tests). This activity was trypsin sensitive and heat stable (56 degrees C) and did not require divalent cations. It did not sediment with the surfactant fraction of lung lavage fluid. Unconcentrated lavage fluid reduced the adherence of C. albicans to serum-coated glass tubes to 2.3 +/- 1.5% of that of control Candida suspensions (n = 5, P less than 0.05 by t test). It did not alter Candida ingestion or intracellular processing by alveolar macrophages. Lavage fluid also killed clinical isolates of Candida tropicalis and Torulopsis glabrata but did not kill Candida krusei or Candida parapsilosis. Lavage fluid was concentrated and passed through an acrylamide-agarose gel matrix. The chromatogram indicated that the candidacidal activity eluted in a peak with a molecular weight range of 29,000 to 40,000. After electrophoresis on 15% sodium dodecyl sulfate-polyacrylamide gels, these fractions resolved into three bands. These were transferred to nitrocellulose and then eluted with Triton X-100; this procedure permitted the isolation of a single band of candidacidal activity with a molecular weight of 29,000. In summary, murine lavage fluid contains a heat-stable protein with direct antifungal activity. This soluble factor may contribute to lung defense processes by reducing fungal viability and adherence to tissue surfaces.
Animals↗
Increased bronchovascular permeability after allergen exposure in sensitive asthmatics.
To investigate the possibility that an increase in bronchovascular permeability is associated with allergen exposure in sensitive asthmatics we evaluated the amounts of serum proteins in bronchoalveolar lavage (BAL) effluents before and after local challenge with allergen. After exposure of sensitive asthmatic airways (n = 15) to allergen significant increases in total protein compared with controls were observed: 0.08 +/- 0.01 mg/ml in control airways and 0.13 +/- 0.02 mg/ml in challenged airways; P less than 0.05. The greatest changes induced by allergen exposure involved small-molecular-weight proteins (less than 345,000) and an inverse correlation was observed between log molecular weight and percent increase in the concentrations of the specific proteins; r = -0.61. BAL-serum distribution coefficients of serum proteins in airway fluids reflected a greater diffusability of low-molecular-weight proteins immediately after allergen exposure. We also evaluated the movement of serum proteins into lung after local allergen exposure using intravenously administered 99mTc-albumin (n = 10) and found an immediate 3.8-fold increase in amounts of radioactive albumin in allergen exposed airways compared with airways exposed to diluent. Most of the radioactivity was recovered in the first 5 ml of aliquot withdrawn, suggesting a marked increase in the permeability of the bronchial (large airway) vascular-epithelial membrane. An increase in serum proteins was also observed in BAL fluid of asthmatics 2-4 h after aerosol challenge (n = 4), including all proteins in the molecular weight range 45,000-900,000. These studies suggest that allergen exposure in sensitive asthmatics causes an acute increase in bronchovascular permeability to serum proteins.
Allergens↗
Bronchoalveolar lavage in allergic asthmatics.
Explore the source record for details and available documents.
Adolescent↗
Immunoglobulin-G subclasses in cystic fibrosis. IgG2 response to Pseudomonas aeruginosa lipopolysaccharide.
Pulmonary macrophage phagocytosis of Pseudomonas aeruginosa is defective when this pathogen is opsonized with IgG antibodies isolated from serum samples from patients with cystic fibrosis (CF). To evaluate this defect further, IgG subclasses in the serum and lung fluids of patients with CF were quantitated. The pattern of IgG subclasses in serum specimens from patients with CF (n = 15) and in patients without CF but with chronic obstructive airway disease and recurrent P. aeruginosa infection (n = 4) was significantly altered from that found in normal subjects (n = 31). Immunoglobulin-G2 and IgG3 expressed as percentages of total IgG subclasses or in micrograms per milliliter of serum were significantly elevated in the serum specimens of these patients (p less than 0.05), and IgG1 was significantly decreased (p less than 0.01). It appears that the increase in IgG2 in the serum of patients with CF and those without CF but with chronic P. aeruginosa infection may be in response to chronic antigenic stimulation by P. aeruginosa lipopolysaccharide. Evidence presented to support this includes: (1) IgG2 is not increased in CF serum if a history of P. aeruginosa infection is absent, (2) IgG2 levels expressed as percentages of total IgG subclasses in CF lung fluids were positively correlated (r = 0.73) with the number of colony-forming units of P. aeruginosa present in CF sputum specimens, and (3) IgG antibodies specifically eluted from P. aeruginosa lipopolysaccharide ligands on affinity gels were largely restricted to IgG2. The opsonic index, ([IgG3] + [IgG1]) divided by ([IgG2] + [IgG4]), is inverted in CF lung fluids (0.73:1; normal, 2:1). Because pulmonary macrophages show surface receptors binding primarily with IgG3 and IgG1, it may be that such an alteration in IgG subclasses in the respiratory secretions of patients with CF further inhibits opsonin-mediated clearance of P. aeruginosa.
Adolescent↗
Antibody to multiple mucoid strains of Pseudomonas aeruginosa in patients with cystic fibrosis, measured by an enzyme-linked immunosorbent assay.
The sera from 32 patients with cystic fibrosis who were chronically colonized with mucoid strains of Pseudomonas aeruginosa (MPA) were tested for anti-MPA antibodies. Using an enzyme-linked immunosorbent assay we measured IgA, IgG, and IgM antibody titers to three MPA strains, extracts of those strains, and seaweed-derived sodium alginate, which is similar chemically to the exopolysaccharide of MPA. These titers were compared with identical tests performed on the sera of eight cystic fibrosis patients who never were colonized with MPA and 10 normal adults. The IgG titers were significantly higher in tests of sera from the colonized patients compared with the other two groups but the IgA and IgM titers were not significantly higher. In colonized patients antibody titers to the different antigens correlated with each other suggesting that the major antibody response was to common antigenic determinants. Using these titers as a data base, eight patients whose clinical status was unknown to the testers, had IgG-enzyme-linked immunosorbent assay tests of their sera and the four colonized patients with cystic fibrosis were correctly identified. Three of them had substantial titers of antibody in the bronchoalveolar lavage fluid.
Adult↗
Complement activation in cystic fibrosis respiratory fluids: in vivo and in vitro generation of C5a and chemotactic activity.
Experiments performed in vitro have demonstrated that leukocyte neutral proteases produce an important mediator of inflammation, C5a, by proteolysis of the C5 component of the complement system. Cystic fibrosis (CF) lung fluids were characterized by high levels of neutrophils (39% of total cells versus 2% in normals) and contained significantly elevated amounts of elastolytic activity (mean 17.7 ng/micrograms total protein) compared to the lung fluids obtained from normal volunteers (0.2 ng elastolytic activity/micrograms protein, p = 0.001). The objective of these studies was to determine if complement activation and complement-derived chemotactic activity are present in CF lung fluids. C3c peptide representing activation of C3 could not be identified in the bronchial-alveolar lung lavage fluids of normal subjects but was readily identified by means of crossed immunoelectrophoresis in CF lung fluids (n = 9, mean 49% of C3); the mean level of C3 was decreased in CF lung specimens. Chemotactic activity was significantly elevated in lung fluids of the CF patients when compared to normal lung fluids. Using gel-filtration chromatography and a sensitive radioimmunoassay the chemotaxin present in CF specimens was identified as the anaphylatoxin C5a. C5a levels in the bronchial-alveolar lavage fluids of CF patients was inversely related to volume in liters expired in 1 s of a forced expiratory maneuver expressed as a percent of vital capacity determined from a forced expiratory maneuver (r = -0.72). Because there was a direct relationship between the total elastolytic activity present in CF airways and the concentration of C5a (r = 0.97, p = 0.03), it was postulated that airway proteases with elastolytic activity also cleave C5, nonimmunologically producing C5a. Detailed inhibition assays revealed that much of the total elastolytic activity had the inhibition profile of a serine proteinase. The levels of the serine proteinases were closely correlated with the numbers of neutrophilic leukocytes present per ml of lavage fluid (r = 0.7, p = 0.05). However, inhibitors of leukocyte serine proteases did not prevent the generation of additional chemotactic activity and the proteolysis of radiolabeled C5 substrate was not prevented by inhibitors of neutrophil elastase. Although the purified metalloelastase of Pseudomonas aeruginosa was active on cell-bound and free C5 yielding C5a, inhibition of this bacterial protease in CF lung fluids only partially blocked cleavage of the alpha- and beta-chains of C5.(ABSTRACT TRUNCATED AT 400 WORDS)
Adolescent↗