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PubMed2003

Intestinal defensin gene expression in human populations.

Defensins are thought to play a major role in the defence of small intestinal crypts against colonisation by potential pathogens. In humans two alpha-defensins, HD5 and HD6 and two beta-defensins, hBD1 and hBD2, probably contribute to the antimicrobial barrier, but there are no data to indicate how the expression of these defensin genes might vary in individuals and in populations. To begin to address this question we developed a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay to quantify HD5 and HD6 mRNA and used it to measure transcripts in small intestinal biopsy tissue from adults living in London, UK, or in Lusaka, Zambia. We also measured alpha- and beta-defensin mRNA in biopsies collected in London from different regions of the small intestine. Jejunal biopsies (n=169) from 83 adults in Lusaka contained approximately one order of magnitude less HD5 and HD6 mRNA than biopsies (n=33) obtained from 27 adults in London. HD5 and HD6 transcript levels were high throughout duodenum, jejunum and ileum. hBD1 and hBD2 mRNA were detected in some, but not all, biopsies from normal small intestine. These data suggest that alpha-defensin expression is down-regulated in tropical populations, and that there are distinct pathways regulating transcription of alpha- and beta-defensins.

Defensins↗
PubMed2002

Defensin-rich granules of human neutrophils: characterization of secretory properties.

The various granule subtypes of the human neutrophil differ in propensity for exocytosis. As a rule, granules formed at late stages of myelopoiesis have a higher secretory potential than granules formed in more immature myeloid cells. Neutrophils contain four closely related alpha-defensins, which are stored in a subset of azurophil granules. These defensin-rich azurophil granules (DRG) are formed later than defensin-poor azurophil granules, near the promyelocyte/myelocyte transition. In order to characterize the secretory properties of DRG, we developed a sensitive and accurate ELISA for detection of the neutrophil alpha-defensins HNP 1-3. This allowed us to quantify the exocytosis of alpha-defensins and markers of azurophil (myeloperoxidase), specific (lactoferrin) and gelatinase (gelatinase) granules from neutrophils stimulated with different secretagogues. The release pattern of alpha-defensins correlated perfectly with the release of myeloperoxidase and showed no resemblance to the exocytosis of lactoferrin or gelatinase. This finding was substantiated through subcellular fractionation experiments. In conclusion, despite a distinct profile of biosynthesis, DRG are indistinguishable from defensin-poor azurophil granules with respect to exocytosis. Thus, in contrast to peroxidase-negative granules, azurophil granules display homogeneity in their availability for extracellular release.

Antibodies↗
PubMed2001

Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae).

We previously purified and determined the partial amino acid sequence of a 4 kDa peptide having high homology with scorpion defensin from the hemolymph of adult fed female soft ticks, Ornithodoros moubata. In this study, the full length sequences of two defensin isoforms were obtained. Deduced amino acid sequences reveal a precursor protein of 73 amino acid residues with a mature portion consisting of 37 amino acid residues. This mature peptide contains six cysteine residues conserved in the same location as other invertebrate defensins. Phylogenetic analysis reveals that Ornithodoros defensin is most closely related to scorpion defensin and other more ancient arthropods. Ornithodoros defensin mRNA is constitutively expressed and up-regulated by blood-feeding and bacterial injection. Ornithodoros defensin gene expression occurs mainly in the midgut. This is the first report of the cloning and gene expression of an antibacterial peptide from the Acari.

Amino Acid Sequence↗
PubMed1999

Levels of human defensin-1, an antimicrobial peptide, in saliva of patients with oral inflammation.

OBJECTIVE: The purpose of this study was to investigate the presence of an antimicrobial peptide, human defensin-1, in the saliva of patients with oral inflammation. STUDY DESIGN: Whole saliva samples were collected from patients with oral inflammation and from healthy volunteers. Human defensin-1 in saliva was isolated and purified by reversed-phase high-performance liquid chromatography. The amino acid sequence and molecular weight of defensin-1 were determined. The concentration of defensin-1 in saliva was quantified by high-performance liquid chromatography. Serum C-reactive protein concentration was measured by particle-enhanced turbidimetric immunoassay. RESULTS: The salivary defensin-1 concentration was significantly higher in patients with oral inflammation than in healthy volunteers; furthermore, in patients with oral inflammation, the concentration was significantly higher before treatment than after treatment. In the patients with oral inflammation, there was a strong positive correlation between salivary defensin-1 concentration and serum C-reactive protein concentration. CONCLUSIONS: The findings suggest that defensin-1 in saliva may be a convenient marker of inflammation associated with oral disease.

Adult↗
PubMed1999

Expression of beta-defensin genes by human salivary glands.

This study investigated expression of genes encoding human beta-defensins 1 and 2 by human salivary glands. Tissues from surgical biopsies were collected fresh onto ice and stored in liquid nitrogen. Total RNA was extracted using Trizol reagent and human beta-defensin messenger RNA detected by reverse transcriptase polymerase chain reaction amplification. DNA sequencing of amplified fragments, after ligation into pGEM-T Easy vector and transformation of competent Escherichia coli, confirmed identities of cloned fragments. Human beta-defensin 1 messenger RNA was detected in all 25 samples that generated amplifiable cDNA, as assessed using abl-specific primers. Three of 13 submandibular gland samples (two normal, one chronically inflamed), and 2 of 2 minor salivary gland samples (one normal, one chronically inflamed) expressed human beta-defensin 2 messenger RNA. All six parotid gland samples studied were negative for human beta-defensin 2 messenger RNA. Thus, human beta-defensin 1 gene expression occurred in all human major and minor salivary glands studied, whereas human beta-defensin 2 expression occurred only in a small number of gland samples.

Defensins↗
PubMed2004

Human beta-defensin 2 is induced by interleukin-1beta in the corneal epithelial cells.

Mammalian epithelia produce the various antimicrobial peptides against the bacterial or viral infection, thereby acting as the active immune modulators in the innate immunity. In this study, we examined the effects of the various proinflammatory cytokines or LPS on cell viability and antimicrobial beta-defensin gene expressions in human corneal epithelial cells. Results showed that the cytokines or LPS did not exert severe cytotoxic effects on the cells, and that beta-defensin 1 was constitutively expressed, while beta-defensin 2 was specifically induced by IL-1beta, supporting the idea that these cytokines or LPS involve the defense mechanism in the cornea. Furthermore, the reporter and gel shift assay to define the induction mechanism of beta-defensin 2 by IL-1beta demonstrated that the most proximal NF-kappaB site on the promoter region of beta-defensin 2 was not critical for the process. Data obtained from the normal or patients with the varying ocular diseases showed that our in vitro results were relevant in the clinical settings. Our results clearly demonstrated that beta-defensin 1 and 2 are important antimicrobial peptides in the corneal tissues, and that the mechanistic induction process of beta-defensin 2 by IL-1beta is not solely dependent on proximal NF-kappaB site activation, thus suggesting that the long distal portion of the promoter is needed for the full responsiveness toward IL-1beta.

Binding, Competitive↗
PubMed1995

Identification of an inhibitor of tissue-type plasminogen activator-mediated fibrinolysis in human neutrophils. A role for defensin.

An inhibitor of tissue-type plasminogen activator (tPA)-mediated and plasminogen-dependent fibrinolysis was isolated from human neutrophils. On a G-50 gel filtration column, the antifibrinolytic activity present in neutrophil homogenates comigrated with proteins of < 13 kDa. The inhibitory fraction had only a slight effect on urokinase with plasminogen- or plasmin-mediated fibrinolysis and no effect on urokinase- or plasmin-mediated cleavage of H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). The neutrophil-derived fraction inhibited tPA with plasminogen activity on S-2251 but not on H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288). The inhibition of tPA-mediated and plasminogen-dependent fibrinolysis or S-2251 cleavage showed a competitive pattern and could be relieved by increasing the concentration of plasminogen. The same fraction also inhibited binding of plasminogen to fibrin. Consecutive purification steps revealed that the molecular mass of the inhibitor was 1-5-kDa. Polylysine-Sepharose affinity chromatography indicated that the inhibitor is a protein of 4 kDa, migrating as one band on SDS-polyacrylamide gel electrophoresis. Amino acid sequence analysis of this band showed the presence of two sequences, differing by one amino acid, which are identical to defensin I and II. Comparison of the sequences of plasminogen and defensin showed homology of defensin to the plasminogen kringles known to contain the lysine binding sites. The close structural similarity between defensin and plasminogen kringles and the ability of defensin to compete with plasminogen on binding to fibrin explain the ability of defensin to inhibit tPA-mediated, plasminogen-dependent fibrinolysis. These results suggest that the antifibrinolytic activity of defensin may have a biological function in preventing the spread of infection.

Amino Acid Sequence↗
PubMed2000

The structure of human beta-defensin-2 shows evidence of higher order oligomerization.

Defensins are small cationic peptides that are crucial components of innate immunity, serving as both antimicrobial agents and chemoattractant molecules. The specific mechanism of antimicrobial activity involves permeabilization of bacterial membranes. It has been postulated that individual monomers oligomerize to form a pore through anionic membranes, although the evidence is only indirect. Here, we report two high resolution x-ray structures of human beta-defensin-2 (hBD2). The phases were experimentally determined by the multiwavelength anomalous diffraction method, utilizing a novel, rapid method of derivatization with halide ions. Although the shape and charge distribution of the monomer are similar to those of other defensins, an additional alpha-helical region makes this protein topologically distinct from the mammalian alpha- and beta-defensin structures reported previously. hBD2 forms dimers topologically distinct from that of human neutrophil peptide-3. The quaternary octameric arrangement of hBD2 is conserved in two crystal forms. These structures provide the first detailed description of dimerization of beta-defensins, and we postulate that the mode of dimerization of hBD2 is representative of other beta-defensins. The structural and electrostatic properties of the hBD2 octamer support an electrostatic charge-based mechanism of membrane permeabilization by beta-defensins, rather than a mechanism based on formation of bilayer-spanning pores.

Amino Acid Sequence↗
PubMed2001

A novel mouse beta-defensin, mBD-6, predominantly expressed in skeletal muscle.

Defensins comprise a family of cationic antimicrobial peptides that is characterized by the conserved 6 cysteine residues. They are expressed in the epithelial cells of various organs and are identified as key elements in the host defense system at the mucosal surface. We isolated a novel mouse beta-defensin gene from the bacterial artificial chromosome DNA containing the mouse beta-defensin-3 gene. The full-length cDNA was cloned from skeletal muscle cDNA and called mouse beta-defensin-6 (mBD-6). The predicted peptide conserved the 6-cysteine motif and had 59% amino acid sequence identity with mouse beta-defensin-3 and 59% identity with mouse beta-defensin-4. We demonstrated the expression of mBD-6 in skeletal muscle in addition to the esophagus, tongue, and trachea. In animal models of endotoxemia, mBD-6 expression was also induced in the lung. mBD-6 showed potent antimicrobial activity against Escherichia coli and would play an important role in host defense in the esophagus, airways, and skeletal muscle. mBD-6 is the first reported beta-defensin predominantly expressed in skeletal muscle. This unique tissue specificity suggests some novel physiological roles of this peptide family.

Amino Acid Sequence↗
PubMed2006

Expression of beta-defensins in the tubotympanum of experimental otitis media.

CONCLUSION: Since the expression levels of beta-defensins 2-4 were up-regulated in experimental otitis media, they may play a protective role in the pathogenesis of otitis media. OBJECTIVES: Defensins are antimicrobial peptides that play a major role in innate immunity. The goal of this study was to identify the expression of defensins in experimental otitis media of the mouse. MATERIALS AND METHODS: The expression of three alpha-defensins (cryptdins, cryptdin-related sequences 1-C (CRS1-C), and CRS4-C) and four beta-defensins (mBD1, mBD2, mBD3, and mBD4) was investigated in the tubotympanum of experimental otitis media in mice by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the expression levels of three beta-defensins (mBD2, mBD3, mBD4) were evaluated by Western blotting. RESULTS: The alpha-defensins were not expressed in the tubotympanum. mBD1 was expressed constitutively in normal middle ear mucosa and Eustachian tube mucosa, but up-regulated expression of mBD2, mBD3, and mBD4 was observed with RT-PCR and Western blotting in the tubotympanums in experimental otitis media, while the normal tubotympanums did not express them.

Animals↗
PubMed2005

Elevated levels of human alpha -defensin in tears of patients with allergic conjunctival disease complicated by corneal lesions: detection by SELDI ProteinChip system and quantification.

PURPOSE: To analyze levels of alpha -defensin in the tears of allergic patients (with/without corneal lesions) comparing the results with those of normal control subjects. METHODS: Screening of the protein profiles of the tears of allergic patients with corneal epithelial lesions and normal controls was performed by surface enhanced laser desorption/ionization (SELDI) ProteinChip array initially. ELISA was then performed to quantify the levels of alpha -defensin in the tears of allergic patients (with/without corneal epithelial lesions) and normal control patients. RESULTS: Proteins expressing significant differences between patients and controls by SELDI analysis were examined. Several peptides with molecular weights similar to alpha -defensins were found to be expressed to a greater extent in allergic patients. ELISA was performed in tears of allergic patients and control subjects to ascertain the presence and increased expression of alpha -defensins in allergic patients. Concentrations of alpha -defensins in allergic patients with corneal epithelial lesions were significantly higher than those of allergic patients without epithelial lesions or normal controls. CONCLUSIONS: Alpha-defensins were found in greater concentrations in tears of allergic patients with corneal lesions. The antimicrobial effects of alpha -defensins may play a role in the prevention of secondary infection of corneal lesions in allergic patients. SELDI ProteinChip technology is a useful and effective tool in profiling the differential expression of proteins in tears.

Conjunctivitis, Allergic↗
PubMed2005

Assessment of the cellular response to the induced expression of defensin sense and antisense cDNA in acute promyelocytic leukemia cell lines.

Defensins are 20-30 amino acid-long, cystine- and arginine-rich peptides that constitute more than 5% of the total cellular proteins in mature granulocytes and at least 30% of proteins in primary granules. Human defensins were reported to have antimicrobial, antifungal, antiviral and tumor lysis activities. Defensin mRNA was isolated using the differential display technique from the well-characterized all-trans retinoic acid (ATRA)-responsive acute promyelocytic leukemia cell line, NB4. The differential display analysis showed an up-regulation of defensin mRNA in NB4 cells after treatment with 10(-7) M ATRA for 24 h. This expression was not seen in an NB4:R2 cell line, an ATRA-resistant subclone of NB4 cells. In order to investigate further the effects of this gene on our cellular model, we virally infected our cells with full-length defensin cDNA in the sense and antisense directions. Sense defensin induced cell growth arrest and cell death in both cell lines. While NB4 cells died within 48-73 h, NB4:R2 cells survived for 96 h before dying in culture. Phenotypic analysis showed high expression of Annexin V in sense-infected cells compared with antisense and uninfected cells in both cell lines. There was not a significant increase in CD11b expression in any of the 2 cell lines used. No cellular response was encountered in antisense-infected cells. Our data suggest that defensin is not only a reliable marker for granulocytic differentiation, but can also be considered a candidate target for molecular therapy in acute promyelocytic leukemia.

Annexin A5↗
PubMed2004

Evidence for duplication of the human defensin gene DEFB4 in chromosomal region 8p22-23 and implications for the analysis of SNP allele distribution.

Defensins constitute a primary mechanism in the innate immune system of humans and all mammals. Defensins are short, processed peptide molecules that are classified by structure into three groups: alpha-defensins, beta-defensins and theta-defensins. In humans, four beta-defensins have been described so far, corresponding to the products of the genes DEFB1 (hBD1, NM_005218), DEFB4 (hBD2, NM_004942.2), DEFB103 (hBD3, NM_018661), and DEFB104 (hBD4, NM_080389), respectively. All these genes have been mapped to chromosome 8p22-23. Much interest has been shown in genetic variation in the population at defensin loci to understand individual differences in disease susceptibility and severity. In this study, we have used an electronic search and then fluorescence in situ hybridization (FISH) on elongated chromosomes to demonstrate that the region containing the DEFB4 gene is duplicated on human chromosome 8p, making difficult the discovery of new SNPs in this gene and compromising the assessment of their allelic distribution in various ethnic populations for disease association studies.

Alleles↗
PubMed2004

alpha-defensins released into stimulated CD8+ T-cell supernatants are likely derived from residual granulocytes within the irradiated allogeneic peripheral blood mononuclear cells used as feeders.

We recently demonstrated the ability of human beta-defensins to inhibit HIV-1 replication in vitro and demonstrated that alpha-defensins account for the great majority of beta-chemokine independent antiretroviral activity in stimulated CD8+ T-cell culture supernatants. In a follow-up study aimed at defining specific subpopulations of CD8+ T-cells that produce alpha-defensins, we have found that in the absence of irradiated allogeneic peripheral blood mononuclear cells (PBMC), stimulated CD8+ T-cell supernatants do not contain alpha-defensins. In our present work, we define residual granulocytes within PBMC fractions as the likely source. In addition, we describe in vitro conditions that promote the internalization of alpha-defensins by cells not natively producing these proteins, thus confounding our ability to define true alpha-defensin producer cells. In light of these findings, alpha-defensins released into stimulated CD8+ T-cell supernatants are unlikely to be derived from the CD8+ T-cells themselves. Moreover, our data imply that under some experimental conditions, a soluble noncytolytic anti-HIV-1 factor other than beta-chemokines is either not produced by CD8+ T-cells or is present in too small quantity to be effective.

Base Sequence↗
PubMed2004

Reassessing the role of defensin in the innate immune response of the mosquito, Aedes aegypti.

Defensin is the predominant inducible immune peptide in Aedes aegypti. In spite of its activity against Gram-positive bacteria in vitro, defensin expression is detected in mosquitoes inoculated with Gram-positive or negative bacteria, or with filarial worms. Defensin transcription and expression are dependent upon bacterial dose; however, translation is inconsistent with transcription because peptide is detectable only in mosquitoes inoculated with large doses. In vitro translation assays provide further evidence for post-transcriptional regulation of defensin. Clearance assays show that a majority of bacteria are cleared before defensin is detected. In gene silencing experiments, no significant difference in mortality was observed between defensin-deficient and control mosquitoes after bacteria inoculation. These studies suggest that defensin may have an alternative function in mosquito immunity.

Aedes↗
PubMed2004

Purification, cDNA cloning and expression of an insect defensin from the great wax moth, Galleria mellonella.

An insect defensin, named Galleria defensin, was purified from the larval haemolymph of Galleria mellonella immunized against E. coli. The peptide was composed of forty-three amino acid residues containing six cysteines that might be engaged in intramolecular disulphide bridges. The primary structure of Galleria defensin shared about 90.7% identity to that of heliomicin, which was an insect defensin isolated from Heliothis virescens. The full-length cDNA encoding Galleria defensin was cloned from the fat body of the immunized G. mellonella larvae. Northern blot analysis revealed that Galleria defensin was expressed not only in the fat body but also in the midgut against invading bacteria into haemocoel. This is the first report presenting cDNA and expression of an insect defensin in the lepidopteran species.

Amino Acid Sequence↗
PubMed2005

A defensin-like gene expressed in the black-legged tick, Ixodes scapularis.

The black-legged tick Ixodes scapularis Linnaeus (Acari: Ixodidae) is an important vector of microbial pathogens. Knowledge of the tick's innate immune response, particularly defensin and other antimicrobial peptides, is important for understanding how microbes survive in this tick. A defensin gene (slnA) from I. scapularis was obtained by reverse transcription-polymerase chain reaction (RT-PCR) using mRNA extracted from tissues of female ticks. RT-PCR indicated the gene was expressed in the midgut, haemocytes, and fat-body, although no evidence of a peptide was found. Sequencing a cloned cDNA fragment revealed a 225 bp open reading frame encoding a 74 amino acid pre-prodefensin, including the putative 38 amino acid mature peptide. Similarity between the defensin amino acid sequences of I. scapularis and Dermacentor variabilis (Say) (Acari: Ixodidae) was 62.2% for the pre-prodefensin region; for the mature defensins from these two species the similarity was 78.9%, with the six cysteine residues being located in the same relative position. PCR amplification and sequencing of chromosomal DNA suggests that slnA, along with vsnA, the defensin gene from D. variabilis, does not contain any introns. This is in contrast to the defensins described for the soft tick, Ornithodoros moubata (sensu Walton) (Acari: Argasidae). The role of defensin in the innate immune response of I. scapularis following microbial invasions is discussed.

Amino Acid Sequence↗
PubMed2006

Human beta-defensin 2 induces a vigorous cytokine response in peripheral blood mononuclear cells.

beta-Defensins are a family of small cationic peptides involved in the innate response to microbial infection. Although their role in microbial killing is well established, the mechanisms through which this occurs remain largely undefined. Here, using protein array technology, we describe a role for human beta-defensins in the induction of an inflammatory cytokine response by human peripheral blood mononuclear cells (PBMCs). Human beta-defensins 1, 2, and 3 were examined for induction of an array of cytokines and chemokines. Some cytokines, such as interleukin 8 (IL-8) and monocyte chemoattractant protein 1, were up-regulated by all three defensins, while others, such as IL-6 and IL-10, were induced more selectively. It was notable that each defensin induced a unique pattern of cytokines. This report documents, for the first time, an analysis of the composite cytokine response of human PBMCs to beta-defensins. The induction or up-regulation of a number of cytokines involved in the adaptive immune response suggests a possible role for these defensins in linking innate and acquired immunity.

Cells, Cultured↗