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PubMed1999

Epithelial defensins impair adenoviral infection: implication for adenovirus-mediated gene therapy.

Epithelial cells have been to participate actively in host defense by producing small cationic peptides called defensins. To investigate the biological activity of epithelial defensins in more detail, we expressed two defensins, hBD-1 and HD-5, in eukaryotic cell lines. Defensins were localized in the cytoplasm and in cell culture medium and exhibited strong microbicidal activity toward Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Moreover, our data indicate that the presence of defensins protected the cells from adenoviral infection. The presence of HD-5 or hBD-1 reduced the infectivity of Av1CF2 three- to fivefold. These results imply that defensins must be considered a serious obstacle whenever adenovirus is used to deliver genes to epithelial cells.

Adenoviridae↗
PubMed1999

Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6.

Defensins contribute to host defense by disrupting the cytoplasmic membrane of microorganisms. This report shows that human beta-defensins are also chemotactic for immature dendritic cells and memory T cells. Human beta-defensin was selectively chemotactic for cells stably transfected to express human CCR6, a chemokine receptor preferentially expressed by immature dendritic cells and memory T cells. The beta-defensin-induced chemotaxis was sensitive to pertussis toxin and inhibited by antibodies to CCR6. The binding of iodinated LARC, the chemokine ligand for CCR6, to CCR6-transfected cells was competitively displaced by beta-defensin. Thus, beta-defensins may promote adaptive immune responses by recruiting dendritic and T cells to the site of microbial invasion through interaction with CCR6.

Antibodies↗
PubMed1990

In vitro sensitivity of oral, gram-negative, facultative bacteria to the bactericidal activity of human neutrophil defensins.

Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria.

Blood Bactericidal Activity↗
PubMed1991

Antimicrobial activity of rabbit leukocyte defensins against Treponema pallidum subsp. pallidum.

Defensins, which are peptides with broad antimicrobial activity, are major constituents of rabbit neutrophils and certain macrophages. We tested six rabbit defensins, NP-1, NP-2, NP-3a, NP-3b, NP-4, and NP-5, for activity against Treponema pallidum subsp. pallidum. Mixtures of T. pallidum and defensin in 10% normal rabbit serum (NRS) or heat-inactivated NRS (HI-NRS) were incubated anaerobically for various time periods ranging between 0 and 16 h and then examined by dark-field microscopy for treponemal motility or inoculated intradermally into rabbits to assess treponemal virulence. Immobilization of T. pallidum by NP-1 (400 micrograms/ml) occurred after 4 and 8 h of coincubation in mixtures containing NRS and HI-NRS, respectively. Similarly, neutralization of T. pallidum by NP-1 occurred more rapidly and was complete when incubations were performed in NRS as compared with that in HI-NRS. Endpoint titration confirmed the augmentation of NP-1 antitreponemal activity by heat-labile serum factors; NP-1 showed neutralizing activity at 4 micrograms/ml (about 1 microM) in NRS and at 40 micrograms/ml in HI-NRS. When NP-1 was tested in serum that was deficient in C6, the T. pallidum neutralizing activity of NP-1 was reduced to levels slightly greater than that observed in HI-NRS. NP-1 that had been reduced and alkylated was inactive against T. pallidum. When NP-2, NP-3a, NP-3b, NP-4, and NP-5 were tested at 400 micrograms/ml, all exerted potent treponemicidal activity, manifested by abrogation or delayed development of cutaneous lesions relative to that of controls. These data suggest that defensins may equip certain macrophages and neutrophils to participate in host defense against T. pallidum, that the direct activity of defensins against T. pallidum is enhanced by heat-labile serum factors (presumably complement), and that conformational factors influence the biological activity of the defensin molecule.

Animals↗
PubMed1994

Inhibition of intracellular Histoplasma capsulatum replication by murine macrophages that produce human defensin.

Although purified defensins are effective microbicides in vitro, their operation within intact phagocytes has not been established. To address this question, we inserted cDNA encoding human defensin HNP-1 into a pBabe/neo retroviral vector and transduced it into RAW 264.7 cells, a murine macrophage line that lacks endogenous defensins. We isolated five independent clones of HNP-1-transduced cells, all of which secreted prodefensin and contained small amounts of fully processed HNP-1. The two clones that produced the largest amounts of defensin (clones 5 and 14), together with wild-type RAW cells and pBabe/neo-transduced RAW cells (control), were used for the present study. All cells were grown in Dulbecco's modified Eagle's medium-F12 medium that contained 10% heat-inactivated fetal bovine serum and gentamicin. The medium used for the transduced cells contained aminoglycoside G418 in lieu of gentamicin. Both wild-type and transduced cells were placed in antibiotic-free medium 96 h prior to challenge with a yeast-phase strain of Histoplasma capsulatum. Phagocytosis of yeast cells was allowed to proceed for 90 min and was followed by washing and further incubation for 18.5 h. Whereas the phagocytic index did not differ significantly among the four cell populations under study, the mean level of intracellular growth of H. capsulatum in the defensin-transduced RAW cells was significantly lower than those observed for any other cell types (P < 0.05). These findings constitute the first instance of xenogeneic expression of an antimicrobial peptide by phagocytes and suggest that macrophages can be armed with defensins to enhance their ability to restrict certain intracellular pathogens.

Animals↗
PubMed2006

Human alpha- and beta-defensins block multiple steps in herpes simplex virus infection.

This study examined the ability of nine human defensins (HD) to protect against herpes simplex virus infection. Noncytotoxic concentrations of all six alpha-defensins (HNP1-4, HD5, and HD6) and human beta-defensin (hBD) 3 inhibited HSV infection. Two other beta-defensins, hBD1 and 2, lacked this protective activity. Synchronized assays revealed that HNP-4, HD6, and hBD3 acted primarily by preventing binding and entry, whereas HNP1-3 and HD5 also inhibited postentry events. Even when added several hours after entry, substantial reduction in viral gene expression ensued. Human cervical epithelial cells incubated with HNP-1 or HD5 accumulated the peptides intracellularly. Surface plasmon resonance studies revealed that HNPs 1, 2, 3, and HD5 bound HSV glycoprotein B (gB) with high affinity, but showed minimal binding to heparan sulfate, the receptor for attachment. In contrast, HNP-4 and HD6 bound heparan sulfate, but not gB. HBD3 bound both gB and heparan sulfate, but hBD1 and hBD2 bound neither. Admixture of HD5 with hydroxyethylcellulose significantly protected mice from a viral challenge lethal to controls receiving an inactive peptide or hydroxyethylcellulose alone. These findings demonstrate that HDs act at multiple steps in the HSV life cycle and support the development of defensins or defensin-like peptides as microbicides.

Animals↗
PubMed2000

Large-scale synthesis and functional elements for the antimicrobial activity of defensins.

Human neutrophil defensins, and their analogues incorporating anionic, hydrophobic or cationic residues at the N- and C-termini, were synthesized by solid-phase procedures. The synthetic defensins were examined for their microbicidal activity against Candida albicans, two Gram-negative bacteria (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) and two Gram-positive bacteria (Streptococcus gordonii and Streptococcus mutans). The human neutrophil peptide 1 (HNP1) and HNP2 were found to be potent candidacidal agents. HNP3, which differs by one amino acid at the N-terminus of its sequence, was totally inactive. The Gram-negative bacteria A. actinomycetemcomitans and P. gingivalis and the Gram-positive bacteria S. gordonii and S. mutans were insensitive to human defensins. However, the insertion of two basic residues, such as arginine, at both the N-terminus and the C-terminus of HNP2 significantly enhanced antifungal and antibacterial activity. The addition of anionic residues, such as aspartic acid, at the N- and C-termini rendered the molecule totally inactive. The presence of two hydrophobic amino acids, such as valine, at the N-terminus of HNP2 and of two basic arginine residues at its C-terminus resulted in molecules that were optimally active against these oral pathogens. The results suggest that the N- and C-terminal residues in defensin peptides are the crucial functional elements that determine their microbicidal potency. The three-dimensional structure of all defensins constitutes the same amphiphilic beta-sheet structure, with the polar face formed by the N- and C-terminal residues playing an important role in defining microbicidal potency and the antimicrobial spectrum. The enhanced microbicidal activity observed for defensin peptides with two basic residues at both the N- and C-termini could be due to optimization of the amphiphilicity of the structure, which could facilitate specific interactions with the microbial membranes.

Aggregatibacter actinomycetemcomitans↗
PubMed1995

The subcellular localization of defensins and myeloperoxidase in human neutrophils: immunocytochemical evidence for azurophil granule heterogeneity.

We used immunogold electron microscopy to examine the distribution of human defensins 1-3 (HNP1-3) and myeloperoxidase (MPO) in the cytoplasmic granules of thin-sectioned, normal human neutrophils. The presence of MPO, defensins, or both was noted in 388 (62.3%) of 623 granule profiles. Whereas MPO and defensins colocalized in 32.5% of such granules, 32.7% demonstrated only MPO and 34.8% demonstrated only defensins. The staining densities of MPO and HNP1-3 showed a low correlation, even when both molecules were present in the same granules (r = 0.09), indicating that they were not present in fixed relative proportions even when colocalized. The low partial correlation coefficient (r = -0.23) between MPO and HNP1-3 for the 388 labeled granules controlling for granule area suggested that the deposition of MPO and defensins might even be negatively correlated. The distribution of MPO and defensins within the cytoplasmic granules of human neutrophils followed a "non-colocalization and multi-compartmental" model that conflicts with the classical notion of azurophil granules as a homogeneous population of organelles marked by the presence of MPO.

Blood Proteins↗
PubMed1990

In vitro antimicrobial activity of defensins against ocular pathogens.

New approaches to antimicrobial therapy for ocular pathogens must overcome organisms that are resistant to current therapeutic modalities. This investigation examined the antimicrobial activity of novel antimicrobial neutrophil peptides (defensins NP-1 and NP-5) against isolates from clinical ocular microbial infections in humans and horses. The test panel of human clinical isolates included Candida albicans, an alpha-hemolytic Streptococcus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Morganella morganii. The test panel of equine pathogens included three clinical isolates of P aeruginosa and two clinical isolates of Staphylococcus aureus. The equine isolates were chosen for their relative resistance to commonly employed antimicrobial therapy. The two defensins differed markedly in their bactericidal activity. Defensin NP-5, at a 50-micrograms/mL concentration, exhibited minimal bactericidal activity against the majority of isolates of the test panel. The inferior microbicidal activity of NP-5 is consistent with previously published results. However, at this concentration, NP-5 did exhibit appreciable bacteriostatic activity against human ocular pathogens M morganii (74%), alpha-hemolytic Streptococcus (57%), and P aeruginosa (93%) during the 2-hour incubation period. In contrast, defensin NP-1 at 10 micrograms/mL exerted potent microbicidal activity against all isolates, effecting a 2 to 3 log10 decrease in colony-forming units within a 60-minute incubation period. Under the assay conditions employed, these findings demonstrate: (1) two distinct mechanisms by which defensins exert their antimicrobial activity against microbial pathogens associated with clinical ocular disease in humans and horses, and (2) that rabbit defensin NP-1 is a potent antimicrobial agent against a wide array of ocular pathogens.

Animals↗
PubMed1990

Insect immunity: expression of the two major inducible antibacterial peptides, defensin and diptericin, in Phormia terranovae.

Injections of low doses of bacteria into larvae of Phormia terranovae induce the appearance of potent bactericidal peptides in the blood, among which predominate the anti-Gram positive insect defensins and the anti-Gram negative diptericins. Insect defensins show significant homologies to mammalian (including human) microbicidal peptides present in polymorphonuclear leukocytes and macrophages. We report the molecular cloning of cDNAs and primer extension studies which indicate that insect defensin is produced as a prepro-peptide yielding mature defensin A (40 residues) after cleavage of a putative signal peptide (23 residues) and a prosequence (34 residues). Previous studies have established that diptericin (82 residues) is matured from a pre-peptide by cleavage of a putative signal peptide (19 residues) and C-terminal amidation. Using oligonucleotide probes complementary to the sequences of the mRNAs for defensin and diptericin, we show by in situ hybridization that both antibacterial peptides are concomitantly synthesized by the same cells: thrombocytoids, a specialized blood cell type, and adipocytes. Transcriptional studies based on hybridization of RNAs to cDNAs of defensin and diptericin indicate that the transcription of both genes is induced regardless of the nature of the stimulus (injection of Gram positive or Gram negative bacteria, lipopolysaccharides). Even a sterile injury applied to axenically raised larvae is efficient in inducing the transcription of both genes suggesting that the local disruption of the integument aspecifically initiates a signalling mechanism which the thrombocytoids and the adipocytes are able to interpret. The transcription of immune genes is relatively short lived and a second challenge yields a response similar to that of the first stimulus, indicating that the experimental insects do not keep a 'memory' of their first injection.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of a new member of the insect defensin family from a beetle, Allomyrina dichotoma.

A new family member of insect defensin, an antibacterial peptide, has been isolated from larvae of a beetle, Allomyrina dichotoma. The peptide consisted of 43 amino acids and 6 cystein residues were conserved in the same position as that of other insect defensins. The new defensin was found to be inducible by bacterial injection. Analysis of the antibacterial spectrum of A. dichotoma defensin indicated that this peptide showed antibacterial activity against Gram-positive bacteria like Staphylococcus aureus and Bacillus subtilis but not against Gram-negative bacteria like Escherichia coli and Pseudomonas aeruginosa, indicating a typical spectrum of the insect defensin family. In addition, A. dichotoma defensin also exhibited antibacterial activity against methicillin-resistant S. aureus (MRSA) isolated from a patient.

Amino Acid Sequence↗
PubMed1997

Coordinated amino acid changes in the evolution of mammalian defensins.

The mammalian defensin molecule is a short, highly cationic peptide cytotoxic to both microbial and mammalian cells which is cleaved from a precursor including a signal peptide and a highly anionic propiece. A phylogenetic analysis of 28 complete sequences from five mammalian species (mouse, rat, guinea pig, rabbit, and human) showed species-specific clusters of sequences, indicating that the genes duplicated after divergence of these species. Comparison of rates of synonymous and nonsynonymous nucleotide substitution suggested that gene duplication has often been followed by a period in which diversification of the mature defensins at the amino acid level has been selectively favored. In some comparisons, it appeared that amino acid differences in this region have appeared in a nonrandom fashion so as to change the pattern of residue charges. Because it has been hypothesized that the negative charge in the propiece serves to balance the positive charge in the mature defensin and thus to prevent cytotoxicity prior to cleavage, we used a maximum likelihood method of reconstructing ancestral states in order to test whether this balance has been maintained over evolutionary time in spite of rapid diversification of the mature defensin at the amino acid level. Reconstructed ancestral sequences always maintained a charge balance between mature defensin and propiece, and changes in the net positive charge of the mature defensin were balanced by corresponding changes in the propiece. The results support the hypothesis that, in the evolution of these proteins, amino acid changes have occurred in a coordinated fashion so as to preserve an adaptive phenotype.

Amino Acid Sequence↗
PubMed2006

Human beta-defensins.

The last decade led to the discovery and characterization of several human beta-defensins. Analysis of genomic information indicates that the number of beta-defensin-like molecules encoded by the human genome may number in the tens. Growing interest in beta-defensins steadily enhances our knowledge about various aspects of their gene location, expression patterns and the transcription factors involved in their regulation in vivo. The hallmark property of beta-defensins, their antimicrobial activity, is clearly only the tip of the iceberg in the extensive network of inter-relations within the immune system in which these peptides function. Structural studies of beta-defensins provide the molecular basis for a better understanding of their properties, functions and their potential for practical applications. In this review, we present some recent advances in the studies of human beta-defensins, with an emphasis on possible correlations between their structural and functional properties.

Amino Acid Sequence↗
PubMed2006

Defensins- and cathepsin G-ANCA in systemic lupus erythematosus.

In this study, we examined the content of antineutrophil cytoplasmic antibodies (ANCA) against defensins and cathepsin G in sera from systemic lupus erythematosus (SLE) patients and their significance in estimating the activity of SLE. Defensins- and cathepsin G-ANCA in sera from 28 patients with SLE, eight patients with rheumatoid arthritis (RA) and eight patients with microscopic polyangitis (mPA) were measured by ELISA. Significantly increased defensins- and cathepsin G-ANCA were found in sera of patients with SLE and mPA when compared with the value of normal controls. Though significantly higher defensins- and cathepsin G-ANCA were detected in both active and inactive SLE patients, the value in active SLE patients was significantly higher than inactive SLE patients. After the therapy with high dose of prednisolone, the serum level of defensins- and cathepsin G-ANCA was decreased, and this decrease was sustained for at least 16 weeks. This study suggests that defensins- and cathepsin G-ANCA may serve as useful markers of the disease activity of SLE.

Adult↗
PubMed1998

Evidence that the role of plant defensins in radish defense responses is independent of salicylic acid.

Radish leaves contain two homologous 5-kDa plant defensins which accumulate systemically upon infection by fungal pathogens (F.R.G. Terras et al., 1995, Plant Cell 7: 573-588). Here we report on the molecular cloning of the cDNAs encoding the two pathogen-inducible plant defensin isoforms from radish (Raphanus sativus L.) leaves. Tissue-print and whole-leaf electroblot immunostaining showed that the plant defensin peptides not only accumulate at high levels at or immediately around the infection sites in leaves inoculated with Alternaria brassicicola, but also accumulate in healthy tissue further away from the infection sites and in non-infected leaves from injected plants. Gel blot analysis of RNA confirmed that expression of plant defensin genes is systemically triggered upon fungal infection whereas radish PR-1 gene expression is only activated locally. In contrast to the radish PR-1 gene(s), expression of the radish plant defensin genes was not induced by external application of salicylic acid. Activation of the plant defensin genes, but not that of PR-1 genes, occurred upon treatment with methyl jasmonate, ethylene and paraquat.

Alternaria↗
PubMed2005

Cytotoxic concentrations of alpha-defensins in the lungs of individuals with alpha 1-antitrypsin deficiency and moderate to severe lung disease.

The lungs of individuals with alpha(1)-antitrypsin (alpha(1)-AT) deficiency have a reduced antiprotease protective shield. Typically, numbers of neutrophils are elevated in the lungs of affected individuals, carrying large amounts of neutrophil elastase (NE) and alpha-defensins that are released upon activation. We hypothesized that in individuals with advanced lung disease associated with alpha(1)-AT deficiency cytotoxic concentrations of alpha-defensins might be present and unopposed, thus further aggravating lung disease. To evaluate this hypothesis, bronchoalveolar lavage was performed in 20 alpha(1)-AT deficient individuals with moderate to severe lung function impairment and 13 healthy volunteers. Cell counts as well as concentrations of NE and alpha-defensins were determined. While concentrations of NE were low (23+/-14nM) and alpha-defensins were undetectable in volunteers, they were significantly elevated in individuals with alpha(1)-AT deficiency (1313+/-723nM and 6605+/-2856nM, both p<0.0001). These concentrations were significantly higher than those found in a historical control of alpha(1)-AT deficient individuals with mild lung disease. Neutrophil numbers and NE concentration correlated with alpha-defensins concentration (r=0.612 and r=0.758, p=0.005 and p=0.0001, respectively). Individuals with alpha(1)-AT deficiency and moderate to severe lung function impairment have lung alpha-defensins concentrations in a range known to induce cytotoxicity in vitro in the absence of normal amounts alpha(1)-AT and thus may contribute to the development of lung disease in this population.

Adult↗
PubMed2004

Differential expression of sheep beta-defensin-1 and -2 and interleukin 8 during acute Mannheimia haemolytica pneumonia.

Beta-defensins are antimicrobial peptides produced by several cell types, including respiratory epithelia and leukocytes. Expression of some beta-defensins is increased by bacterial-induced inflammatory responses whereas expression of other beta-defensins is constitutive. Two beta-defensins are expressed in lungs of sheep (sheep beta-defensin-1 and -2; SBD-1/-2) and expression of SBD-1 is increased during parainfluenza virus type 3 (PI-3) infection. The effect of Mannheimia haemolytica, a Gram-negative bacteria known to induce expression of bovine beta-defensins and NF-kappa B in lung, has not been determined for SBD-1/-2. In this study, different concentrations of M. haemolytica were inoculated into pulmonary bronchi of lambs. SBD-1 and SBD-2 mRNA levels detected by real time reverse transcriptase polymerase chain reaction in lung homogenates did not increase. In fact, SBD-1 mRNA levels were significantly decreased with the highest administered inoculum concentration (10(9)). In contrast, mRNA levels of interleukin-8 (IL-8) were significantly increased over controls and progressively increased with M. haemolytica concentrations. Co-inoculation of M. haemolytica with xylitol, an osmotic agent, did not alter mRNA levels of SBD-1, SBD-2 or IL-8. SBD-1 mRNA expression was detected in lung epithelia, but not in leukocytes. This study suggests that SDB-1 expression occurs in epithelia and decreases during severe bacterial pneumonia, which is in contrast to the increase that occurs with PI-3 infection.

Animals↗
PubMed1997

Detection of human defensin 5 in reproductive tissues.

OBJECTIVES: This study was undertaken to determine whether tissue-specific defensins are expressed within female reproductive tissues. STUDY DESIGN: Messenger ribonucleic acid from amnion, chorion, endometrium, endocervix, myometrium, placenta, small intestine, peripheral blood lymphocytes, and cervical, endometrial, and trophoblast cell lines was reverse transcribed with a 3'-RACE adapter. 3'-RACE polymerase chain reaction was conducted with an upstream human defensin 5 primer and 3'-RACE adapter primer. Polymerase chain reaction products hybridizing to a human defensin 5 probe were cloned for sequence analysis. Sequence data were compared against a nucleotide sequence database, and secondary structure predictions were made. RESULTS: Chorionic tissue, endocervical tissue, endometrial tissue, and an endometrial cell line all demonstrated a single hybridizing 362 bp polymerase chain reaction product. Sequence analysis of all clones demonstrated near-perfect identity with human defensin 5. CONCLUSIONS: Human endocervix, endometrium, and chorion express defensin 5 at the level of transcription. These findings suggest that a previously unrecognized mechanism of protecting female reproductive tissues against infection, by means of a natural antimicrobial system (defensins), may be present.

Amnion↗