lactoferrin and Cystic-Fibrosis

The review presents the pathobiochemical and molecular mechanisms of sputum formation in patients with cystic fibrosis associated with the pathophysiological features of the disease. Statistical data on the prevalence of this pathology in the world and in the Russian Federation are presented. The mechanisms of sputum formation and disorders of the mucociliary apparatus, leading to the accumulation of viscous bronchopulmonary secret in cystic fibrosis, are considered. The principles of the relationship between the rheological properties of sputum and the formation of inflammation in the lungs with the addition of a concomitant specific microflora in the bronchopulmonary system in patients with cystic fibrosis are presented. Describes the opportunities for biochemical studies of sputum of patients with this pathology: determining the activity of enzymes (myeloperoxidase), the content of proteinase inhibitors (α2-macroglobulin and α1-antitrypsin) and proinflammatory cytokines (IL-8 and TNFa), concentrations of iron and ferriferous proteins (lactoferrin and ferritin), which makes biochemical studies of sputum available, non-invasive, quick and cost-effective method of diagnosis, which can be widely used as an auxiliary laboratory method and makes it possible to use these metabolites as diagnostic markers to assess the severity of inflammation and infection of the lower respiratory tract and predict the development of respiratory complications in patients with cystic fibrosis.

Topics: Cystic Fibrosis; Cytokines; Ferritins; Humans; Inflammation; Lactoferrin; Peroxidase; Protease Inhibitors; Sputum

Topics: Animals; Burkholderia; Burkholderia gladioli; Burkholderia Infections; Burkholderia mallei; Burkholderia pseudomallei; Computational Biology; Cystic Fibrosis; Ferritins; Glanders; Heme; Horses; Humans; Iron; Lactoferrin; Lung; Melioidosis; Siderophores; Virulence

The aerobic Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen responsible for life-threatening acute and chronic infections in humans. As part of chronic infection P. aeruginosa forms biofilms, which shield the encased bacteria from host immune clearance and provide an impermeable and protective barrier against currently available antimicrobial agents. P. aeruginosa has an absolute requirement for iron for infection success. By influencing cell-cell communication (quorum sensing) and virulence factor expression, iron is a powerful regulator of P. aeruginosa behaviour. Consequently, the imposed perturbation of iron acquisition systems has been proposed as a novel therapeutic approach to the treatment of P. aeruginosa biofilm infection. In this review, we explore the influence of iron availability on P. aeruginosa infection in the lungs of the people with the autosomal recessive condition cystic fibrosis as an archetypal model of chronic P. aeruginosa biofilm infection. Novel therapeutics aimed at disrupting P. aeruginosa are discussed, with an emphasis placed on identifying the barriers that need to be overcome in order to translate these promising in vitro agents into effective therapies in human pulmonary infections.

Topics: Anti-Infective Agents; Biofilms; Chelating Agents; Cystic Fibrosis; Gene Expression Regulation, Bacterial; Homeostasis; Humans; Iron; Lactoferrin; Lung; Pseudomonas aeruginosa; Pseudomonas Infections; Quorum Sensing; Respiratory Tract Infections; Thiocyanates

The airway provides numerous defense mechanisms to prevent microbial colonization by the large numbers of bacteria and viruses present in ambient air. An important component of this defense is the antimicrobial peptides and proteins present in the airway surface fluid (ASF), the mucin-rich fluid covering the respiratory epithelium. These include larger proteins such as lysozyme and lactoferrin, as well as the cationic defensin and cathelicidin peptides. While some of these peptides, such as human beta-defensin (hBD)-1, are present constitutively, others, including hBD2 and -3 are inducible in response to bacterial recognition by Toll-like receptor-mediated pathways. These peptides can act as microbicides in the ASF, but also exhibit other activities, including potent chemotactic activity for cells of the innate and adaptive immune systems, suggesting they play a complex role in the host defense of the airway. Inhibition of antimicrobial peptide activity or gene expression can result in increased susceptibility to infections. This has been observed with cystic fibrosis (CF), where the CF phenotype leads to reduced antimicrobial capacity of peptides in the airway. Pathogenic virulence factors can inhibit defensin gene expression, as can environmental factors such as air pollution. Such an interference can result in infections by airway-specific pathogens including Bordetella bronchiseptica, Mycobacterium tuberculosis, and influenza virus. Research into the modulation of peptide gene expression in animal models, as well as the optimization of peptide-based therapeutics shows promise for the treatment and prevention of airway infectious diseases.

Topics: Animals; Antimicrobial Cationic Peptides; Cathelicidins; Cystic Fibrosis; Defensins; Disease Models, Animal; Humans; Lactoferrin; Muramidase; Proteinase Inhibitory Proteins, Secretory; Proteins; Respiratory System; Toll-Like Receptors; Tuberculosis, Pulmonary; Virus Diseases

Cystic Fibrosis (CF) has prominent gastrointestinal and pancreatic manifestations. The aim of this study was to determine the effect of Cystic fibrosis transmembrane conductance regulator (CFTR) modulation on, gastrointestinal inflammation, pancreatic function and gut microbiota composition in people with cystic fibrosis (CF) and the G551D-CFTR mutation.. Fourteen adult patients with the G551D-CFTR mutation were assessed clinically at baseline and for up to 1 year after treatment with ivacaftor. The change in gut inflammatory markers (calprotectin and lactoferrin), exocrine pancreatic status and gut microbiota composition and structure were assessed in stool samples.. There was no significant change in faecal calprotectin nor lactoferrin in patients with treatment while all patients remained severely pancreatic insufficient. There was no significant change in gut microbiota diversity and richness following treatment.. There was no significant change in gut inflammation after partial restoration of CFTR function with ivacaftor, suggesting that excess gut inflammation in CF is multi-factorial in aetiology. In this adult cohort, exocrine pancreatic function was irreversibly lost. Longer term follow-up may reveal more dynamic changes in the gut microbiota and possible restoration of CFTR function.

Topics: Adult; Aminophenols; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Inflammation; Lactoferrin; Leukocyte L1 Antigen Complex; Microbiota; Mutation; Prospective Studies; Quinolones

To determine the antimicrobial activity of ALX-009, a combination of bovine lactoferrin and hypothiocyanite, in sputum against Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc), key pathogens causing infection in the lungs of cystic fibrosis (CF) patients.. The antimicrobial activity of ALX-009 against clinical respiratory P. aeruginosa isolates was determined by time-kill assay. Sputum from CF patients was treated with ALX-009, either alone or in combination with tobramycin, and the effect on P. aeruginosa, Bcc and total sputum density was determined.. Time-kill assay indicated that ALX-009 was bactericidal at 24 h against 4/4 P. aeruginosa isolates under aerobic conditions, and against 3/4 isolates under anaerobic conditions. ALX-009 was also bactericidal against P. aeruginosa in sputum samples at 6 h (n = 22/24 samples) and 24 h (n = 14/24 samples), and demonstrated significantly greater activity than tobramycin at both timepoints. Activity against Bcc in sputum samples (n = 9) was also demonstrated, but the magnitude of change in Bcc density was less than for P. aeruginosa. To determine the effect of treating sputum with two doses of ALX-009, similar to current regimens for inhaled antibiotics, aliquots of a further 10 sputum samples positive for P. aeruginosa were treated with one (t = 0 h) or two doses (t = 0 h, t = 12 h) of ALX-009; treatment with two doses resulted in bactericidal activity in 7/10 samples at 34 h compared with only 3/10 samples when treatment was with one dose.. ALX-009 demonstrates promise as a novel antimicrobial that could be used to decrease P. aeruginosa density in the lungs of people with CF.

Topics: Anti-Infective Agents; Burkholderia cepacia complex; Cystic Fibrosis; Humans; Lactoferrin; Microbial Sensitivity Tests; Microbial Viability; Pseudomonas aeruginosa; Sputum; Thiocyanates

Conflicting data are reported on pro- or anti-inflammatory activity of bovine lactoferrin (bLf) in different cell models as phagocytes or epithelial cell lines infected by bacteria. Here we evaluated the bLf effect on epithelial models mimicking two human pathologies characterized by inflammation and infection with specific bacterial species. Primary bronchial epithelium from a cystic fibrosis (CF) patient and differentiated intestinal epithelial cells were infected with Pseudomonas aeruginosa LESB58 isolated from a CF patient and Adherent-Invasive Escherichia coli LF82 isolated from a Crohn's disease patient. Surprisingly, bLf significantly reduced the intracellular bacterial survival, but differently modulated the inflammatory response. These data lead us to hypothesize that bLf differentially acts depending on the epithelial model and infecting pathogen. To verify this hypothesis, we explored whether bLf could modulate ferroportin (Fpn), the only known cellular iron exporter from cells, that, by lowering the intracellular iron level, determines a non permissive environment for intracellular pathogens. Here, for the first time, we describe the bLf ability to up-regulate Fpn protein in infected epithelial models. Our data suggest that the mechanism underlying the bLf modulating activity on inflammatory response in epithelial cells is complex and the bLf involvement in modulating cellular iron homeostasis should be taken into account.

Topics: Animals; Antimicrobial Cationic Peptides; Bronchi; Cation Transport Proteins; Cattle; Cells, Cultured; Crohn Disease; Cystic Fibrosis; Epithelial Cells; Escherichia coli; Humans; Inflammation Mediators; Iron; Lactoferrin; Models, Biological; Pseudomonas aeruginosa; Respiratory Mucosa

Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl(-) and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca(2+) potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus.

Topics: Amiloride; Animals; Biomarkers; Calcium; Cells, Cultured; Chlorides; Cyclic AMP; Cystic Fibrosis; Diffusion Chambers, Culture; Disease Models, Animal; Electric Impedance; Epithelial Cells; Exocrine Glands; Humans; Ion Transport; Lactoferrin; Methacholine Chloride; Mucin-5B; Muramidase; Swine; Tight Junctions; Trachea

To determine the feasibility of formulating and aerosolizing powders containing bacteriophages KS4-M and ΦKZ for lung delivery and treatment of pulmonary Burkholderia cepacia complex and Pseudomonas aeruginosa infections.. Endotoxin-removed bacteriophages KS4-M and ΦKZ were lyophilized in lactose/lactoferrin 60 : 40 w/w matrix and deagglomerated in a mixer mill (without beads) to formulate respirable powders. The powders were then aerosolized using an Aerolizer(®) capsule inhaler. Mass median aerodynamic diameter (MMAD) of this inhalable aerosol was determined using Andersen cascade impactor at 60 l min(-1). Measured MMAD for both types of powders was 3·4 μm, and geometric standard deviation was 1·9-2·0. Viability of bacteriophages delivered distal to an idealized mouth-throat replica was determined from bioassays of samples collected on filters placed after the idealized replica. As a percentage of inhaler load, amount of powder delivered distal to the mouth-throat replica, which is a measure of lung delivery, was 33·7 ± 0·3% for KS4-M and 32·7 ± 0·9% for ΦKZ. Titres collected downstream of the mouth throat were (3·4 ± 2·5) × 10(6) PFU for KS4-M with an Aerolizer capsule load of (9·8 ± 4·8) × 10(6) and (1·9 ± 0·6) × 10(7) for ΦKZ with an Aerolizer capsule load of (6·5 ± 1·9) × 10(7).. Bacteriophages KS4-M and ΦKZ can be lyophilized without significant loss of viability in a lactose/lactoferrin 60 : 40 w/w matrix. The resulting powders can be aerosolized to deliver viable bacteriophages to the lungs..   Development of lactoferrin-based bacteriophage aerosol powders solidifies the ground for future research on developing novel formulations as an alternative to inhaled antibiotic therapy in patients with cystic fibrosis.

Topics: Aerosols; Bacteriophages; Burkholderia cepacia complex; Burkholderia Infections; Cystic Fibrosis; Dry Powder Inhalers; Freeze Drying; Humans; Lactoferrin; Lung; Nebulizers and Vaporizers; Pseudomonas aeruginosa; Pseudomonas Infections

Antimicrobial proteins are important in lung defense and are potential therapeutic agents in chronic airways infection such as seen in cystic fibrosis (CF). In preparation for future clinical studies, we sought (1) to determine levels of three antimicrobial proteins [lactoferrin, lysozyme, and secretory leukoprotease inhibitor (SLPI)] in the CF airway and (2) to examine the relationships between these antimicrobial proteins and airway inflammation and infection. We examined bronchoalveolar lavage fluid (BALF) from 45 individuals with CF and 23 disease control individuals. Airway inflammation was measured through BALF neutrophil counts and neutrophil elastase activity. Infection was assessed through quantitative counts of CF-related bacterial pathogens. BALF lysozyme activity and lactoferrin levels were elevated in individuals with CF compared to controls whereas SLPI levels were not different between the groups. Among the CF subjects, lysozyme activity and lactoferrin increased with age while SLPI decreased with age. Lysozyme activity and lactoferrin concentrations correlated positively with neutrophil counts but not with bacterial colony counts. SLPI levels were inversely related to both neutrophil counts and bacterial colony counts. This study provides information concerning the levels of antimicrobial proteins present in the CF airway that are relevant to future clinical trials of these compounds and demonstrates clear relationships between antimicrobial protein-specific levels and airway inflammation and infection.

Topics: Adolescent; Adult; Biomarkers; Bronchitis; Bronchoalveolar Lavage Fluid; Case-Control Studies; Child; Child, Preschool; Cross-Sectional Studies; Cystic Fibrosis; Female; Humans; Immunity, Innate; Infant; Lactoferrin; Leukocyte Count; Leukocyte Elastase; Male; Muramidase; Respiratory Tract Infections; Secretory Leukocyte Peptidase Inhibitor; Young Adult

The cystic fibrosis (CF) pathogen Burkholderia cepacia complex (Bcc) is innately resistant to antibiotics and the development of effective therapeutic strategies to treat these infections is a major challenge. The objectives of this study were to investigate the effects of recombinant human lactoferrin (rHL) on planktonic and biofilm cultures of Bcc organisms and to establish whether lactoferrin alters the susceptibility of these cultures to a range of antibiotic therapies.. Planktonic and biofilm cultures of strains representative of three species of Bcc, Burkholderia multivorans, Burkholderia cenocepacia and Burkholderia dolosa, were exposed to 0-900 mg/L lactoferrin over 0-48 h. Growth was determined using both spectrophotometric and plate counting methods. The ability of these strains to form and develop biofilms in vitro was also examined. Antimicrobial susceptibility in the presence/absence of lactoferrin was assessed using conventional MICs and a modified method was used to determine biofilm susceptibility to various antibiotics.. We found that physiological concentrations of lactoferrin inhibited the growth of both planktonic and biofilm cultures of Bcc in vitro. The addition of lactoferrin to rifampicin enhanced the antibiotic susceptibility of the Bcc strains when grown planktonically and as biofilms.. The present study demonstrates the growth inhibitory and antibiofilm activity of rHL against different species of Bcc. Furthermore, the enhanced susceptibility of both planktonic and biofilm cultures to rifampicin in the presence of lactoferrin offers the potential for novel uses of antibiotics in combination with lactoferrin to treat Bcc infections in CF patients.

Topics: Biofilms; Burkholderia cepacia complex; Colony Count, Microbial; Cystic Fibrosis; Drug Resistance, Bacterial; Lactoferrin; Microbial Sensitivity Tests; Recombinant Proteins

Lactoferrin-induced cell depolarization and a delayed tobramycin-killing effect on Pseudomonas aeruginosa cells were correlated. This antibiotic tolerance effect (ATE) reflects the ability of a defense protein to modify the activity of an antibiotic as a result of its modulatory effect on bacterial physiology. P. aeruginosa isolates from cystic fibrosis patients showed higher ATE values (< or = 6-fold) than other clinical strains.

Topics: Anti-Bacterial Agents; Cystic Fibrosis; Drug Resistance, Bacterial; Humans; Lactoferrin; Pseudomonas aeruginosa; Pseudomonas Infections; Tobramycin

Topics: Anti-Bacterial Agents; Cystic Fibrosis; Drug Therapy, Combination; Gentamicins; Gram-Negative Bacterial Infections; Humans; Lactoferrin; Microbial Sensitivity Tests; Recombinant Proteins; Rifampin; Stenotrophomonas maltophilia

Oxidative stress has been proposed as a mechanism of injury underlying obliterative bronchiolitis. Catalytically reactive iron is a potential source of reactive oxygen species in transplanted tissue. Using samples acquired from surveillance bronchoalveolar lavage (BAL), we tested the postulate that there is a disruption of iron equilibrium in transplanted lung, which can worsen with time.. A control group of 5 healthy, non-smoking volunteers underwent BAL. Five bilateral lung transplant patients underwent surveillance BAL with transbronchial lung biopsies. The BAL fluid concentrations of protein, albumin, total iron, lactoferrin, ferritin, transferrin receptor and total iron binding capacity were measured.. The mean ages in the control and transplant groups were 25.0 +/- 2.4 and 34.6 +/- 5.0 years, respectively. Patients were transplanted for cystic fibrosis (n = 3), primary ciliary dyskinesia (n = 1) and bronchiolitis obliterans (n = 1). Surveillance bronchoscopies were performed at 100.6 +/- 63.3, 175.0 +/- 87.7 and 259.2 +/- 82 days post-transplant. No significant differences were noted in BAL protein, albumin and total iron binding capacity (TIBC) levels between the 2 groups. The BAL iron, transferrin, transferrin receptor, lactoferrin and ferritin levels were significantly elevated in transplant patients relative to controls. With time after transplantation, there were increases in lavage iron, transferrin receptor, lactoferrin and ferritin concentrations.. Abnormally high levels of iron and its homeostatic proteins were found in the lung allografts, and levels appeared to increase with time. This supports a disruption in the normal homeostasis of this metal after transplantation and a potential role for a catalyzed oxidative stress in bronchiolitis obliterans. The use of iron-depleting therapy is a possible means for preventing injury in the lung allograft.

Topics: Adolescent; Adult; Bronchoalveolar Lavage; Cystic Fibrosis; Female; Ferritins; Homeostasis; Humans; Iron; Lactoferrin; Lung Transplantation; Male; Oxidative Stress

Intractable formation of biofilm by and infection with the opportunistic pathogen Pseudomonas aeruginosa are hallmarks of cystic fibrosis (CF). Lactoferrin, an innate immunity protein, has recently been shown to inhibit the formation of P. aeruginosa biofilm. Partial cleavage of lactoferrin by the proteases neutrophil elastase and Pseudomonas elastase has previously been described in CF. Here, we show that cathepsins in CF secretions are responsible for complete and rapid cleavage of lactoferrin. We demonstrate that levels of lactoferrin in P. aeruginosa-positive sputum samples are decreased when corrected for inflammatory burden and that P. aeruginosa-positive sputum samples have significantly higher cathepsin activity and significantly reduced ability to inhibit formation of biofilm, compared with P. aeruginosa-negative sputum samples. We also show that cleavage of lactoferrin by cathepsin results in loss of both its microbicidal and antibiofilm activity. Loss of such a vital innate immunity protein clearly has important implications for the pathogenesis of chronic P. aeruginosa lung infection in patients with CF.

Topics: Adolescent; Anti-Infective Agents; Biofilms; Bronchoalveolar Lavage Fluid; Cathepsins; Child; Cystic Fibrosis; Female; Humans; Lactoferrin; Male; Pseudomonas aeruginosa; Sputum

Antimicrobial peptides are part of the innate host defense system, and inactivation of these peptides is implicated in airway infections in cystic fibrosis (CF). The sputum of patients with CF contains anionic polyelectrolytes, including F-actin and DNA not found in normal airway fluid. These anionic filaments aggregate to contribute to the altered viscoelastic properties of CF sputum. We hypothesized that the airway components stabilizing bundles of F-actin and DNA are in part cationic antimicrobial agents, and that appropriate modification of diseased airway fluid of patients with CF might dissociate these bundles and restore antimicrobial activity. We demonstrate that the human cathelicidin peptide LL37 forms bundles with F-actin and DNA, which are dissolved by gelsolin and DNase, respectively. Coincident with bundle formation, antimicrobial activity of LL37 is inhibited by F-actin and DNA. Pseudomonas bacteria were killed by low concentrations of LL37, but killing was significantly reduced in the presence of F-actin. The actin filament-fragmenting protein gelsolin restored bactericidal activity nearly completely. In a growth inhibition assay, the effects of F-actin were confirmed, and DNA was also shown to inhibit the activity of LL37. When added to CF sputum, gelsolin significantly reduced the growth of bacteria, suggesting activation of endogenous antimicrobial factors. These findings may have therapeutic implications for treatments previously thought to alter only the viscoelastic properties of airway secretions and amplify the possible advantage of gelsolin in CF treatment.

Topics: Actin Cytoskeleton; Actins; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria; Cathelicidins; Cystic Fibrosis; Deoxyribonucleases; DNA; Gelsolin; Humans; Lactoferrin; Light; Macromolecular Substances; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Scattering, Radiation; Sputum

Matrix metalloproteinases (MMPs) are involved in the remodelling and degradation of extracellular matrix and may play a role in pulmonary tissue destruction in cystic fibrosis (CF).. Bronchoalveolar lavage (BAL) fluid levels of MMP-8, MMP-9, and their natural inhibitor TIMP-1 were measured on two occasions within 18 months in 23 children with mild CF, 13 of whom were treated with DNase.. MMP-8 (39.3 (6.8) v 0.12 (0.01) ng/ml), MMP-9 (58.0 (11.4) v 0.5 (0.02) ng/ml), and the molar ratio of MMP-9/TIMP-1 (0.36 (0.05) v 0.048 (0.01)) were significantly higher in patients with CF than in control children without lung disease. Gelatine zymography showed the typical banding pattern of neutrophil derived MMP-9, including 130 kDa NGAL-MMP-9 complex and 92 kDa latent MMP-9 bands; 85 kDa bands (corresponding to active MMP-9) were seen in all patients. There was a close correlation between BAL fluid concentrations of MMPs and alpha(2)-macroglobulin, a marker of alveolocapillary leakage. After 18 months MMP levels were increased in untreated patients and decreased in patients treated with DNase.. Uninhibited MMPs may contribute to pulmonary tissue destruction even in CF patients with mild lung disease that may be positively affected by treatment with DNase.

Topics: Adolescent; alpha-Macroglobulins; Bronchoalveolar Lavage Fluid; Child; Cystic Fibrosis; Deoxyribonuclease I; Enzyme-Linked Immunosorbent Assay; Gelatinases; Humans; Lactoferrin; Matrix Metalloproteinase 8; Matrix Metalloproteinase 9; Tissue Inhibitor of Metalloproteinase-1

The cystic fibrosis transmembrane conductance regulator (CFTR) mediates secretion of mucins and serous proteins. The aim was to correct pharmacologically the CFTR defect in protein secretion in airway gland cells and so to correct the viscous mucous secretions in cystic fibrosis (CF) airways and lungs. The strategies tested included direct activation of CFTR, bypass of CFTR-mediated protein secretion and movement of the mutated form of CFTR (DeltaF(508)-CFTR) to the cell membrane. Compounds related to 3-isobutyl-1-methylxanthine (IBMX), including a selective type-IV phosphodiesterase inhibitor and the adenosine receptor antagonists 8-cyclopentyltheophylline (CPT) and 8-cyclopentyl-1,3-dipropylxanthine (CPX), corrected the defective beta-adrenergic stimulation of mucin secretion in CFTR antibody-inhibited submandibular gland cells. CPT also corrected lactoferrin secretion in DeltaF(508)/DeltaF(508)-CFTR nasal gland cells. The data suggest that correction of CFTR protein secretion activity is not mediated by excessive increase in cyclic AMP, involves direct interaction with CFTR but does not require increase in CFTR Cl(-) channel activity. Regulated glycoprotein secretion was characterised in the airway gland cell line Calu-3 to investigate whether a CFTR bypass is present. Studies of DeltaF(508)-CFTR trafficking using confocal imaging showed that some DeltaF(508)-CFTR colocalised with the apical membrane protein CD59; however a large amount was mislocalised within the cell. The results showing pharmacological correction of the defective CFTR-mediated protein secretion afford promise for the development of a rational drug therapy for CF patients.

Topics: Adrenergic beta-Agonists; Animals; Cell Line; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Inhibitors; Humans; Isoproterenol; Lactoferrin; Mucins; Nasal Mucosa; Rats; Submandibular Gland; Theophylline; Thionucleotides

The presence of lactoferrin at the concentration found in cystic fibrosis (CF) sputum (0.9 g/L) reduced MICs and MBCs of doxycycline for Burkholderia cepacia and Pseudomonas aeruginosa strains. MICs for B. cepacia fell by 32- to 64-fold, from highly resistant to clinically achievable values. Rifampicin MICs for B. cepacia strains were reduced by lactoferrin and for some strains MBCs were reduced. These findings suggest new therapeutic approaches to infections and question the relevance of standard sensitivity tests for CF pathogens. Addition of lactoferrin to media for the routine sensitivity testing of CF isolates might give more relevant results.

Topics: Anti-Bacterial Agents; Antibiotics, Antitubercular; Burkholderia cepacia; Cystic Fibrosis; Doxycycline; Drug Interactions; Drug Therapy, Combination; Humans; Lactoferrin; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Rifampin; Sputum

Cystic fibrosis (CF) is characterized by the production of abnormally thick secretions in the airways, chronic bacterial endobronchial infections and a chronic, predominantly neutrophilic inflammatory response. Therefore, myeloperoxidase (MPO) and lactoferrin are frequently used as inflammatory markers. Recently, a new protein in the neutrophil granules, human neutrophil lipocalin (HNL) has been discovered. The aim of the present study was to investigate HNL in sera of patients with CF and its relation to MPO and lactoferrin as well as to acute pulmonary exacerbation. Serum concentrations of HNL, MPO and lactoferrin were determined in 42 patients with CF and in 25 healthy subjects. Patients with CF were divided into groups with and without acute pulmonary exacerbation (APE) and also with and without colonization with Pseudomonas aeruginosa (Pa). Median serum levels of HNL (200.5 microg x L(-1)), MPO (595 microg x L(-1)) and lactoferrin (1,356.5 microg x L(-1)) were significantly increased in patients with CF compared to control subjects (57.7, 178 and 478 microg x L(-1), respectively; p<0.0001). CF patients with APE had significantly increased serum concentrations of HNL (321 versus 97.7 microg x L(-1); p<0.0001), MPO (1,125 versus 300 microg x L(-1); p<0.005) and lactoferrin (4,936 versus 980 microg x L(-1); p<0.001) compared with patients in stable clinical condition. Similarly, patients colonized with Pa had significantly higher concentrations of HNL, MPO and lactoferrin than Pa negative patients. These results indicate that in patients with cystic fibrosis, serum concentrations of human neutrophil lipocalin are markedly increased with a strong relationship to myeloperoxidase and lactoferrin. Thus, determination of serum human neutrophil lipocalin concentrations may be another useful diagnostic tool to monitor neutrophil inflammation in cystic fibrosis. The more marked difference in human neutrophil lipocalin compared with myeloperoxidase concentrations with no overlap between patients with acute pulmonary exacerbation and those in stable condition even suggests that human neutrophil lipocalin may be a more sensitive and specific discriminator.

Topics: Acute-Phase Proteins; Adolescent; Biomarkers; Carrier Proteins; Case-Control Studies; Cystic Fibrosis; Female; Humans; Lactoferrin; Lipocalin-2; Lipocalins; Male; Neutrophils; Oncogene Proteins; Peroxidase; Proto-Oncogene Proteins; Sensitivity and Specificity; Severity of Illness Index

Previous studies have implicated the novel peptide antibiotic human beta-defensin 1 (hBD-1) in the pathogenesis of cystic fibrosis. We describe in this report the isolation and characterization of the second member of this defensin family, human beta-defensin 2 (hBD-2). A cDNA for hBD-2 was identified by homology to hBD-1. hBD-2 is expressed diffusely throughout epithelia of many organs, including the lung, where it is found in the surface epithelia and serous cells of the submucosal glands. A specific antibody made of recombinant peptide detected hBD-2 in airway surface fluid of human lung. The fully processed peptide has broad antibacterial activity against many organisms, which is salt sensitive and synergistic with lysozyme and lactoferrin. These data suggest the existence of a family of beta-defensin molecules on mucosal surfaces that in the aggregate contributes to normal host defense.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Anti-Infective Agents; beta-Defensins; Blood Proteins; Bronchoalveolar Lavage; Cloning, Molecular; Cystic Fibrosis; Defensins; Humans; In Situ Hybridization, Fluorescence; Lactoferrin; Lung; Molecular Sequence Data; Muramidase; Peptide Fragments; Proteins; Recombinant Proteins; RNA, Messenger; Salts; Sequence Analysis, DNA

MlCs of rifampicin and chloramphenicol for mucoid strains of Pseudomonas aeruginosa were lower in the presence of human lactoferrin (0.9 mg/mL, the concentration found in cystic fibrosis sputum) than in its absence. MICs for some strains were lowered to clinically achievable levels of the antibiotics, which is compatible with impressions of greater clinical efficacy in pseudomonas infections than would be predicted by standard sensitivity tests. The routine addition of lactoferrin to sensitivity media for testing of cystic fibrosis isolates may give more useful results than conventional tests as in-vivo conditions are more closely simulated.

Topics: Anti-Bacterial Agents; Chloramphenicol; Cystic Fibrosis; Drug Synergism; Humans; Lactoferrin; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Rifampin

Patients with cystic fibrosis (CF) of the same age differ significantly in their degree of pulmonary disease. Based on preliminary observations, we postulated that the activity of myeloperoxidase would be significantly increased in patients with greater structural lung damage than in those with less lung damage. Acid extracts of weighed sputum samples were assayed for lactoferrin concentrations by ELISA. Activities of peroxidase, cathespsin G, and elastase (with and without proteinase 3) were determined by kinetic analysis using chromogenic substrates. The patients were divided into quartiles based on their Brasfield chest-radiograph score. Patients in the first quartile (least amount of structural lung abnormality) were compared to those in the fourth quartile. The concentration of lactoferrin, a specific (secondary) granule protein of neutrophils, did not differ between the two patient groups. However, the activities of the neutrophil primary granule proteins, peroxidase, elastase, and elastase plus proteinase 3, were significantly elevated in the group with the most structural lung abnormality. Sputum albumin concentration was used to estimate leakages of plasma proteins into the airways. Peroxidase activity, but not the activity of cathepsin G, of elastase, or of elastase plus proteinase 3, correlated significantly with albumin/g sputum in both quartile groups. To confirm the association of sputum peroxidase activity with differences in lung structure and to test its correlation with lung function, spirometry was performed in a second group of patients during the week prior to the time of sputum sampling. In this second group, increased sputum peroxidase activity was associated with worse Brasfield scores and with decreased percent-predicted forced expiratory volume in 1 sec.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Analysis of Variance; Cathepsin G; Cathepsins; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lactoferrin; Lung Diseases; Male; Pancreatic Elastase; Peroxidase; Respiratory Function Tests; Serine Endopeptidases; Severity of Illness Index; Sputum

The acquisition of Pseudomonas cepacia in patients with cystic fibrosis is associated with increasing deterioration in lung function and more frequent hospital admissions. Pseudomonas cepacia is usually resistant to several antibiotics in vitro, but the response of patients colonised with the organism has not been extensively studied in vivo.. A three month prospective study was performed to investigate the response of 14 Ps cepacia positive patients and 10 Ps cepacia negative patients to a two week course of intravenous antibiotics. All those who were Ps cepacia negative and six of the 14 Ps cepacia positive patients had Ps aeruginosa in their sputum which was sensitive to the prescribed therapy. The inflammatory markers C-reactive protein, white blood cell count, serum lactoferrin, neutrophil elastase/alpha 1-antitrypsin complex, and tumour necrosis factor alpha were measured at the start and end of each antibiotic course.. The median (range) % improvement in baseline FEV1 and FVC following treatment in the group as a whole was 15.2% (-23.5% to 156.3%) and 23.9% (-36.8% to 232.7%) respectively. There was no statistical difference in improvement in lung function, body weight, or inflammatory markers between individuals who were Ps cepacia positive and those who were Ps cepacia negative.. Patients who are Ps cepacia positive appear to respond as well to intravenous antibiotics as those who are Ps cepacia negative, despite having lower lung function and a bacterium in their sputum which is resistant in vitro to the antibiotics used.

Topics: Adult; Anti-Bacterial Agents; Biomarkers; Burkholderia cepacia; C-Reactive Protein; Cystic Fibrosis; Female; Humans; Lactoferrin; Leukocyte Count; Lung; Male; Prospective Studies; Pseudomonas Infections; Tumor Necrosis Factor-alpha

Bacterium- and neutrophil-derived proteases have been suggested to contribute to tissue injury at sites of Pseudomonas aeruginosa infection. Pseudomonas elastase cleavage of transferrin enhances in vitro iron removal from this protein by the P. aeruginosa siderophore pyoverdin. This cleavage also generates new iron chelates which, in contrast to iron bound to transferrin, are able to catalyze formation of the highly cytotoxic hydroxyl radical from neutrophil-derived superoxide and hydrogen peroxide via the Haber-Weiss reaction. In order to determine whether this cleavage occurs in vivo, a chemiluminescence immunoblot system was developed to detect the presence of proteolysis products of transferrin or the related iron-binding protein, lactoferrin. Using this immunoblot system, we detected transferrin and lactoferrin cleavage products in bronchoalveolar lavage (BAL) samples from 21 of 22 and 20 of 21 cystic fibrosis (CF) patients, respectively. Three of eleven and two of nine BAL samples from individuals with other forms of chronic inflammatory lung disease had transferrin and lactoferrin cleavage products, respectively. Each patient in whom such products were detected was also infected with P. aeruginosa. No such products were detected in normal individuals. In the CF patients, there was no clear correlation between the extent of transferrin or lactoferrin cleavage and BAL neutrophil or P. aeruginosa concentration or the disease status of the patient. In contrast, in the non-CF patients with chronic inflammatory lung disease, transferrin and lactoferrin cleavage products were detected only in those BAL samples which contained the greatest concentration of both neutrophils and P. aeruginosa. These data provide evidence that P. aeruginosa- and/or human-derived protease cleavage of transferrin and lactoferrin occurs in vivo in the airways of individuals with CF and other forms of chronic lung disease, suggesting that this process could contribute to P. aeruginosa-associated lung injury in these patients.

Topics: Adult; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Endopeptidases; Humans; Lactoferrin; Middle Aged; Neutrophils; Pneumonia; Pseudomonas Infections; Transferrin

Plasma neutrophil elastase-alpha 1 antiproteinase complex, lactoferrin and C-reactive protein (CRP) were determined over a 15-month period in 26 patients with cystic fibrosis, of whom 21 were chronically infected with Pseudomonas aeruginosa. Median concentrations of both neutrophil products and CRP were greater in patients who were clinically stable than in healthy subjects without cystic fibrosis. CRP concentrations increased further at the onset of symptomatic exacerbations. Thirty-five courses of intravenous antibiotics and 22 courses of oral ciprofloxacin were reviewed and revealed similar improvements in clinical scores and lung function tests for both forms of treatment. Intravenous antibiotics reduced the plasma concentrations of both neutrophil products and CRP, while oral ciprofloxacin only significantly reduced the concentration of neutrophil elastase-alpha 1 antiproteinase complex. Plasma concentrations of inflammatory markers were significantly greater in exacerbations associated with fever and leukocytosis. Statistical modelling demonstrated negative within-patient relationships between lung function and both CRP and lactoferrin, and positive relationships between the three inflammatory markers. Neutrophil granule products and CRP reflect the pulmonary inflammatory state in cystic fibrosis and may be of value in monitoring treatment.

Topics: Adolescent; Adult; alpha 1-Antitrypsin; Anti-Bacterial Agents; C-Reactive Protein; Cystic Fibrosis; Humans; Lactoferrin; Leukocyte Elastase; Pancreatic Elastase; Pneumonia; Respiratory Function Tests; Time Factors

Cellular mechanisms regulating airway secretion, secretory products of individual airway cell types, and control of airway cell growth and differentiation are poorly understood. In order to aid studies of these questions, we have established a system for culturing human tracheobronchial submucosal gland cells. Gland acini were isolated by enzymatic disaggregation from submucosal tissue obtained postmortem from patients without pulmonary diseases and from patients with cystic fibrosis. In culture, acini attached to a collagen substratum, and gland cells proliferated and formed confluent monolayers which were homogeneous by phase microscopy. In contrast to cells of freshly disaggregated acini which expressed either serous or mucous gland cell secretory antigens, in culture virtually all cells (greater than or equal to 95%) concurrently expressed both antigens as assessed by immunocytochemical staining with serous and mucous cell-specific antibodies. Similarly, electron microscopy revealed cells with serous- or mucous-type secretory granules, and cells containing both types of granules. Cultures incorporated 35S into high (greater than 10(6) D) and lower (greater than 700 kD; 150 kD) molecular weight molecules. Cholinergic and adrenergic agonists increased release of radio-labeled secretions. These findings demonstrate that human tracheal gland cells in culture retain immunocytochemical, ultrastructural, and functional features of both differentiated serous and mucous gland cells. This culture system will be useful for studying the biology and pathology of human tracheobronchial submucosal gland cells.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bronchi; Cell Differentiation; Cells, Cultured; Child; Child, Preschool; Cystic Fibrosis; Exocrine Glands; Female; Glycoconjugates; Humans; Infant; Infant, Newborn; Lactoferrin; Male; Microscopy, Electron; Middle Aged; Muramidase; Trachea

Plasma lactoferrin concentrations were measured in blood of cystic fibrosis patients, heterozygotes and controls using a specific and sensitive enzyme immunoassay. 67 plasmas were studied (26 controls, 23 heterozygotes, 18 cystic fibrosis patients) and the results showed a statistically significant increase (p less than 0.05) of the level of plasma lactoferrin in cystic fibrosis patients (265 +/- 224 micrograms/l) compared to controls (168 +/- 100 micrograms/l) and heterozygotes (150 +/- 72 micrograms/l). Since it is well established that plasma lactoferrin level could be influenced by the number of neutrophils, a second set of experiments was performed on 20 cystic fibrosis patients on whom leukocyte counts were also made. When the 15 plasmas with normal neutrophils (in the range 2 to 6 giga/l) were considered, the mean lactoferrin level was 318 +/- 116 micrograms/l, still far above the normal values. For serum, a similar significant increase of lactoferrin concentration was observed in 33 cystic fibrosis patients (610 +/- 551 micrograms/l) compared to the values observed for 25 controls (237 +/- 155 micrograms/l) and 37 heterozygotes (272 +/- 231 micrograms/l). Cystic fibrosis protein (CFP) was identified in the same sera by isoelectric focusing and the intensity of the band was closely related to the increase of lactoferrin concentration in cystic fibrosis patients. In contrast, no difference in serum lactoferrin concentrations was observed between heterozygotes with or without CFP, indicating that the increased CFP concentration cannot be due only to altered granulocyte function.

Topics: Blood Proteins; Calgranulin A; Cystic Fibrosis; Heterozygote; Humans; Lactoferrin; Lactoglobulins; Leukocyte Count; Neutrophils; Plasma

The effect of Pseudomonas aeruginosa alkaline protease (AP), elastase (Ela), and the elastase from polymorphonuclear leukocytes (PMN Ela) on iron acquisition of pyoverdin from human transferrin and lactoferrin at physiologic pH was investigated. Incubation of iron-loaded transferrin with iron-free pyoverdin for 10 h at 40 degrees C in the presence of Ela yielded pyoverdin-iron(III) complex, in contrast to incubations of transferrin with pyoverdin alone, AP, or PMN Ela. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the incubations revealed fragmentation of transferrin by Ela in peptides smaller than 14,000 daltons, whereas AP and PMN Ela cleaved transferrin in fragments of 49,000 d and 43,000 d, respectively. Incubations of lactoferrin with the proteases and pyoverdin or pyoverdin alone did not result in iron acquisition by pyoverdin; however, lactoferrin was fragmented by Ela and AP.

Topics: Bacterial Proteins; Cystic Fibrosis; Endopeptidases; Humans; In Vitro Techniques; Iron Chelating Agents; Lactoferrin; Lactoglobulins; Metalloendopeptidases; Neutrophils; Oligopeptides; Pigments, Biological; Pseudomonas aeruginosa; Serine Endopeptidases; Transferrin

Serum samples from cystic fibrosis homozygotes and heterozygotes contain elevated levels of a protein, known as cystic fibrosis antigen, which is synthesized primarily in granulocytes. We have produced monoclonal antibodies against cystic fibrosis antigen and developed a sensitive two-site immunoassay. Antigen levels were evaluated in serum samples from 50 cystic fibrosis homozygotes, 34 heterozygotes, 60 healthy controls and 25 disease controls. Simultaneous measurement was made of another granulocyte-derived serum protein, lactoferrin, and also of the acute-phase reactant, C-Reactive Protein. Attempts to use the concentrations of cystic fibrosis antigen in serum to distinguish cystic fibrosis patients from heterozygotes were unsuccessful, even when these concentrations were expressed as a ratio with lactoferrin or with C-Reactive Protein. However, examination of the ratio of cystic fibrosis antigen to lactoferrin in serum samples from cystic fibrosis heterozygotes suggests that there is some specific association between this antigen and the cystic fibrosis gene.

Topics: Antibodies, Monoclonal; Blood Proteins; C-Reactive Protein; Calcium-Binding Proteins; Calgranulin A; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Granulocytes; Heterozygote; Humans; Lactoferrin

Plasma levels of lactoferrin (LF) have been found to be increased in a few patients with cystic fibrosis (CF). This study was aimed at investigating plasma LF levels in children with CF (26 cases) and in controls (C) (19 cases). Plasma LF was measured by a radioimmunoassay method. Plasma LF levels were not significantly different in CF and in C, even though 10 CF patients showed LF levels above the mean + 2 SD value of the controls. Neither the duration of the disease nor the age of the controls was correlated with LF or with the exocrine pancreatic capacity. A significant relationship between the presence of an acute lung inflammation and LF levels was found. This study shows that LF is increased in CF only in the presence of an acute inflammatory state. Further studies are necessary to establish the usefulness of an LF assay as an index of the presence of an acute inflammatory process.

Topics: Adolescent; Child; Child, Preschool; Cystic Fibrosis; Humans; Infant; Inflammation; Lactoferrin; Lactoglobulins; Lung Diseases

Plasma lactoferrin levels were determined by radioimmunoassay for the different weeks of normal pregnancy, in normal healthy adults and in children with and without cystic fibrosis. The lactoferrin levels were higher in pregnancy than in both male and female normal adults and showed a slight progressive increase up to week 29 and thereafter remained high. Five our of seven children with cystic fibrosis had markedly raised plasma lactoferrin levels from six to 16 times higher than the mean of a control group of children.

Topics: Adolescent; Adult; Child; Child, Preschool; Cystic Fibrosis; Female; Humans; Infant; Lactoferrin; Lactoglobulins; Male; Pregnancy; Radioimmunoassay; Reference Values

Bronchial secretions from 207 children suffering from various pulmonary diseases and from 15 healthy controls were tested concentration of IgA, IgG, lactoferrin and lysozyme. The results obtained suggest that in many cases of chronic lung diseases in children the levels of lactoferrin and immunoglobulins, especially secretory IgA, are very low. In severe infections (cystic fibrosis, bronchiectases) significant increase of IgG concentration was observed.

Topics: Adolescent; Bronchi; Bronchiectasis; Bronchitis; Child; Child, Preschool; Chronic Disease; Cystic Fibrosis; Humans; Immunoglobulin A; Immunoglobulin A, Secretory; Immunoglobulin G; Lactoferrin; Lactoglobulins; Muramidase; Recurrence; Respiratory Tract Diseases; Respiratory Tract Infections

The concentration of immunoglobulins, lactoferrin and lysozyme we compared in bronchial secretions obtained from children with various chronic lung diseases. The IgG, lactoferrin and lysozyme, but not secretory IgA, concentrations were shown to be increased during chronic inflammatory response.

Topics: Adolescent; Bronchi; Bronchiectasis; Bronchitis; Bronchography; Bronchoscopy; Child; Child, Preschool; Chronic Disease; Cystic Fibrosis; Humans; Immunoglobulin A; Immunoglobulin A, Secretory; Immunoglobulin G; Lactoferrin; Lactoglobulins; Muramidase; Respiratory Tract Diseases

The concentrations of nine plasma proteins were determined by quantitative immunoelectrophoresis in sputum specimens from 29 patients with cystic fibrosis (CF) and from 24 patients with severe asthma and chronic bronchitis. The results suggested that the population of CF patients could be divided into two groups in spite of an absence of difference in clinical status between the groups. Average concentrations of seven plasma proteins in sputum of group I CF patients were identical with those in sputum of patients with bronchitis, but the average concentrations of six of these proteins in sputum from group II CF patients were higher than those in specimens from the bronchitic patients and were similar to corresponding concentrations in sputum from patients with asthma, all of whom were examined while in status asthmaticus. The average concentrations of 14 secretory proteins were the same in all sputum specimens whether or not they were produced by patients with cystic fibrosis, asthma or bronchitis. It was concluded that the concentrations in the bronchopulmonary secretions of proteins associated with host defence were not diminished in patients with cystic fibrosis, and failure to produce adequate concentrations of proteins with antimicrobial activity was unlikely to be responsible for the above average susceptibility to chest infection in cystic fibrosis. It is suggested that there exists a group of CF patients in whom a pulmonary allergic reaction generates an inflammatory response as severe as that characterizing status asthmaticus and that this response could be detrimental.

Topics: Adult; Aged; Albumins; Asthma; Beta-Globulins; Blood Proteins; Bronchitis; Child; Child, Preschool; Cystic Fibrosis; Female; Glycoproteins; Haptoglobins; Humans; Immunoelectrophoresis; Immunoglobulin A; Immunoglobulin G; Lactoferrin; Male; Middle Aged; Muramidase; Sputum; Transferrin