Β-defensin Research Articles

Evaluation of beta defensin 2 production by chicken heterophils using direct MALDI mass spectrometry

Beta defensins (BD) are cysteine rich, cationic antimicrobial peptides (AMP) produced mainly by epithelial and myeloid cells such as neutrophils. In birds, the neutrophil equivalent heterophils produce avian beta defensins (AvBD) of which AvBD2 is the major isoform. Heterophils recognize pathogens or their derived products through a series of pattern recognition receptors called toll-like receptors (TLR) leading to their antimicrobial activities. This work is the first report of TLR modulation of AvBD2 expression in chickens. To measure the effect of TLR activation on AvBD2 production, the heterophils were cultured with different TLR agonists for 6h. Modulation of AvBD2 levels by TLR activation was measured using direct MALDI mass spectrometry without stable isotopic labeling or chromatographic separation. Chemical modification of the conditioned media was performed using reduction/alkylation with dithiothreitol/iodoacetamide to distinguish TLR treated AvBD2 (reduced/alkylated) from controls (non-reduced). Changes in corrected ion intensity ratios were assumed to reflect AvBD2 modulation in heterophils upon activation with different TLR agonists. In general, TLR agonists increased AvBD2 production with LPS showing the greatest induction and CpG-ODN showing little or no effect. These data show that the direct MALDI-MS coupled with reduction/alkylation may provide a rapid relative quantitative approach to the measurement of agonist-induced differential expression of AvBD2.

Human beta defensin-3 engineered keratinocyte sheets constructed by a magnetic force-based tissue engineering technique

A multilayered keratinocyte sheet overexpressing human beta defensin-3 (HBD-3) gene was prepared by the magnetic force-based tissue engineering technique. The cell sheet demonstrated significant antimicrobial activity, indicating that the therapeutic introduction of HBD-3 gene into cell sheets may provide a new gene therapy strategy for infectious diseases.

Transcriptional profiling avian beta-defensins in chicken oviduct epithelial cells before and after infection with Salmonella enterica serovar Enteritidis

BackgroundSalmonella enterica serovar Enteritidis (SE) colonizes the ovary and oviduct of chickens without causing overt clinical signs which can lead to SE-contamination of the content and membrane of shell-eggs as well as hatchery eggs. The organism utilizes the Salmonella Pathogenicity Island-2 encoded type III secretion system (T3SS-2) to promote persistence in the oviduct of laying hens. In this study, reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out to determine the expression profiles of 14 known avian beta defensins (AvBDs) in primary chicken oviduct epithelial cells (COEC) before and after infections with a wild type SE strain and T3SS mutant SE strains carrying an inactivated sipA or pipB gene.ResultsBased on the expression levels in uninfected COEC, AvBDs can be loosely grouped into three categories with AvBD4-5 and AvBD9-12 being constitutively expressed at high levels; AvBD1, AvBD3, and AvBD13-14 at moderate levels; and AvBD2 and AvBD6-8 at minimal levels. Infection with the wild type SE strain temporarily repressed certain highly expressed AvBDs and induced the expression of minimally expressed AvBDs. The pipB mutant, compared to the wild type strain, had reduced suppressive effect on the expression of highly expressed AvBDs. Moreover, the pipB mutant elicited significantly higher levels of the minimally expressed AvBDs than the wild type SE or the sipA mutant did.ConclusionChicken oviduct epithelial cells express most of the known AvBD genes in response to SE infection. PipB, a T3SS-2 effector protein, plays a role in dampening the β-defensin arm of innate immunity during SE invasion of chicken oviduct epithelium.

Keloid pathogenesis—Do Human Beta Defensins have a role?

Keloid pathogenesis—Do Human Beta Defensins have a role?

The universal detection of antigens from one skin biopsy specimen

Immunohistochemistry is an important tool in dermatology but is limited. Certain antigens can only be preserved in formalin-fixed paraffin-embedded sections, while others can only be detected on frozen sections, resulting in situations where two biopsies are needed. We aimed to develop a technique for universal detection of different antigens out of just one biopsy specimen. Single biopsies were obtained from lesional skin of patients with psoriasis. Standard sample procedures for frozen and paraffin-embedded sections were used. To convert frozen tissue into paraffin-embedded sections, the biopsy specimen was disposed of the embedding medium and subsequently fixed in 10% neutral buffered formalin. We applied various antigen retrieval techniques with alkaline solutions. The differential expression of keratin 10, keratin 15, CD3, CD26 and human beta defensin-2 (HBD-2) was examined using immunohistochemical staining. We showed that keratin 10 and 15 can be stained on both frozen and paraffin-embedded sections. Staining of paraffin-embedded sections required unmasking with trypsin and Tris-buffered saline Tween solution, respectively. CD3 and CD26 can only be detected on frozen sections, while HBD-2 can only be detected on paraffin-embedded sections. We have described a straightforward technique that gives us the opportunity to use just one biopsy specimen to obtain frozen sections as well as paraffin-embedded sections.

Hemoglobin Transcript Abundance in a cDNA Library from Bone Marrow of Crested Ducks (Lophonetta specularioides) in the Peruvian High Andes

ABSTRACT. Hemoglobins play a key role in oxygen transport. High-oxygen-affinity hemoglobins are adaptive in hypoxic environments. To better understand adaptation to high-altitude hypoxia, we extracted RNA from the bone marrow of six Crested Ducks (Lophonetta specularioides) inhabiting the central high Andes of Peru (4,218–4,605 m elevation) and sequenced >2,000 expressed sequence tags (EST) from a non-normalized complementary-DNA (cDNA) library. Overall, we identified 1,692 ESTs in the expression profile representing 462 different genes. Among those, the ESTs that occurred at the highest frequency were the αA (major) hemoglobin subunit (HBA2; 22.7%), leukocyte cell-derived chemotaxin 2 (LECT2; 10%), αD (minor) hemoglobin subunit (HBA1; 9.6%), beta defensins (DEFB; 6.3%), and the βA hemoglobin subunit (HBB; 3.7%). These results provide the first quantitative identification of gene expression in bone marrow of individuals inhabiting high-altitude regions and are in agreement with the known hemopoietic and i...

The role of human beta defensins 2 and 3 in the second trimester amniotic fluid in predicting preterm labor and premature rupture of membranes

Human beta defensins 2 (HBD2) and 3 (HBD3) are peptides expressed in the amnion and chorion. This is a matched case control study conducted in our Department to determine whether second trimester amniotic fluid HBD2 and HBD3 concentrations measured at the time of genetic amniocentesis could be potential markers of preterm labor prediction. Amniotic fluid HBD2 and HBD3 were determined by an enzyme-linked immunosorbent assay (ELISA) Women with preterm labor were defined as cases (N=41) while for each case a woman matched for age delivering at term served as control (N=41). Subgroup analysis was conducted to examine possible associations of HBD2 and HBD3 in cases of premature rupture of membranes. Nineteen women with preterm labor and premature rupture of membranes were defined as cases while for every case a woman matched for maternal age delivering at term served as control (N1=19). Results were presented as odds ratios (OR) and 95% confidence intervals. Statistical analysis used STATA 8.2 and SPSS 11.5 edition. A P-value of <0.05 was considered statistically significant. Amniotic fluid concentrations of HBD2 at the time of genetic amniocentesis were positively associated with preterm premature rupture of membranes (P=0.028), but not with preterm labour. No association of HBD3 and preterm birth was documented. Second trimester amniotic fluid HBD2 might be a predictor of premature rupture of membranes.

Structure, Dynamics, and Activity of an All-Cysteine Mutated Human β Defensin-3 Peptide Analogue

Human beta defensin-3 (HBD-3) is a unique potent antimicrobial peptide. To explore the importance of the three-dimensional structure of HBD-3 in its activity and selectivity, we have mutated all six cysteine residues of HBD-3 to other amino acids, expressed the mutant (named as Def-A) in Escherichia coli, and analyzed the mutant's activity, structure, and dynamics. Def-A is active against several bacterial strains, but the activity is influenced by the ionic strength of the environment. When subjected to vesicles like POPG or to micelles like SDS, Def-A is changed from a random coil structure to an ordered helical form. We have determined the structure of Def-A in SDS micelle and found that it is folded into two distinct helices separated by a proline kink. We propose that the long N-terminal helix with many hydrophobic residues is inserted inside the micelle while the C-terminal helix with one large positive charge patch is located outside the micelle and interacts with the charged head groups of the micelle. The model is supported by NMR relaxation and H-D exchange data. Our results indicate that in addition to the number of positively charged residues and hydrophobic residues, the arrangement of these residues in the three-dimensional space is important to the antimicrobial selectivity and salt-dependent activity of human beta defensins.

Antibiotic resistance in clinical isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus does not impact sensitivity to human beta defensin 4

Antibiotic usage is essential for infection control but hastens emergence of antibiotic resistant microbes. In particular, Acinetobacter baumannii is an important pathogen because of its heightened ability to acquire drug resistance. The need for novel antibacterial agents led us to evaluate the sensitivity of drug-resistant bacteria to the antimicrobial activity of human beta defensin 4 (HBD-4). Clinical isolates of A. baumannii ( N = 14), Pseudomonas aeruginosa ( N = 15), and Staphylococcus aureus ( N = 20), including 10 methicillin-resistant (MRSA) isolates, were examined. All bacterial strains were susceptible to HBD-4 antimicrobial activity, with no correlation between antibiotic resistance and HBD-4 sensitivity. The results demonstrate that antibiotic resistant microorganisms, including MRSA, can be inhibited by HBD-4, which may represent an effective therapeutic agent for infections involving drug-resistant microorganisms.

Convergence of IL-1β and VDR Activation Pathways in Human TLR2/1-Induced Antimicrobial Responses

Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1β and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1β activity, involving the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1β was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.

S1684 Human Beta Defensin 3 Protein Accumulation Is Increased and Redirected in Crohn's Ileitis In Vivo and Modulates T Cell Responses In Vitro

S1684 Human Beta Defensin 3 Protein Accumulation Is Increased and Redirected in Crohn's Ileitis In Vivo and Modulates T Cell Responses In Vitro

Role of beta-dsfensin-3 in lung tissue in a rat model of ventilator-associated pneumonia

Objective To investigate the role of beta-defensin-3 (BD-3) in lung tissue in a rat model of ventilator-associated pneumonia. Methods Twenty pathogen-free male SD rats weighing 240-290 g were randomly divided into 2 groups (n=10 each): non-ventilator-associated pneumonia group (group N) and wentilator-associated pneumonia group (group V). The animals were anesthetized with intraperitoneal (IP) 20% urethane 1.3 g/kg. Oral tracheal intubation was performed under direct vision. In group N the animals kept spontaneous breathing. MRSA suspension (1010 cfu/ml) 0.2 ml was introduced into bronchus immediately after tracheal intubation. The animals were then extubated. In group V the animals were mechanically ventilated (VT 6 ml/kg, RR 88 bpm, PEEP 5 cm H2O, FiO2 21%) for 4 h. MRSA suspension (1010 cfu/ml) 0.2 ml was introduced into bronchus at the end of 4 h mechanical ventilation. The animals were killed at 48 h after inoculation in both groups. The lungs were removed for microscopic examination of pathologic changes which were scored. Wet lung weight/body weight ratio (WW/BW) was measured. Lung bacterial counts and spleen eulture were performed. The expression of BD-3 in lung tissue was detected by immuno-histochemical staining. Results WW/BW, lung pathology scores, lung bacterial counts and the incidence of bacteremia were significantly higher in group V than in group N, while BD-3 expression in lung tissue was significantly lower in group V than in group N. Conclusion The pathogenesis of ventilator-associated pneumonia is related to the down-regulation of BD-3 expression in rat lung tissue. Key words: Respiration, artificial; Pneumonia, baeterial; beta-Defensins

CCR6 Regulation of the Actin Cytoskeleton Orchestrates Human Beta Defensin-2- and CCL20-mediated Restitution of Colonic Epithelial Cells

Intestinal inflammation is exacerbated by defects in the epithelial barrier and subsequent infiltration of microbes and toxins into the underlying mucosa. Production of chemokines and antimicrobial peptides by an intact epithelium provide the first line of defense against invading organisms. In addition to its antimicrobial actions, human beta defensin-2 (HBD2) may also stimulate the migration of dendritic cells through binding the chemokine receptor CCR6. As human colonic epithelium expresses CCR6, we investigated the potential of HBD2 to stimulate intestinal epithelial migration. Using polarized human intestinal Caco2 and T84 cells and non-transformed IEC6 cells, HBD2 was equipotent to CCL20 in stimulating migration. Neutralizing antibodies confirmed HBD2 and CCL20 engagement to CCR6 were sufficient to induce epithelial cell migration. Consistent with restitution, motogenic concentrations of HBD2 and CCL20 did not induce proliferation. Stimulation with those CCR6 ligands leads to calcium mobilization and elevated active RhoA, phosphorylated myosin light chain, and F-actin accumulation. HBD2 and CCL20 were unable to stimulate migration in the presence of either Rho-kinase or phosphoinositide 3-kinase inhibitors or an intracellular calcium chelator. Together, these data indicate that the canonical wound healing regulatory pathway, along with calcium mobilization, regulates CCR6-directed epithelial cell migration. These findings expand the mechanistic role for chemokines and HBD2 in mucosal inflammation to include immunocyte trafficking and killing of microbes with the concomitant activation of restitutive migration and barrier repair.

Ontogeny of mRNA expression of pulmonary innate immune factors

Prematurely born infants are at an increased risk of developing respiratory infections due in part to decreased function of their innate immune system. It was our goal to examine the ontogeny of innate immune gene expression by respiratory epithelia in our ovine model. Targets of interest are surfactant proteins A and D (SP‐A and SP‐D), sheep beta defensin 1 (SBD‐1), dual oxidases 1 and 2 (Duox1 and Duox2), lactoperoxidase (LPO), SPLUNC1, LPLUNC2, Toll‐like receptors 3, 7, and 8 (TLR 3, 7, and 8). Lung from sheep was collected at 115 and 130 days gestation, full term, 15 days post partum, and adulthood. Levels of mRNA for the target innate immune genes were measured by real‐time RT‐qPCR. LPO, SPLUNC1, LPLUNC2, TLR‐8, SBD‐1, and Duox1 expression increased progressively with age. Surfactant proteins A and D, previously reported to be decreased in preterm lambs, did not follow a steady trend, although SP‐A expression was lower at 130 days gestation than at full term. Duox2 levels were lower in gestational and perinatal lambs than in adults, but did not show a linear increase with age. These results are significant in that they may indicate that reduced expression in fetal lung may underlie susceptibility of the lung to infection with preterm birth. Conversely, upregulation of these genes may have therapeutic potential.

Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1

Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins—18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue “demidefensin” precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.

Differences in virulence and immune response induced in a murine model by isolates ofMycobacterium ulceransfrom different geographic areas

Buruli ulcer (BU) is the third most common mycobacterial disease in immunocompetent hosts. BU is caused by Mycobacterium ulcerans, which produces skin ulcers and necrosis at the site of infection. The principal virulence factor of M. ulcerans is a polyketide-derived macrolide named mycolactone, which has cytotoxic and immunosuppressive activities. We determined the severity of inflammation, histopathology and bacillary loads in the subcutaneous footpad tissue of BALB/c mice infected with 11 different M. ulcerans isolates from diverse geographical areas. Strains from Africa (Benin, Ghana, Ivory Coast) induced the highest inflammation, necrosis and bacillary loads, whereas the strains collected from Australia, Asia (Japan, Malaysia, New Guinea), Europe (France) and America (Mexico) induced mild inflammation. Subsequently, animals were infected with the strain that exhibited the highest (Benin) or lowest (Mexico) level of virulence in order to analyse the local immune response generated. The Mexican strain, which does not produce mycolactone, induced a predominantly T helper type 1 (Th1) cytokine profile with constant high expression of the anti-microbial peptides beta defensins 3 and 4, in co-existence with low expression of the anti-inflammatory cytokines interleukin (IL)-10, IL-4 and transforming growth factor (TGF)-beta. The highly virulent strain from Benin which produces mycolactone A/B induced the opposite pattern. Thus, different local immune responses were found depending on the infecting M. ulcerans strain.

Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1 (133.49)

Abstract Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins - 18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature stop codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature stop codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. Since retrocyclins are broadly antiviral, the ability to reawaken retrocyclin genes from their 7 million years of slumber could provide a novel way to secure enhanced resistance to several major viral pathogens. AI052017; AI065430

Are defensin beta 1 gene polymorphisms associated with HIV infection and virus replication?

Are defensin beta 1 gene polymorphisms associated with HIV infection and virus replication?

Response to Segat et al. ‘Are DEFB1 gene polymorphisms associated with HIV-1 infection and virus replication?’

Response to Segat et al. ‘Are DEFB1 gene polymorphisms associated with HIV-1 infection and virus replication?’

High-Level Expression and Novel Antifungal Activity of Mouse Beta Defensin-1 Mature Peptide in Escherichia coli

Mouse beta defensin-1 (mBD-1) is a cationic 37-amino acid antimicrobial peptide with three conserved cysterine disulfied bonds. It exhibits a broad antimicrobial spectrum, but mBD-1 against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) is poorly understood. This study describes the mBD-1 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released mature mBD-1. By constructing the expression plasmid (pET32a-mBD1), high yields of soluble mBD-1 fusion protein (0.67 g/L) could be obtained in E. coli and cleaved by enterokinase. The digested product was further purified and desalted with the final amount of pure mature mBD-1 being 0.14 g/L. Classical fungi growth inhibition assay showed clear antifungal activity against C. albicans and C. neoformans with IC(50) of 5 and 2 microM, respectively. The results show that the mBD-1 control fungal colonization through hyphal induction, direct fungicidal activity, and the activity is suppressed by increasing NaCl concentration. Successful expression of the mBD-1 peptide in E. coli offers a basis for further studying its antifungal mechanisms and may provide significance in developing this peptide to an antifungal drug.