Biomedical subjects
T Ganz
Publications and source records attributed to T Ganz.
Defensin promotes the binding of lipoprotein(a) to vascular matrix.
Retention of lipoproteins within the vasculature is a central event in the pathogenesis of atherosclerosis. However, the signals that mediate this process are only partially understood. Prompted by putative links between inflammation and atherosclerosis, we previously reported that alpha-defensins released by neutrophils are present in human atherosclerotic lesions and promote the binding of lipoprotein(a) [Lp(a)] to vascular cells without a concomitant increase in degradation. We have now tested the hypothesis that this accumulation results from the propensity of defensin to form stable complexes with Lp(a) that divert the lipoprotein from its normal cellular degradative pathways to the extracellular matrix (ECM). In accord with this hypothesis, defensin stimulated the binding of Lp(a) to vascular matrices approximately 40-fold and binding of the reactants to the matrix was essentially irreversible. Defensin formed stable, multivalent complexes with Lp(a) and with its components, apoprotein (a) and low-density lipoprotein (LDL), as assessed by optical biosensor analysis, gel filtration, and immunoelectron microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited (>90%) by heparin and by antibodies to fibronectin (>70%), but not by antibodies to vitronectin or thrombospondin. Defensin increased the binding of Lp(a) (10 nmol/L) to purified fibronectin more than 30-fold. Whereas defensin and Lp(a) readily traversed the endothelial cell membranes individually, defensin/Lp(a) complexes lodged on the cell surface. These studies demonstrate that alpha-defensins released from activated or senescent neutrophils stimulate the binding of an atherogenic lipoprotein to the ECM of endothelial cells, a process that may contribute to lipoprotein accumulation in atherosclerotic lesions.
A 450-kb contig of defensin genes on human chromosome 8p23.
Defensins are a large family of host defense peptides expressed in leukocytes and epithelia. Using P1 and BAC clones, we have determined the organization of the human alpha-defensin genes and the beta-defensin gene HDEFB1 on chromosome 8p23. From the telomere, the order of the genes (with encoded peptides in parentheses) is HDEFA5 (HD-5), HDEFA1/1A (HNP-1/3), HDEFA4 (HNP-4), HDEFA6 (HD-6), and HDEFB1 (HBD-1). These genes span a region of approximately 450kb. Genes encoding intestinal Paneth cell defensins (HDEFA5 and HDEFA6) flank the myeloid defensin gene cluster (HDEFA1, HDEFA1A, HDEFA4). Based on our previous studies, the remaining known defensin gene, HDEFB2 (HBD-2), is about 400kb centromeric to HDEFB1. This map supports the hypothesis, originally proposed because of sequence similarities, that myeloid alpha-defensin genes evolved by reduplication and divergence from Paneth cell defensin genes, and identifies regions and clones, which should be useful in the search for new defensin genes.
Antimicrobial peptides in mammalian and insect host defence.
During the past year, additional insights into systems that regulate antimicrobial peptide production in Drosophila were reported. Granulysin, a peptide stored in the cytoplasmic granules of human natural killer cells and cytolytic T cells, was shown to kill Mycobacterium tuberculosis. More data implicating antimicrobial peptides in the pathogenesis of bronchopulmonary infections in cystic fibrosis appeared. Studies that examined the potential contributions of antimicrobial peptides to regional innate immunity gained in prominence. Efforts to design peptide analogues to prevent or treat infections continued.
Antibiotic peptides from higher eukaryotes: biology and applications.
Gene-encoded antibiotic peptides are increasingly being recognized as effector molecules of host defense in plants and animals. Studies of antimicrobial peptides are providing new insights into the dynamic interactions between microbes and their hosts, and are generating new paradigms for the pathogenesis and treatment of diseases. Because antimicrobial peptides of higher eukaryotes differ structurally from conventional antibiotics produced by bacteria and fungi, they offer novel templates for pharmaceutical compounds that could be effective against increasingly resistant microbes.
Eukaryotic antibiotic peptides: not only a membrane business.
Explore the source record for details and available documents.
Immunomagnetic recovery of human neutrophil defensins from the human gingival crevice.
The human neutrophilic protein defensins are cationic, antimicrobial, cytotoxic, corticostatic chemotactic, opsonic peptides found in azurophilic granules and constitute about 5% of the total protein in human neutrophils. In the present study, defensins were recovered from the human gingival crevice using paramagnetic microspheres (beads), coated with anti-defensin antibodies. The bead-bound defensins were assayed using an enzyme-linked immunosorbent assay developed in this laboratory. Twenty sites were sampled; defensin was found in 100% of the sites and ranged in amount from 270-2000 ng/site. The large local concentrations of defensins, estimated in the mg/ml range, may have major effects on the microbiology of the gingival crevice.
Porcine epithelial beta-defensin 1 is expressed in the dorsal tongue at antimicrobial concentrations.
Epithelial cells and phagocytes contain antimicrobial polypeptides that participate in innate host defense. A recently cloned porcine beta-defensin, PBD-1, was detected by Northern organ blots exclusively in the tongue epithelium. We generated recombinant PBD-1 peptide by using a baculovirus-insect cell expression system and obtained two forms (PBD-142 and PBD-138), which differed by N-terminal truncation. Only PBD-142 was found in scrapings of the surface of the dorsal tongue or the buccal mucosa. Immunohistochemical staining with antibody to PBD-142 revealed that PBD-1 was highly concentrated in an approximately 0.1-mm-thick layer in the cornified tips of the filiform (but not fungiform) papillae of the dorsal tongue and in the superficial squamous cell layers of the buccal mucosa. By scraping, extraction, and semiquantitative Western blotting, the concentration of PBD-1 in the dorsal tongue surface and the buccal mucosa was estimated at 20 to 100 micrograms/ml. PBD-1 had antibacterial activity against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, and Candida albicans in 10 mM sodium phosphate buffer (pH 7.4). Added NaCl progressively inhibited the activity of PBD-1 against E. coli and C. albicans. In 10 mM sodium phosphate with 125 mM NaCl, the combinations of sublethal concentrations of PBD-1 and the porcine neutrophil peptide PG-3, PR-39, or PR-26 showed synergistic activity against E. coli or the multidrug-resistant S. typhimurium DT104. At its physiologic concentration, PBD-1 has antimicrobial effects under both low- and high-salt conditions encountered in the oral cavity and may contribute to the antimicrobial barrier properties of the dorsal tongue and oral epithelium.
Innate antimicrobial activity of nasal secretions.
Minimally manipulated nasal secretions, an accessible form of airway surface fluid, were tested against indigenous and added bacteria by using CFU assays. Antimicrobial activity was found to vary between donors and with different target bacteria and was markedly diminished by dilution of the airway secretions. Donor-to-donor differences in electrophoresis patterns of nasal secretions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and acid urea-PAGE analyses were readily observed, suggesting that polymorphic genes encode the secreted proteins. Three donors (of twenty-four total), whose nasal fluid yielded similar protein band patterns and did not kill indigenous bacteria, were determined to be heavy nasal carriers of Staphylococcus aureus. Their fluid was deficient in microbicidal activity toward a colonizing strain of S. aureus but the defect was corrected in vitro by a 1:1 addition of nasal fluid from noncarriers. The microbicidal activity of normal fluid was inactivated by heating it for 10 min to 100 degrees C and could not be restored solely by the addition of two major nasal antimicrobial proteins, lysozyme and lactoferrin. Several other known antimicrobial proteins and peptides, including statherin, secretory phospholipase A2, and defensins, were identified in nasal secretions and likely contribute to their total antimicrobial properties. Nasal fluid may serve as a useful model for the analysis of lower-airway secretions and their role in host defense against airway colonization and pulmonary infections.
Defensins and innate host defence of the gastrointestinal tract.
Explore the source record for details and available documents.
Impaired innate immunity in the newborn: newborn neutrophils are deficient in bactericidal/permeability-increasing protein.
OBJECTIVE: The mechanisms by which newborns are at increased risk for invasive bacterial infections have been incompletely defined. A central element of innate immunity to bacterial infection is the neutrophil-a cell that contains cytoplasmic granules replete with antibiotic proteins and peptides. The activity of adult neutrophils against gram-negative bacteria is believed to depend to a significant degree on the presence in neutrophil primary (azurophilic) granules of the 55-kDa bactericidal/permeability-increasing protein (BPI), which binds with high affinity to bacterial lipopolysaccharides and kills gram-negative bacteria. In light of the importance of BPI to antibacterial host defense and to investigate possible factors underlying the risk of neonatal bacterial infections, we determined the relative content of BPI in the neutrophils of adults and newborns. DESIGN: The cellular content of BPI was determined by Western blotting of neutrophils derived from full-term newborn cord blood (n = 21; mean gestational age: 38.6 weeks) and from adult peripheral blood (n = 22; mean age: 29 years). Extracellular levels of BPI in adult and newborn plasma were assessed by enzyme-linked immunosorbent assay. Neutrophil content of other azurophil granule markers also was assessed: myeloperoxidase by Western blotting and defensin peptides by acid-urea polyacrylamide gel electrophoresis and Coomassie staining. Acid extracts of newborn and adult neutrophils were analyzed for antibacterial activity against serum-resistant encapsulated isolate Escherichia coli K1/r. RESULTS: The neutrophils of newborns contain at least threefold to fourfold less BPI per cell than adult neutrophils (67 +/- 13 ng per 10(6) cells vs 234 +/- 27 ng per 10(6) cells). The relative BPI-deficiency of newborn neutrophils apparently was not attributable to perinatal stress-related degranulation of intracellular BPI stores because: 1) newborn and adult neutrophils contained nearly identical amounts of 2 microbicidal constituents derived from the same primary (azurophil) granule compartment as BPI (the enzyme myeloperoxidase as well as defensin peptides), and 2) levels of extracellular BPI in newborn plasma, measured by enzyme-linked immunosorbent assay, represent only approximately 2% of cellular BPI content. As predicted by their lower BPI content, newborn neutrophil acid extracts demonstrated significantly lower antibacterial activity against E coli K1/r than did adult neutrophil acid extracts. CONCLUSION: These data suggest that the neutrophils of newborns are selectively deficient in BPI, a central effector of antibacterial activity against gram-negative bacteria. BPI deficiency correlates with decreased antibacterial activity of newborn neutrophil extracts against serum-resistant E coli and could contribute to the increased incidence of gram-negative sepsis among newborns relative to healthy adults.neonatal sepsis, gram-negative bacteria, endotoxin, neutrophil, polymorphonuclear leukocyte, innate immunity, bactericidal/permeability-increasing protein, defensin, myeloperoxidase.
Production of beta-defensins by human airway epithelia.
Human beta-defensins (HBDs) are antimicrobial peptides that may play a role in mucosal defense. Diminished activity of these peptides has been implicated in the pathogenesis of cystic fibrosis (CF) lung disease. We show that HBD-1 and HBD-2 mRNAs are expressed in excised surface and submucosal gland epithelia from non-CF and CF patients. The pro-inflammatory cytokine interleukin-1beta stimulated the expression of HBD-2 but not HBD-1 mRNA and peptide in primary cultures of airway epithelia. HBD-1 was found in bronchoalveolar lavage (BAL) fluid from normal volunteers, CF patients, and patients with inflammatory lung diseases, whereas HBD-2 was detected in BAL fluid from patients with CF or inflammatory lung diseases, but not in normal volunteers. Both HBD-1 and HBD-2 were found in BAL fluid in concentrations of several ng/ml, and both recombinant peptides showed salt-sensitive bactericidal activity. These data suggest that in the lung HBD-2 expression is induced by inflammation, whereas HBD-1 may serve as a defense in the absence of inflammation.
Structure and mapping of the human beta-defensin HBD-2 gene and its expression at sites of inflammation.
We cloned a second human beta-defensin gene, HBD-2, and determined its gene structure and expression in inflamed tissue sections. The entire gene spanned about 2 kb with two small exons and one intron. Radiation hybrid studies confirmed the location on chromosome 8p, were consistent with the order HNP-1, HBD-1 and HBD-2, and located HBD-2 as the most centromeric of the genes. By three-color fluorescence in situ hybridization on both free chromatin fiber mapping and interphase mapping, HBD-1, HBD-2 and HNP-1 were mapped to chromosome 8p23. HBD-1 was within 40-100kb of HNP-1, while HBD-2 was about 500-600 kb from HBD-1, with the most likely order HNP-1, HBD-1, HBD-2. The expression of HBD-2 was locally regulated by inflammation. HBD-2 mRNA was markedly increased in the epidermis surrounding inflamed regions, but not detectable in adjacent non-inflamed areas, a distribution that was confirmed at the peptide level by immunostaining with HBD-2 antibody. The HBD-2 gene is the first member of the human defensin family that is locally inducible by inflammation.
An antimicrobial activity of cytolytic T cells mediated by granulysin.
Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.
Isolation of human intestinal defensins from ileal neobladder urine.
We describe the isolation of naturally occurring human intestinal defensins HD-5 and HD-6 from ileal neobladder urine and ileal mucosa. Using an antibody-based detection assay, we found multiple N-terminally processed forms of HD-5. The predominant HD-5 forms in tissue were longer than those in neobladder urine (amino acid (aa) 23-94 and 29-94 versus aa 36-94, 56-94 and 63-94) suggesting that Paneth cells store prodefensin that is processed to mature defensin during or after degranulation. Search for mature HD-6 yielded aa 69-100 as the predominant form in both sources. The ileal neobladder is a promising model to study human Paneth cell secretion.
Human beta-defensin-1: an antimicrobial peptide of urogenital tissues.
Antimicrobial peptides are widely distributed mediators of innate host defense in animals and plants. A 36 amino acid antimicrobial peptide belonging to the defensin family, and named human beta-defensin-1 (HBD-1), was purified recently from hemodialysate fluid, but its tissue sources were not identified. By Northern blotting, we found the highest concentrations of HBD-1 mRNA in the kidney and the female reproductive tract. In situ hybridization localized the HBD-1 mRNA in the epithelial layers of the loops of Henle, distal tubules, and the collecting ducts of the kidney and the epithelial layers of the vagina, ectocervix, endocervix, uterus, and fallopian tubes in the female reproductive tract. Using a novel technique designed to detect cationic peptides in urine, we recovered several forms of HBD-1 ranging in length from 36 to 47 amino acid (aa) residues and differing from each other by amino terminal truncation. The total concentration of HBD-1 forms in voided urine was estimated at 10-100 microg/liter, with individual variations in the total amount of HBD-1 peptides and the relative proportion of HBD-1 forms. Multiple forms of HBD-1 (size 36-47 aa) were also found in the blood plasma, bound to carrier macromolecules that released the peptide under acid conditions, and in vaginal mucosal secretions (39, 40, and 44 aa). By immunostaining, HBD-1 was located in the kidney within the lumen of the loops of Henle, but no intracellular storage sites were identified in renal or female reproductive tissues. Recombinant HBD-1 forms (36, 39, and 42 aa) and natural HBD-1 forms were antimicrobial to laboratory and clinical strains of Escherichia coli at micromolar concentrations. HBD-1 activity was not changed appreciably by low pH, but was inhibited by high salt conditions. Some of the HBD-1 peptides retained their activity against E. coli in unconcentrated (low conductance) urine, and the 36 aa form was microbicidal even in normal (high conductance) urine. Production of HBD-1 in the urogenital tract could contribute to local antimicrobial defense.
Molecular cloning and tissue expression of porcine beta-defensin-1.
Beta-defensins constitute an emerging family of cysteine-rich antimicrobial peptides, which are particularly prominent at mucosal epithelial sites in mammals. Here we report the identification of a novel beta-defensin from porcine tissues, porcine beta-defensin-1 (pBD-1). The cDNA sequence of pBD-1 encoded a 64 amino acid prepro-peptide, which contained the beta-defensin consensus sequence of six invariantly spaced cysteine residues. Northern blot analysis showed that pBD-1 was expressed abundantly in tongue epithelia and that the expression was regulated developmentally. Using RT-PCR, pBD-1 mRNA was detected throughout the respiratory and digestive tracts and also in thymus, spleen, lymph node, brain, liver, kidney, urinary bladder, testis, skin, heart, muscle, bone marrow, peripheral blood neutrophils, alveolar macrophages, and umbilical cord. The wide expression of pBD-1 suggests that this endogenous peptide antibiotic may contribute to both mucosal and systemic host defenses in pigs, which may have implications for the use of porcine tissues and organs in xenotransplantation.
Antimicrobial peptides of vertebrates.
The past year brought several discoveries that focused attention on antimicrobial peptides on epithelial surfaces. The malfunction of these substances was implicated as a cause of airway infections in cystic fibrosis. Other highlights included new insights into the relative selectivity of antimicrobial peptides for microbial membranes, their primary site of action.