Complementation of methionine auxotrophs of Pseudomonas aeruginosa from cystic fibrosis

Auxotrophic Pseudomonas aeruginosa are exclusive to respiratory infections in cystic fibrosis (CF) and bronchiectatic patients, and isolates require specific amino acids for growth on minimal media, particularly methionine. Since auxotrophic and prototrophic P. aeruginosa from CF are identical by genotyping, we investigated the genetic events leading to methionine auxotrophy (Met-). Most (10/13) Met- strains had the same pattern of growth on methionine precursors and required methionine exclusively for growth. Back mutation to prototrophy was very low (frequencies 10(-8) to <10(-10)). Complementation of the mutations leading to auxotrophy was achieved for five strains with a genomic library of P. aeruginosa PAO1. Strains with different patterns of growth on methionine precursors were complemented by clones with different restriction patterns, while identical clones complemented strains with the same pattern of growth on methionine precursors. Methionine auxotrophy in P. aeruginosa from CF results from stable chromosomal mutations, and the commonest defect is probably in gene(s) encoding enzymes that convert homocysteine to methionine.     

Immunohistologic quantification of Pseudomonas aeruginosa in the tracheobronchial tree from patients with cystic fibrosis

Pseudomonas aeruginosa has been recognized as a pathogen of major importance in the patient with cystic fibrosis (CF). However, no information is available regarding the histologic quantification of P. aeruginosa organisms in the CF tracheobronchial tree. We retrieved all formalin-fixed paraffin-embedded lung blocks from 20 consecutive autopsies of cystic fibrosis patients. Serial histologic sections were made and stained by three methods: hematoxylin and eosin, immunoperoxidase with anti-P. aeruginosa rabbit serum as the primary antibody, and immunoperoxidase with normal rabbit serum as the primary antibody. By studying the hematoxylin and eosin section, we classified five areas in the lung as bronchi, large bronchioles, small bronchioles, bronchioloectatic areas, and abscess/airways with destroyed epithelium. The areas stained by an anti-P. aeruginosa immunoperoxidase method were examined under high-power magnification, and the bacteria within random fields were counted. Pseudomonas aeruginosa organisms were identified in 14 of 20 cases, including 13 of 16 cases in which P. aeruginosa was specifically cultured at autopsy. Quantification of organisms within the lumens of all five airway types showed that the bacterial density in cystic fibrosis airways is highest in bronchi.     

Direct sputum analysis of Pseudomonas aeruginosa macrorestriction fragment genotypes in patients with cystic fibrosis

The distribution of bacterial populations in the airways of 13 patients with cystic fibrosis who were colonized for 6-23 years with Pseudomonas aeruginosa was investigated by genotyping of bacterial chromosomes directly isolated from 21 sputa. After removal of host material from sputum by hypotonic cell lysis and repetitive washing and centrifugation steps, agarose-embedded bacterial cells were lysed, residual eukaryotic DNA separated by field inversion gel electrophoresis, and the purified bacterial chromosomes subjected to macrorestriction fragment pattern and Southern analyses. Bacterial populations consisted of a single P. aeruginosa clone in 17 sputa, of which more than one clonal variant was apparent in two SpeI fragment fingerprints. Two clones of P. aeruginosa and another species co-existed in four samples. Genomically homogeneous populations of P. aeruginosa are characteristic for chronically colonized lungs in most cases of cystic fibrosis.     

Immunization of cystic fibrosis patients with a Pseudomonas aeruginosa O-polysaccharide-toxin A conjugate vaccine

Healthy, non-colonized cystic fibrosis (CF) patients (N = 26) were immunized with an octavalent Pseudomonas aeruginosa O-polysaccharide-toxin A conjugate vaccine. Vaccination was well tolerated and induced anti-lipopolysaccharide (LPS) antibodies of a high affinity capable of promoting the opsonophagocytic killing of P. aeruginosa by human peripheral lymphocytes. In contrast, anti-LPS antibodies acquired after natural infection possessed a very low affinity and were non-opsonic. To determine if immunization could prevent or delay infections due to P. aeruginosa, the infection rate among immunized patients was compared retrospectively to age and gender-matched controls. After 6 years of clinical follow-up, 15/20 (75%) of control and 8/23 (35%) of immunized subjects were classified as infected (p = 0.022). The persistence of high-affinity antibodies among immunized patients correlated with a significantly lower rate of infection after 4-6 years of observation. Infection of immunized patients was correlated with a dramatic decline in total antibody titer between year 2 and 3 of follow-up. Smooth, typeable strains of P. aeruginosa predominated among immunized patients. In contrast, rough, nontypeable strains were most frequently isolated from nonimmunized patients. Mucoid P. aeruginosa strains were isolated from 6 nonimmunized patients versus only I immunized subject.     

Comprehensive analysis of risk factors for acquisition of Pseudomonas aeruginosa in young children with cystic fibrosis

Racial differences in insulin secretion and insulin sensitivity in healthy children were studied by administering a 2-hour hyperglycemic clamp (225 mg/dL) to 14 black and 16 white healthy adolescents (Tanner II-V), and 12 black and 11 white prepubertal children, matched for age, body mass index, and Tanner I pubertal development. In prepubertal children, fasting and first-phase insulin concentrations were higher in blacks compared with whites (14.7+/-1.3 vs 10.4+/-1.2, P=0.02, and 76.9+/-6.8 vs 52.1+/-6.4 microu/mL, P=0.016). There were no differences in second-phase insulin levels and insulin sensitivity index. In pubertal adolescents, first-phase and second-phase insulin concentrations were higher in blacks compared with whites (first-phase: 157.3+/-18.3 vs 77.0+/-8.7 microu/mL, P=0.0003; second-phase: 175.0+/-24.3 vs 108.7+/-8.8 microu/mL, P=0.012). Insulin sensitivity index was 35% lower in black adolescents compared with whites (P=0.02). These findings indicate that significant differences in insulin secretion and sensitivity are detectable early in childhood in healthy African-American vs American whites. However, genetic (race) vs environmental factors (physical activity/fitness, energy balance) should be carefully scrutinized as potential factors responsible for such differences.     

Lung disease in mice with cystic fibrosis

The leading cause of mortality and morbidity in humans with cystic fibrosis is lung disease. Advances in our understanding of the pathogenesis of the lung disease of cystic fibrosis, as well as development of innovative therapeutic interventions, have been compromised by the lack of a natural animal model. The utility of the CFTR-knockout mouse in studying the pathogenesis of cystic fibrosis has been limited because of their failure, despite the presence of severe intestinal disease, to develop lung disease. Herein, we describe the phenotype of an inbred congenic strain of CFTR-knockout mouse that develops spontaneous and progressive lung disease of early onset. The major features of the lung disease include failure of effective mucociliary transport, postbronchiolar over inflation of alveoli and parenchymal interstitial thickening, with evidence of fibrosis and inflammatory cell recruitment. We speculate that the basis for development of lung disease in the congenic CFTR-knockout mice is their observed lack of a non-CFTR chloride channel normally found in CFTR-knockout mice of mixed genetic background.     

Rationale for development of immunotherapies that target mucoid Pseudomonas aeruginosa infection in cystic fibrosis patients

Despite a complex sputum bacteriology, the progressive decline in pulmonary function that is the hallmark of the genetic disease cystic fibrosis (CF) is attributable to a single infecting pathogen, mucoid Pseudomonas aeruginosa. Therefore, active and passive immunotherapies that target this particular variant of the bacterium should be of value in attenuating infection and interfering with the decline in pulmonary function. The major surface antigen of mucoid P. aeruginosa is referred to as either mucoid exopolysaccharide (MEP) or alginate, a random polymer of D-mannuronic and L-guluronic acid residues linked beta 1-4. During chronic infection CF patients make antibodies to MEP that fail to mediate opsonic killing of bacteria in vitro. These antibodies can be elicited by vaccination in 35-40% of plasma donors given a preparation of MEP comprised of only the highest molecular-weight polymers; inclusion in human vaccines of smaller polymers normally produced by the bacterium fails to elicit opsonic antibodies, just like in infected CF patients. Opsonic, but not non-opsonic, antibodies to MEP protect animals against chronic endobronchial infection. CF patients do produce opsonic antibodies to mucoid P. aeruginosa that are in a planktonic or suspended state, but these antibodies are not directed at the MEP antigen and they fail to kill P. aeruginosa growing in a biofilm. This is the state that the bacteria grow in the lung. Therefore immunoglobulin G preparations with opsonic antibodies to MEP could provide CF patients with antibodies that they normally do not produce during chronic lung infection and may improve their clinical course.     

Activation of NF-kappaB by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells

We have generated proliferating cell nuclear antigen (PCNA) mutants by low fidelity PCR and screened for lethal mutations by testing for lack of complementation of a Schizosaccharomyces pombe strain disrupted for the pcn1 + gene. We thus identified eight lethal mutants out of the 50 cDNAs tested. Six were truncated in their C-terminal region due to the introduction of a stop codon within their coding sequences. Two were full-length with a single point mutation at amino acid 68 or 69. The two latter mutants were overexpressed in insect cells via a recombinant baculovirus and were purified. They were unable to stimulate DNA polymerase delta DNA replication activity on a poly(dA).oligo(dT) template. Cross-linking experiments showed that this was due to their inability to form trimers. Since these two mutations are adjacent and not located in a domain of the protein putatively involved in inter-monomer interactions, our results show that the beta-sheet betaF1 to which they belong must play an essential role in maintaining the 3-dimensional structure of S.pombe PCNA.     

Interleukin-4 enhances pulmonary clearance of Pseudomonas aeruginosa

To determine the effects of interleukin-4 (IL-4) on bacterial clearance from the mouse lung, transgenic mice expressing IL-4 in respiratory epithelial cells under the control of the Clara cell secretory protein promoter (CCSP-IL-4 mice) were infected intratracheally with Pseudomonas aeruginosa. Survival of CCSP-IL-4 mice following bacterial administration was markedly improved compared with that of control mice. While bacteria proliferated in lungs of wild-type mice, a rapid reduction in the number of bacteria was observed in the IL-4 mice as early as 6 h postinfection. Similarly, intranasal administration of IL-4 enhanced bacterial clearance from the lungs of wild-type mice. While acute and chronic IL-4 increased the numbers of neutrophils in bronchoalveolar lavage fluid, bacterial infection was associated with acute neutrophilic pulmonary infiltration, and this response was similar in the presence or absence of IL-4. Local administration or expression of IL-4 in the mouse lung enhanced pulmonary clearance of P. aeruginosa in vivo and decreased mortality following infection.     

Airway clearance techniques in the treatment of cystic fibrosis

A one-year-old girl with a giant cell fibroblastoma (GCF) of the skin in the left arm is described. The tumor presented as a small, asymptomatic, subcutaneous mass that enlarged relatively slowly. GCF is a rare mesenchymal tumor occurring predominantly in young children. Its unique histopathological feature can lead to a misdiagnosis as sarcoma very easily. We review briefly the clinical and pathological information of 68 documented cases in the literature and discuss the pathogenesis of this peculiar neoplasm.     

Finding evidence to support airway clearance techniques in cystic fibrosis

Chest physiotherapy, aiming to clear bronchopulmonary secretions, has become a mainstay in the respiratory management of cystic fibrosis (CF). Early diagnosis and new therapeutic interventions have dramatically improved the outlook for patients with this disorder and it is no longer a disease of childhood. Along with these changes chest physiotherapy has also progressed, with the development of several treatment modalities that are more effective and can be performed by the patient without assistance. This allows older children and adults with CF to lead more normal and independent lifestyles. Despite this progress questions remain regarding the efficacy and consequences of airway clearance techniques, the scientific evidence available to support the selection of the most appropriate treatment modality and, not least, the problems associated with the treatment-related burden that is placed on patients and their families.     

Pseudomonas aeruginosa isolates from patients with cystic fibrosis have different beta-lactamase expression phenotypes but are homogeneous in the ampC-ampR genetic region

Pseudomonas aeruginosa isolates from 1 of 17 cystic fibrosis patients produced secondary beta-lactamase in addition to the ampC beta-lactamase. Isolates were grouped into three beta-lactamase expression phenotypes: (i) beta-lactam sensitive, low basal levels and inducible beta-lactamase production; (ii) beta-lactam resistant, moderate basal levels and hyperinducible beta-lactamase production; (iii) beta-lactam resistant, high basal levels and constitutive beta-lactamase production. Apart from a base substitution in the ampR-ampC intergenic region of an isolate with moderate-basal-level and hyperinducible beta-lactamase production, sensitive and resistant strains were identical in their ampC-ampR genetic regions. Thus, enhanced beta-lactamase expression is due to mutations in regulatory proteins other than AmpR.     

Mucociliary clearance and respiratory function in foundry workers

The aim of the study was to establish whether changes occur in respiratory function, particularly mucociliary clearance, among second fusion smeltery workers. The research covered 93 male smelters employed in steel forming and casting and 116 male workers of an electric power station, considered as non-exposed. Physiological, pathological and occupational histories of all subjects under study were available. An ECCS respiratory symptoms questionnaire was administered to all subjects ad the two groups also underwent a general medical examination, a spirometry and a chest X-ray. During the medical examination sputum was collected from the subjects to measure mucus transport rate on frog palate, expressed as Normalised Frog Palate Transport Rate (NFPTR). For the environmental research, dust, fumes and gas samplings were taken either at a fixed station or by means of personal dosimeters. Environmental research revealed very low concentrations of respiratory irritants (total dust: 0.2-6.8 mg/m3; respirable dust: 0.1-4.9 mg/m3; total silica: < 2-15.5%; respirable silica: < 0.004-0.3 mg/m3; iron: 0.008-0.085 mg/m3; chromium and manganese: < 0.001 mg/m3; fumes and gases: well below the TLV. The two groups were homogeneous with regard to age and smoking habits. Exposed workers showed rales, dyspnoea and spontaneous phlegm more frequently than non-exposed workers. NFPTR alterations were checked in 49 out of 81 exposed and in 18 out of 81 non-exposed subjects (chi squared = 22.9; p < 0.001). Stratification of the results according to smoking habits further confirmed the strong association between occupational exposure and NFPTR alterations. Smelters showed significantly lower mean NFPTR values compared to non-exposed subjects; also, the mean value of NFPTR in the exposed was below 0.70, which is considered the lowest individual limit in normal subjects. The only variable which explains a large part of the variability of NFPTR is past work in a smeltery rather than in an electric power station. The spirometries showed that only the mean PEF values were significantly lower among the exposed. Stratified analysis of the results according to smoking habits in the two groups revealed a close association between smeltery work and reduction of PEF to under 80% of the ECCS 1983 theoretical values, independently of smoking habits. We also compared the mean PEF values, both as measured values and as percent values of the ECCS 1983 theoretical values, stratified for occupational exposure and smoking; the results again showed that differences between these mean values were mainly due to current or past work in the foundry.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant DeltaF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.     

Amphotericin B-induced resistance to Pseudomonas aeruginosa infection in mice

We evaluated the effects of amphotericin B (AmB) against Pseudomonas aeruginosa (P. aeruginosa) infection in mice. Pretreatment with 2 mg/kg of AmB 24 hours before infection significantly increased the survival rates of mice intraperitoneally infected with either P. aeruginosa or Escherichia coli. To evaluate the mechanism of this AmB-induced resistance to infection, we conducted a number of experiments. Peritoneal macrophages exposed in vitro to AmB showed superior bactericidal activity compared to that of control macrophages. Interleukin-1 production by peritoneal macrophages from mice pretreated with 2 mg/kg of AmB was significantly higher than that in control mice. Serum tumor necrosis factor level after intravenous injection of P. aeruginosa was also higher in mice pretreated with 2 mg/kg of AmB than in control mice. These data indicate that AmB induces resistance to P. aeruginosa in mice. Furthermore AmB-induced activation of peritoneal macrophages and their production of interleukin-1 and tumor necrosis factor appeared to play important roles in this phenomenon.