The role of cardiovascular and pulmonary limitation in performing maximal exercise is contrasted ... more The role of cardiovascular and pulmonary limitation in performing maximal exercise is contrasted in patients with mitral valve disease (MVD) and chronic obstructive pulmonary disease (COPD). The ventilatory (V.) gas exchange (Vo, and Vco1) and heart rate (HR) responses to an incremental cycle ergometer exercise were measured in six patients with MVD, seven patients with COPD, and six normal subjects. The VE requirement for the work (VE-Vo1 14.15
Experimental and Molecular Pathology, Apr 30, 2003
The skin presents a mechanical, as well as an immunological barrier to infection, and displays co... more The skin presents a mechanical, as well as an immunological barrier to infection, and displays considerable innate immune capacity. Recently, cultured human keratinocytes were described to produce and export a microbicidal peptide human β-defensin 2 (HBD-2). Immunogold was used to label ultrathin cryosections of stimulated, cultured human epidermis. HBD-2 was found to be stored in the lamellar bodies (LBs) of
Human defensins are 29 to 30 amino acid (aa) antimicrobial peptides that are among the principal ... more Human defensins are 29 to 30 amino acid (aa) antimicrobial peptides that are among the principal constituents of the neutrophil's azurophil granules. To determine the tissue specificity of posttranslational processing and subcellular targeting of defensins, the cDNA for a 94 aa human preprodefensin was transduced into murine cell lines (NIH 3T3 embryonic fibroblasts, AtT-20 pituitary adenoma, J774.1 and RAW 264.7 macrophages, and 32D and 32D cl3 granulocytes) using retroviral vectors. All transduced cell types expressed and to a variable extent constitutively secreted a 75 aa prodefensin formed by the removal of the amino terminal signal sequence. In AtT-20 cells, the 75 aa form accumulated intracellularly in granules and was releasable by secretagogues. Proteolytic processing to mature defensins was seen only in myeloid cells (J774.1, RAW 264.7, 32D, and 32D cl3). Newly formed mature defensin was rapidly degraded in J774.1 and RAW 264.7 macrophages, but accumulated stably in mu...
Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells... more Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells but recently also found to be produced by epithelial cells. We biosynthesized recombinant human intestinal defensin 5 (rHD-5) using the baculovirus-insect cell expression system. Since insect cells process defensin incompletely and secrete the precursor proHD-5, we substituted a methionine for an alanine at a likely processing site to allow selective chemical cleavage with cyanogen bromide, and rHD-5 was used to elicit polyclonal antibodies. By the immunoperoxidase-staining technique, the antibodies selectively stained Paneth cells of the normal adult small intestine. Immunogold electron microscopy further localized HD-5 to the Paneth cell secretory granules. Since some defensins exert activity cytotoxic to mammalian cells, we assayed the effect of rHD-5 on the human intestinal cell lines Caco2 and Int407. proHD-5 did not exert cytotoxic activity, and rHD-5 showed only minimal activity ...
We developed a sensitive and quantitative radial diffusion method to ascertain the susceptibility... more We developed a sensitive and quantitative radial diffusion method to ascertain the susceptibility of six strains of Neisseria gonorrhoeae to antimicrobial peptides derived from mammalian leukocytes. The test organisms included the well-characterized serum-resistant FA19 and serum-sensitive F62 strains plus four antibiotic-resistant clinical isolates. Although each N. gonorrhoeae strain was resistant to human neutrophil defensins, all six were exquisitely sensitive to protegrins, a family of small beta-sheet antimicrobial peptides recently identified in porcine leukocytes. Protegrin-treated N. gonorrhoeae became vacuolated and had striking membrane changes when viewed by transmission and scanning electron microscopy. Because low concentrations of protegrins can also inactivate Chlamydia trachomatis and human immunodeficiency virus, they show promise for development as topical agents to avert sexually transmitted diseases.
The CO2 rebreathing method can be very useful to test the hypercapnic ventilatory response in pat... more The CO2 rebreathing method can be very useful to test the hypercapnic ventilatory response in patients, including those with chronic acid-base changes (e.g. chronic metabolic acidosis due to renal failure). The ventilatory response to hypercapnia (CO2-R) was measured in 4 normal men by the rebreathing method under control conditions (CaCO3: 0.1 g.kg-1.day-1) and with induced metabolic acidosis (NH4Cl: 0.3 g.kg-1.day-1) and alkalosis (NaHCO3: 0.7 g.kg-1.day-1). The slope of the CO2-R did not change as a result of the acid-base alterations, but was shifted to the left of normal by metabolic acidosis, and to the right by metabolic alkalosis. These results compare favorably with previous reports on the CO2-R as measured by the steady-state technique, and validate the rebreathing method as a reliable and useful technique for evaluating CO2-R in man with altered acid-base states.
In patients with pulmonary disease, the diffusing capacity for carbon monoxide has been used to p... more In patients with pulmonary disease, the diffusing capacity for carbon monoxide has been used to predict abnormal gas exchange in the lung. However, abnormal values for arterial blood gases during exercise are likely to be the most sensitive manifestations of lung disease. We compared the single-breath diffusing capacity for carbon monoxide at rest with measurements of gas exchange during exercise, including arterial oxygen tension, the alveolar-arterial difference in oxygen tension, the arterial-end-tidal difference in carbon dioxide tension, and the dead-space/tidal-volume ratio in 276 current and former shipyard workers. Sixteen workers had a diffusing capacity for carbon monoxide below 70 percent of predicted; one or more measurements of gas exchange during exercise were abnormal in 14. In contrast, of 96 men who had abnormal gas exchange during exercise, only 14 had a diffusing capacity for carbon monoxide below 70 percent of predicted. Neither the type nor the degree of abnormality in gas exchange could be predicted from the diffusing capacity. We conclude that diffusing capacity for carbon monoxide at rest is a specific but insensitive predictor of abnormal gas exchange during exercise and that, if indicated, measurements of arterial blood gases should be obtained during exercise.
Healthy men (N = 1424, age 20-70 yr) underwent a progressive incremental treadmill exercise test ... more Healthy men (N = 1424, age 20-70 yr) underwent a progressive incremental treadmill exercise test to volitional maximum. Cardiopulmonary variables were measured breath-by-breath. The aerobic power (VO2max) declined at an average yearly rate of 0.33 ml.kg.-1min-1, HRmax declined 0.685 beats.min-1.yr-1, and max O2 pulse declined at an annual rate of 0.115 ml.beat-1.kg-1*100. Gas exchange threshold (GET) expressed as percentage of VO2max was 58% and 69% in the youngest (20-30 yr) and oldest (61-70 yr) decades, respectively. The average decline in VE, Vt, f, and PETCO2 over the entire age range was 29%, 10%, 21%, and 7%, respectively. There were increases in VE/VO2, and VE/VECO2, from age 20-70 yr of 13% and 14%, respectively, but no changes across 5 decades in PETO2. Physical (height and weight) as well as life-style characteristics (leisure time activity, place of residency, smoking), were found to be potent predictors in most of the cardiopulmonary values at maximal exercise and therefore should be incorporated in the predictive equations for such variables. Normal response patterns of most cardiopulmonary variables throughout the range of exercise intensities were shown to be age-affected and thus should be standardized for age decades.
... Tomas Ganz, Ami Oren, and Robert I. Lehrer Will Rogers Institute Pulmonary Research Laborator... more ... Tomas Ganz, Ami Oren, and Robert I. Lehrer Will Rogers Institute Pulmonary Research Laboratory, Department of Medicine, UCLA School of Medicine ... were incubated first with mouse monoclonal antibodies to human defensins HNP 1-3, then with 10-nm gold-conjugated goat ...
Protegrins are broad spectrum antibiotic peptides isolated from porcine leukocytes. In this study... more Protegrins are broad spectrum antibiotic peptides isolated from porcine leukocytes. In this study, we (i) examine the sensitivity of Gram-negative, anaerobic periodontal pathogens to synthetic protegrins; (ii) determine the relative potencies of protegrin congeners against these bacteria; and (iii) compare the potency of protegrins with other antibiotic peptides, including magainin MSI-78, tachyplesin I, cecropin P1, human defensins HNP-1-3, and clavanin A. Synthetic Land D-enantiomers of protegrin 1 (PG-1 and D-PG-1, respectively), and L-enantiomers of protegrins 2, 3 and 5 (PG-2, PG-3 and PG-5) were tested against Fusobacteriurn nucleatum, and black-pigmented organisms including Porphyromonas gingivalis and Prevotella intermedia. Strains of both F. nucleatum and the black-pigmented organisms were sensitive to PG-1, and exhibited mean ED,, of 2.2-2.3 pg/ml and 3.4-9.9 pg/ml, respectively. The D-form was statistically more potent than the L-form against these oral anaerobes, and although this difference in potency is unlikely to be of decisive therapeutic significance, the D-form may be of value given ability to resist microbial and host-derived proteases. PG-1 was more potent than magainin, tachyplesin, cecropin, defensins and clavanin under test conditions. Hypertonic saIt concentrations and heat-inactivated serum were found to be inhibitory to the bactericidal activity of PG-1. PG-1 was found to induce morphologic alterations in the ultrastructural appearance of F. nucleatum consistent with damage to the bacterial membranes. We conclude that protegrins may be useful antimicrobial agents in therapy against Gram-negative anaerobic bacteria believed to be involved in chronic, adult forms of periodontal infections.
Granulocytes expose phagocytized microbes to microbicidal substances that are stored in cytoplasm... more Granulocytes expose phagocytized microbes to microbicidal substances that are stored in cytoplasmic granules and delivered by fusion to the phagocytic vacuoles (phagosomes). To determine if the murine myeloid cell line 32D cl3 is suitable as a genetic model of protein translocation to granules and phagosomes, we permanently transduced 32D cl3 cells with human HNP-1 defensin cDNA, incubated them with opsonized
The skin presents a mechanical, as well as an immunological barrier to infection, and displays co... more The skin presents a mechanical, as well as an immunological barrier to infection, and displays considerable innate immune capacity. Recently, cultured human keratinocytes were described to produce and export a microbicidal peptide human β-defensin 2 (HBD-2). Immunogold was used to label ultrathin cryosections of stimulated, cultured human epidermis. HBD-2 was found to be stored in the lamellar bodies (LBs) of
Nasal carriage of Staphylococcus aureus has been identified as a risk factor for community-acquir... more Nasal carriage of Staphylococcus aureus has been identified as a risk factor for community-acquired and nosocomial infections. We screened 230 donors of diverse ethnic and socioeconomic backgrounds and identified 62 (27%) whose nasal secretions were colonized by S.aureus. In 18 donors in whom the various regions of the nasal luminal surface were separately sampled, the predominant region of S. aureus colonization was the moist squamous epithelium on the septum adjacent to the nasal ostium. Nasal fluid from carriers was defective in killing endogenousS. aureus and nasal carrier isolates ofS. aureus but not a laboratoryS. aureus strain. Transmission electron microscopy revealed that S.aureus isolates incubated in nasal fluid from carriers for 2 h at 37°C were less damaged than those incubated in noncarrier fluid and were coated with an electron-dense layer. Compared with that from healthy donors and patients with acute rhinitis, nasal fluid from carriers contained elevated concentrati...
More than 70 years ago, Alexander Fleming discovered lysozyme and proposed that nonpathogenic bac... more More than 70 years ago, Alexander Fleming discovered lysozyme and proposed that nonpathogenic bacteria fail to cause disease because they are very susceptible to destruction by lysozyme, an enzyme that is one of the principal proteins of phagocytes. Although much has been learned about the effects of lysozyme in vitro, its biological role in vivo has not been determined. We examined transgenic mice deficient in lysozyme M after challenge by the normally nonpathogenic and highly lysozyme-sensitive bacterium Micrococcus luteus. Despite partial compensation by newly expressed lysozyme P in macrophages, lysozyme M–deficient mice developed much more severe lesions than wild-type mice. The tissue injury was due to the failure of lysozyme M–deficient mice to inactivate peptidoglycan, resulting in an intense and prolonged inflammatory response. Our data indicate that tissue injury is normally limited by prompt degradation of bacterial macromolecules that trigger innate immunity and inflamma...
Antimicrobial polypeptides including lysozymes (Ly) have membrane perturbing activity and are wel... more Antimicrobial polypeptides including lysozymes (Ly) have membrane perturbing activity and are well documented effector molecules of innate immunity. In cystic fibrosis, a hereditary disease with frequent lung infection with Pseudomonas aeruginosa, the free fatty acid docosahexaenoic acid (DA), but not oleic acid (OA), is decreased and DA supplementation has been shown to improve the clinical condition in these patients. We hypothesized that DA may alone, or in conjunction with Ly, exert antibacterial action against P. aeruginosa. We found that DA and Ly synergistically inhibit the metabolic activity of P. aeruginosa, in contrast to OA. Electron microscopy and equilibrium dialysis suggest that DA accumulates in the bacterial membrane in the presence of Ly. Surface plasmon resonance with live bacteria and differential scanning calorimetry studies with bacterial model membranes reveal that, initially, DA facilitates lysozyme incorporation into the membrane, which in turn allows influx of more DA leading to bacterial cell death. Our study elucidates a molecular basis for the synergistic action of free fatty acids and antimicrobial polypeptides, which may be dysfunctional in cystic fibrosis.
The role of cardiovascular and pulmonary limitation in performing maximal exercise is contrasted ... more The role of cardiovascular and pulmonary limitation in performing maximal exercise is contrasted in patients with mitral valve disease (MVD) and chronic obstructive pulmonary disease (COPD). The ventilatory (V.) gas exchange (Vo, and Vco1) and heart rate (HR) responses to an incremental cycle ergometer exercise were measured in six patients with MVD, seven patients with COPD, and six normal subjects. The VE requirement for the work (VE-Vo1 14.15
Experimental and Molecular Pathology, Apr 30, 2003
The skin presents a mechanical, as well as an immunological barrier to infection, and displays co... more The skin presents a mechanical, as well as an immunological barrier to infection, and displays considerable innate immune capacity. Recently, cultured human keratinocytes were described to produce and export a microbicidal peptide human β-defensin 2 (HBD-2). Immunogold was used to label ultrathin cryosections of stimulated, cultured human epidermis. HBD-2 was found to be stored in the lamellar bodies (LBs) of
Human defensins are 29 to 30 amino acid (aa) antimicrobial peptides that are among the principal ... more Human defensins are 29 to 30 amino acid (aa) antimicrobial peptides that are among the principal constituents of the neutrophil's azurophil granules. To determine the tissue specificity of posttranslational processing and subcellular targeting of defensins, the cDNA for a 94 aa human preprodefensin was transduced into murine cell lines (NIH 3T3 embryonic fibroblasts, AtT-20 pituitary adenoma, J774.1 and RAW 264.7 macrophages, and 32D and 32D cl3 granulocytes) using retroviral vectors. All transduced cell types expressed and to a variable extent constitutively secreted a 75 aa prodefensin formed by the removal of the amino terminal signal sequence. In AtT-20 cells, the 75 aa form accumulated intracellularly in granules and was releasable by secretagogues. Proteolytic processing to mature defensins was seen only in myeloid cells (J774.1, RAW 264.7, 32D, and 32D cl3). Newly formed mature defensin was rapidly degraded in J774.1 and RAW 264.7 macrophages, but accumulated stably in mu...
Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells... more Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells but recently also found to be produced by epithelial cells. We biosynthesized recombinant human intestinal defensin 5 (rHD-5) using the baculovirus-insect cell expression system. Since insect cells process defensin incompletely and secrete the precursor proHD-5, we substituted a methionine for an alanine at a likely processing site to allow selective chemical cleavage with cyanogen bromide, and rHD-5 was used to elicit polyclonal antibodies. By the immunoperoxidase-staining technique, the antibodies selectively stained Paneth cells of the normal adult small intestine. Immunogold electron microscopy further localized HD-5 to the Paneth cell secretory granules. Since some defensins exert activity cytotoxic to mammalian cells, we assayed the effect of rHD-5 on the human intestinal cell lines Caco2 and Int407. proHD-5 did not exert cytotoxic activity, and rHD-5 showed only minimal activity ...
We developed a sensitive and quantitative radial diffusion method to ascertain the susceptibility... more We developed a sensitive and quantitative radial diffusion method to ascertain the susceptibility of six strains of Neisseria gonorrhoeae to antimicrobial peptides derived from mammalian leukocytes. The test organisms included the well-characterized serum-resistant FA19 and serum-sensitive F62 strains plus four antibiotic-resistant clinical isolates. Although each N. gonorrhoeae strain was resistant to human neutrophil defensins, all six were exquisitely sensitive to protegrins, a family of small beta-sheet antimicrobial peptides recently identified in porcine leukocytes. Protegrin-treated N. gonorrhoeae became vacuolated and had striking membrane changes when viewed by transmission and scanning electron microscopy. Because low concentrations of protegrins can also inactivate Chlamydia trachomatis and human immunodeficiency virus, they show promise for development as topical agents to avert sexually transmitted diseases.
The CO2 rebreathing method can be very useful to test the hypercapnic ventilatory response in pat... more The CO2 rebreathing method can be very useful to test the hypercapnic ventilatory response in patients, including those with chronic acid-base changes (e.g. chronic metabolic acidosis due to renal failure). The ventilatory response to hypercapnia (CO2-R) was measured in 4 normal men by the rebreathing method under control conditions (CaCO3: 0.1 g.kg-1.day-1) and with induced metabolic acidosis (NH4Cl: 0.3 g.kg-1.day-1) and alkalosis (NaHCO3: 0.7 g.kg-1.day-1). The slope of the CO2-R did not change as a result of the acid-base alterations, but was shifted to the left of normal by metabolic acidosis, and to the right by metabolic alkalosis. These results compare favorably with previous reports on the CO2-R as measured by the steady-state technique, and validate the rebreathing method as a reliable and useful technique for evaluating CO2-R in man with altered acid-base states.
In patients with pulmonary disease, the diffusing capacity for carbon monoxide has been used to p... more In patients with pulmonary disease, the diffusing capacity for carbon monoxide has been used to predict abnormal gas exchange in the lung. However, abnormal values for arterial blood gases during exercise are likely to be the most sensitive manifestations of lung disease. We compared the single-breath diffusing capacity for carbon monoxide at rest with measurements of gas exchange during exercise, including arterial oxygen tension, the alveolar-arterial difference in oxygen tension, the arterial-end-tidal difference in carbon dioxide tension, and the dead-space/tidal-volume ratio in 276 current and former shipyard workers. Sixteen workers had a diffusing capacity for carbon monoxide below 70 percent of predicted; one or more measurements of gas exchange during exercise were abnormal in 14. In contrast, of 96 men who had abnormal gas exchange during exercise, only 14 had a diffusing capacity for carbon monoxide below 70 percent of predicted. Neither the type nor the degree of abnormality in gas exchange could be predicted from the diffusing capacity. We conclude that diffusing capacity for carbon monoxide at rest is a specific but insensitive predictor of abnormal gas exchange during exercise and that, if indicated, measurements of arterial blood gases should be obtained during exercise.
Healthy men (N = 1424, age 20-70 yr) underwent a progressive incremental treadmill exercise test ... more Healthy men (N = 1424, age 20-70 yr) underwent a progressive incremental treadmill exercise test to volitional maximum. Cardiopulmonary variables were measured breath-by-breath. The aerobic power (VO2max) declined at an average yearly rate of 0.33 ml.kg.-1min-1, HRmax declined 0.685 beats.min-1.yr-1, and max O2 pulse declined at an annual rate of 0.115 ml.beat-1.kg-1*100. Gas exchange threshold (GET) expressed as percentage of VO2max was 58% and 69% in the youngest (20-30 yr) and oldest (61-70 yr) decades, respectively. The average decline in VE, Vt, f, and PETCO2 over the entire age range was 29%, 10%, 21%, and 7%, respectively. There were increases in VE/VO2, and VE/VECO2, from age 20-70 yr of 13% and 14%, respectively, but no changes across 5 decades in PETO2. Physical (height and weight) as well as life-style characteristics (leisure time activity, place of residency, smoking), were found to be potent predictors in most of the cardiopulmonary values at maximal exercise and therefore should be incorporated in the predictive equations for such variables. Normal response patterns of most cardiopulmonary variables throughout the range of exercise intensities were shown to be age-affected and thus should be standardized for age decades.
... Tomas Ganz, Ami Oren, and Robert I. Lehrer Will Rogers Institute Pulmonary Research Laborator... more ... Tomas Ganz, Ami Oren, and Robert I. Lehrer Will Rogers Institute Pulmonary Research Laboratory, Department of Medicine, UCLA School of Medicine ... were incubated first with mouse monoclonal antibodies to human defensins HNP 1-3, then with 10-nm gold-conjugated goat ...
Protegrins are broad spectrum antibiotic peptides isolated from porcine leukocytes. In this study... more Protegrins are broad spectrum antibiotic peptides isolated from porcine leukocytes. In this study, we (i) examine the sensitivity of Gram-negative, anaerobic periodontal pathogens to synthetic protegrins; (ii) determine the relative potencies of protegrin congeners against these bacteria; and (iii) compare the potency of protegrins with other antibiotic peptides, including magainin MSI-78, tachyplesin I, cecropin P1, human defensins HNP-1-3, and clavanin A. Synthetic Land D-enantiomers of protegrin 1 (PG-1 and D-PG-1, respectively), and L-enantiomers of protegrins 2, 3 and 5 (PG-2, PG-3 and PG-5) were tested against Fusobacteriurn nucleatum, and black-pigmented organisms including Porphyromonas gingivalis and Prevotella intermedia. Strains of both F. nucleatum and the black-pigmented organisms were sensitive to PG-1, and exhibited mean ED,, of 2.2-2.3 pg/ml and 3.4-9.9 pg/ml, respectively. The D-form was statistically more potent than the L-form against these oral anaerobes, and although this difference in potency is unlikely to be of decisive therapeutic significance, the D-form may be of value given ability to resist microbial and host-derived proteases. PG-1 was more potent than magainin, tachyplesin, cecropin, defensins and clavanin under test conditions. Hypertonic saIt concentrations and heat-inactivated serum were found to be inhibitory to the bactericidal activity of PG-1. PG-1 was found to induce morphologic alterations in the ultrastructural appearance of F. nucleatum consistent with damage to the bacterial membranes. We conclude that protegrins may be useful antimicrobial agents in therapy against Gram-negative anaerobic bacteria believed to be involved in chronic, adult forms of periodontal infections.
Granulocytes expose phagocytized microbes to microbicidal substances that are stored in cytoplasm... more Granulocytes expose phagocytized microbes to microbicidal substances that are stored in cytoplasmic granules and delivered by fusion to the phagocytic vacuoles (phagosomes). To determine if the murine myeloid cell line 32D cl3 is suitable as a genetic model of protein translocation to granules and phagosomes, we permanently transduced 32D cl3 cells with human HNP-1 defensin cDNA, incubated them with opsonized
The skin presents a mechanical, as well as an immunological barrier to infection, and displays co... more The skin presents a mechanical, as well as an immunological barrier to infection, and displays considerable innate immune capacity. Recently, cultured human keratinocytes were described to produce and export a microbicidal peptide human β-defensin 2 (HBD-2). Immunogold was used to label ultrathin cryosections of stimulated, cultured human epidermis. HBD-2 was found to be stored in the lamellar bodies (LBs) of
Nasal carriage of Staphylococcus aureus has been identified as a risk factor for community-acquir... more Nasal carriage of Staphylococcus aureus has been identified as a risk factor for community-acquired and nosocomial infections. We screened 230 donors of diverse ethnic and socioeconomic backgrounds and identified 62 (27%) whose nasal secretions were colonized by S.aureus. In 18 donors in whom the various regions of the nasal luminal surface were separately sampled, the predominant region of S. aureus colonization was the moist squamous epithelium on the septum adjacent to the nasal ostium. Nasal fluid from carriers was defective in killing endogenousS. aureus and nasal carrier isolates ofS. aureus but not a laboratoryS. aureus strain. Transmission electron microscopy revealed that S.aureus isolates incubated in nasal fluid from carriers for 2 h at 37°C were less damaged than those incubated in noncarrier fluid and were coated with an electron-dense layer. Compared with that from healthy donors and patients with acute rhinitis, nasal fluid from carriers contained elevated concentrati...
More than 70 years ago, Alexander Fleming discovered lysozyme and proposed that nonpathogenic bac... more More than 70 years ago, Alexander Fleming discovered lysozyme and proposed that nonpathogenic bacteria fail to cause disease because they are very susceptible to destruction by lysozyme, an enzyme that is one of the principal proteins of phagocytes. Although much has been learned about the effects of lysozyme in vitro, its biological role in vivo has not been determined. We examined transgenic mice deficient in lysozyme M after challenge by the normally nonpathogenic and highly lysozyme-sensitive bacterium Micrococcus luteus. Despite partial compensation by newly expressed lysozyme P in macrophages, lysozyme M–deficient mice developed much more severe lesions than wild-type mice. The tissue injury was due to the failure of lysozyme M–deficient mice to inactivate peptidoglycan, resulting in an intense and prolonged inflammatory response. Our data indicate that tissue injury is normally limited by prompt degradation of bacterial macromolecules that trigger innate immunity and inflamma...
Antimicrobial polypeptides including lysozymes (Ly) have membrane perturbing activity and are wel... more Antimicrobial polypeptides including lysozymes (Ly) have membrane perturbing activity and are well documented effector molecules of innate immunity. In cystic fibrosis, a hereditary disease with frequent lung infection with Pseudomonas aeruginosa, the free fatty acid docosahexaenoic acid (DA), but not oleic acid (OA), is decreased and DA supplementation has been shown to improve the clinical condition in these patients. We hypothesized that DA may alone, or in conjunction with Ly, exert antibacterial action against P. aeruginosa. We found that DA and Ly synergistically inhibit the metabolic activity of P. aeruginosa, in contrast to OA. Electron microscopy and equilibrium dialysis suggest that DA accumulates in the bacterial membrane in the presence of Ly. Surface plasmon resonance with live bacteria and differential scanning calorimetry studies with bacterial model membranes reveal that, initially, DA facilitates lysozyme incorporation into the membrane, which in turn allows influx of more DA leading to bacterial cell death. Our study elucidates a molecular basis for the synergistic action of free fatty acids and antimicrobial polypeptides, which may be dysfunctional in cystic fibrosis.
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