Alexander M. Cole

Cutting edge: IFN-inducible ELR- CXC chemokines display defensin-like antimicrobial activity.

Recent reports highlighted the chemotactic activities of antimicrobial peptide defensins whose structure, charge, and size resemble chemokines. By assaying representative members of the four known families of chemokines we explored the obverse: whether some chemokines exert antimicrobial activity. In a radial diffusion assay, only recombinant monokine induced by IFN-γ (MIG/CXCL9), IFN-γ-inducible protein of 10 kDa (IP-10/CXCL10), and IFN-inducible T cell α chemoattractant (I-TAC/CXCL11), members of the IFN-γ-inducible tripeptide motif Glu-Leu-Arg (ELR)− CXC chemokines, were antimicrobial against Escherichia coli and Listeria monocytogenes. Similar to human defensins, antimicrobial activities of the chemokines were inhibited by 50 and 100 mM NaCl. The concentration of MIG/CXCL9 and IP-10/CXCL10 released from IFN-γ-stimulated PBMC in 24 h were, respectively, 35- and 28-fold higher than from unstimulated cells. Additionally, the amounts of chemokines released per monocyte suggest that, in tissues with mononuclear cell infiltration, IFN-γ-inducible chemokines may reach concentrations necessary for microbicidal activity. IFN-γ-inducible chemokines may directly inactivate microbes before attracting other host defense cells to the area of infection.

Retrocyclin: A primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1

Human bone marrow expresses a pseudogene that encodes an antimicrobial peptide homologous to rhesus monkey circular minidefensins (θ-defensins). We prepared the putative ancestral human peptide by solid-phase synthesis and named it “retrocyclin.” Retrocyclin did not cause direct inactivation of HIV-1, and its modest antibacterial properties resembled those of its rhesus homologs. Nevertheless, retrocyclin had a remarkable ability to inhibit proviral DNA formation and to protect immortalized and primary human CD4+ lymphocytes from in vitro infection by both T-tropic and M-tropic strains of HIV-1. Confocal fluorescent microscopy studies performed with BODIPY-FL-labeled RC-101, a close analog of retrocyclin, showed that the peptide formed patch-like aggregates on the surface of CD4+ cells. These findings suggest that retrocyclin interferes with an early stage of HIV-1 infection and that retrocyclin-like agents might be useful topical agents to prevent sexually acquired HIV-1 infections.

Determinants of Staphylococcus aureus nasal carriage.

ABSTRACT Nasal carriage of Staphylococcusaureus 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 concentrations of the neutrophil-derived defensins human neutrophil peptides 1 to 3 (47- and 4-fold increases, respectively), indicative of a neutrophil-mediated inflammatory host response to S.aureus colonization. The concentration of the inducible epithelial antimicrobial peptide human β-defensin 2 was also highly elevated compared to that in healthy donors, in whom the level was below the detection limit, or patients with acute rhinitis (sixfold increase). Thus, nasal carriage of S.aureus takes hold in nasal fluid that is permissive for colonization and induces a local inflammatory response that fails to clear the colonizing bacteria.

Cationic Polypeptides Are Required for Antibacterial Activity of Human Airway Fluid

In a search for direct evidence leading to the biological relevance of airway secretions in innate host defense, we characterized the antibacterial function of cationic polypeptides within minimally manipulated nasal fluid. In this study, we show that cationic antimicrobial polypeptides are responsible for most of the bactericidal activity of whole nasal fluid. The removal of cationic polypeptides using a cation-exchange resin ablated the activity of nasal fluid against Escherichia coli, Listeria monocytogenes, and Pseudomonas aeruginosa. By using a novel proteomic approach, we identified a dozen cationic peptides and proteins within nasal fluid, all of which either are known antimicrobial polypeptides or have other proposed roles in host defense. Of the three most abundant cationic polypeptides in nasal fluid, lysozyme was more effective than either lactoferrin or secretory leukoprotease inhibitor in restoring the antibacterial activity of the cationic polypeptide-depleted fluid against a mucoid cystic fibrosis isolate of P. aeruginosa.

Activity of α- and θ-Defensins against Primary Isolates of HIV-1

θ-Defensins are lectin-like, cyclic octadecapeptides found in the leukocytes of nonhuman primates. They are also homologues of the more familiar α-defensins expressed by humans and certain other mammals. This study compares the ability of six θ-defensins (hominid retrocyclins 1–3 and rhesus θ-defensins 1–3) and four human α-defensins (human neutrophil peptides (HNPs) 1–4) to bind gp120 and CD4. In addition, we compared the ability of these θ-defensins and HNP-1 to protect J53-BL cells (an indicator cell line) from primary HIV-1 isolates that varied in subtype and coreceptor usage. The most potent θ-defensin, retrocyclin-2, bound with exceptionally high affinity to gp120 (KD, 9.4 nM) and CD4 (KD, 6.87 nM), and its effectiveness against subtype B isolates (IC50, 1.05 ± 0.28 μg/ml; 520 ± 139 nM) was approximately twice as great as that of HNP-1 on a molar basis. We also show, for the first time, that human α-defensins, HNPs 1–3, are lectins that bind with relatively high affinity to gp120 (KD range, 15.8–52.8 nM) and CD4 (KD range, 8.0–34.9 nM). Proteins found in human and FBS bound exogenous HNP-2 and retrocyclin-1, and competed with their ability to bind gp120. However, even the low concentrations of α-defensins found in normal human serum suffice to bind over half of the gp120 spikes on HIV-1 and a higher percentage of cell surface CD4 molecules. Although this report principally concerns the relationship between carbohydrate-binding and the antiviral properties of α- and θ-defensins, the lectin-like behavior of defensins may contribute to many other activities of these multifunctional peptides.

Calcitermin, a novel antimicrobial peptide isolated from human airway secretions

The human airways are protected from pathogenic colonization by a blanket of fluid impregnated with innate antimicrobial effector molecules. Among several previously uncharacterized components, we isolated a peptide that had activity primarily targeting Gram‐negative bacteria. We named the peptide ‘calcitermin’ since its amino acid sequence and mass were equivalent to the 15 C‐terminal residues of the S100 protein, calgranulin C. The antimicrobial activity of calcitermin was enhanced in acidic buffers (pH 5.4) and in the presence of micromolar concentrations of ZnCl2. Analysis revealed a putative zinc‐binding consensus sequence as well as an α‐helical conformation in structure‐promoting solvents.

The Role of Defensins in Lung Biology and Therapy

Innate host defence, involving both cellular and humoral mediators, is a prominent function of the human airways. Cellular mediators of innate immunity include dendritic cells, natural killer cells, cytotoxic T cells, macrophages and neutrophils, while humoral mediators of innate immunity consist of components of the epithelial lining fluid (ELF) covering the airways. Microbicidal substances in the ELF can selectively disrupt bacterial cell walls and membranes, sequester microbial nutrients or act as decoys for microbial attachment. Antimicrobial components of airway secretions include lysozymes, lactoferrin, secretory leukoprotease inhibitor, defensins and cathelicidins.Defensins are the most widely studied family of antimicrobial peptides present in airway fluid. Humans produce at least 10 different defensin molecules, six α-defensins and four β-defensins similar in structure and function. Direct evidence that defensins have central roles in host defense has only recently become available.Some defensins and defensin-like molecules could serve as templates for the development of pulmonary Pharmaceuticals. As potential therapeutics, they possess several desirable properties, including the ability to kill a broad spectrum of micro-organisms while permitting little development of microbial resistance. Many peptides can also neutralize effects of lipopolysaccharide on macrophages and other host defense cells and decrease the release of proinflammatory cytokines thereby giving protection against septic shock.Protegrin-1 is a minidefensin isolated from pig leukocytes and has proved to be an attractive template for large-scale development of antibacterials. One such protegrin analog, iseganan is in phase III clinical trials for the treatment of oral mucositis secondary to systemic chemotherapy. Other prospective uses of iseganan include control of respiratory pathogens in patients with cystic fibrosis and reduction of oral bacteria to prevent ventilator-associated pneumonia.However, in order to advance the production and clinical testing of peptide-based therapeutics, technical hurdles of synthesizing large quantities of complexly folded peptides must be first overcome. Strategies to develop potent peptide-based microbicides are promising in the struggle against increasingly resistant pathogens.

Lysozyme levels in the nasal secretions of patients with perennial allergic rhinitis and recurrent sinusitis

BACKGROUND The association of perennial allergic rhinitis (PAR) with recurrent sinusitis (RS) is well recognized. Anatomic abnormalities at the osteomeatal complex or ciliary dysfunction may play a significant role in some patients. However, for most patients with allergy, the determinants of RS are unknown. OBJECTIVE To determine whether altered concentrations of antimicrobial peptides and proteins, such as lysozyme, lactoferrin, human beta-defensin-2 (HBD-2), and human neutrophil peptides 1 to 3 (HNP-1 to 3), contribute to the development of RS in patients with PAR. METHODS Nasal secretions were collected by vacuum aspiration from 15 individuals with PAR+RS, 16 with PAR alone, and 16 controls. Lysozyme and lactoferrin levels were determined in nasal secretions by using quantitative enzyme-linked immunosorbent assay, and HBD-2 and HNP-1 to 3 levels were determined in nasal secretions by using semiquantitative Western blot analysis. Eosinophil-derived neurotoxin (EDN) levels were measured by using enzyme-linked immunosorbent assay as a marker of nasal eosinophilia in all 3 groups. RESULTS Levels of EDN were elevated significantly in patients with PAR+RS compared with controls. Lysozyme levels were decreased significantly in patients with PAR+RS compared with PAR alone or controls. Mean lysozyme levels were significantly lower in patients with EDN levels greater than 1,000 ng/mL vs those with levels of 1,000 ng/mL or less in the PAR+RS group. There were no statistically significant differences in lactoferrin, HBD-2, and HNP-1 to 3 levels among the 3 groups. CONCLUSIONS The presence of eosinophils and their products and reduced lysozyme concentrations may be critical factors that predispose the airways of patients with PAR to RS.

Microanalysis of antimicrobial properties of human fluids

Host defense responses of animals and plants to pathogenic microbes are mediated in part by the release of antimicrobial substances into tissue fluids. Exploration of the antimicrobial properties of tissue fluids is often limited by their small quantity. We have developed assays of antimicrobial activity that require only 1 microl of fluid. Using normal nasal secretions as a model mucosal fluid we demonstrated that the kinetics of the 1 microl colony-forming unit (CFU) assays were equivalent to the larger CFU assays. The handling of viscous mucin-containing fluids was facilitated by pretreatment with N-acetylcysteine (NAC), a treatment that did not alter the performance of the assay. This low-volume assay will facilitate studies of the antimicrobial properties of scarce biological fluids.

Inducibility of HBD-2 in acute burns and chronic conditions of the lung

The respiratory tract produces a number of molecules that act in the first line of host defense to protect against pathogenic colonization and tissue invasion. Most of the innate antimicrobial activity can be attributed to airway fluid proteins, such as lysozyme, lactoferrin, and secretory leukoproteinase inhibitor, and peptides, such as defensins. Human beta-defensins are cationic antimicrobial peptides with broad and potent microbicidal activity that have been shown to play a role in protecting the healthy lung from infection. To determine the effect of thermal injury on the production of the inducible beta-defensin, human beta-defensin-2 (HBD-2), we measured the concentration of HBD-2 by Western blot analysis in bronchoalveolar lavage samples from the lungs of burned patients with and without inhalation injury. Our data demonstrates an increased amount of HBD-2 in the pulmonary airways with thermal injury compared to normal lung. A further substantial increase in levels was noted in chronic lung conditions.