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Recent reports have identified defensins as a new type of potassium channel inhibitors; differential binding mechanisms of human β-defensins hBD1 and hBD2 point to complex interactions between human β-defensins and potassium channels. We investigated the inhibitory effects of human defensins hBD3 and hBD4 on potassium channels. The data indicate that hBD3 is a voltage-gated channel subfamily A member 3 (Kv1.3) inhibitor with an IC50 value of 187.6 ± 25.7 nM; 1 μM hBD4 inhibited 34.0 ± 0.2% of Kv1.3 channel currents. Moreover, 1 μM hBD3 inhibited 50.6 ± 3.6% of Kv1.2 channel currents and had smaller effects on Kv1.1, SKCa3, and IKCa channel currents; these effects differed from the Kv1.3 channel-specific inhibitors hBD1 and hBD2. Similar to the pharmacological profiles of hBD1 and hBD2, hBD4 had lower inhibitory effects on Kv1.1, Kv1.2, SKCa3, and IKCa channels. Subsequent mutagenesis and channel activation experiments confirmed that hBD3 binds in a manner similar to that of hBD1, interacting with the outer pore region of the Kv1.3 channel without affecting Kv1.3 channel activation. Thus, the data indicate that the human β-defensin family is a novel group of potassium channel inhibitors with diverse types of human β-defensin-potassium channel interactions.

Antimicrobial peptides (AMPs) are naturally occurring promising candidates which can be used as antibiotics against a wide variety of bacteria. The key component for using them as a potent antibiotic is that their mechanism of action is less prone to bacterial resistance. However, the molecular details of their mechanism of action is not yet fully understood. In this study, we try to shed light on the mode of action of AMPs, possible reason behind it, and their interaction with lipid bilayers through experimental as well as molecular dynamics (MD) simulation studies. The focal of our study was Human beta defensin 3 (hBD-3) which is a naturally occurring AMP. We chose three derivatives of hBD-3, namely CHRG01, KSR, and KLR for the detailed analysis presented in this study. These three peptides are evaluated for their antibacterial potency, secondary structure analysis and mechanism of action. The experimental results reveal that these peptides are active against gram positive as well as gram negative bacteria and kill bacteria by forming membrane pores. The MD simulation results correlate well with the antibacterial activity and shed light into the early membrane insertion dynamics. Moreover, the specific amino acids responsible for membrane disruptions are also identified from the MD simulations. Understanding the molecular level interaction of individual amino acids with the lipid bilayer will greatly help in the design of more efficient antimicrobial peptides.

Respiratory disease is an important cause of morbidity and mortality in cetaceans, which are also threatened by environmental degradation caused by crude oil spills. Following oil spills, cetaceans at the water surface may inhale droplets of oil containing toxic polycyclic aromatic hydrocarbons (PAHs), which could potentially alter respiratory immunity via activation of the aryl hydrocarbon receptor (AHR) and its subsequent interaction with nuclear factor kappa B (NF-κB). β-defensins are antimicrobial peptides secreted by airway epithelial cells and their expression is known to be dependent on NF-κB. We hypothesized that PAHs may suppress the expression of β-defensins, and thereby contribute to the pathogenesis of pneumonia. This hypothesis was modeled by measuring the in vitro effects of benzo(a)pyrene (BAP), phenanthrene, and naphthalene on tracheal antimicrobial peptide (TAP) gene expression in bovine tracheal epithelial cells. Stimulation with lipopolysaccharide (LPS) induced 20 ± 17-fold (mean ± SD) increased TAP gene expression. Exposure of tracheal epithelial cells to 5 μM BAP for 4 or 8 h, followed by incubation with a combination of LPS and 5 μM BAP for another 16 h, significantly (P = 0.002) suppressed LPS-induced TAP gene expression by 40.6 ± 21.8% (mean ± SD) in tracheal epithelial cells from 9 calves tested. BAP-induced suppression of TAP gene expression coincided with induction of cytochrome P450 1A1 gene expression. In contrast, phenanthrene and naphthalene had no consistent effect, and exposure to PAHs did not significantly affect constitutive TAP gene expression (i.e. without LPS). These findings characterize the suppressive effects of BAP—a toxic pollutant found in crude oil—on this respiratory innate immune response.

Objective To investigate the change of Th1/Th2/Th17 cytokines and human beta defensin 2 (HBD-2) in HIV-infected patients with oral candidiasis (OC) and gather information about OC-specific immunity. Design During the 1st year of highly active anti-retroviral therapy (HAART), 25 HIV-infected patients were followed up at the baseline, 3rd, 6th, 12th month. At each visit, oral manifestations were examined; oral rinses were collected and cultured for Candida; peripheral venous blood was taken to determine CD4 + T cell counts and HIV RNA viral load (VL); both unstimulated whole saliva and peripheral venous blood were taken to determine cytokine (IL-4, IL-17(A/F), IFN-γ) and HBD-2 levels. Twenty-five healthy individuals were enrolled as control. Results HIV-infected patients displayed lower levels of IL-17(A/F) and IFN-γ but higher level of IL-4 and HBD-2 compared with healthy controls. During the 1st year of HAART, salivary IL-17(A/F) and IFN-γ were in uptrend, whereas salivary IL-4 and salivary HBD-2 were in downtrend. Serum cytokines all show no significant changes. After 1 year of HAART, serum, salivary IL-4 level and salivary IL-17(A/F) showed no significant difference from healthy controls. HIV-infected patients with OC had a higher IL-4 level but lower IFN-γ and IL-17(A/F) levels than those without OC since the 3rd month of HAART. The occurrence of OC was negatively correlated to IL-17(A/F) and IFN-γ, but positively correlated to IL-4. Salivary HBD-2 expression was up-regulated in HIV and might associate with Candida albicans. Conclusions In HIV-infected patients, the decrease of IL-17 and IFN-γ, and the increase of IL-4 in local and systemic level could influence the prevalence of OC. Salivary HBD-2 may also play an important role against OC.

β-Defensins are cationic antimicrobial peptides showing little sequence similarity but highly conserved tertiary structure stabilized by a six-cysteines-motif. Using a PCR approach, we described β-defensin sequences with two exons in three species of Colubridae snakes with high sequence similarity between them. The deduced amino acid sequence presented the characteristics of β-defensin family. The phylogenetic analysis using β-defensin coding sequences of different snakes grouped them in two main branches: genes organized in three or two exons.

Background The number of intermediate monocytes (CD14++CD16+) increases in many inflammatory conditions. However, it is not yet known which functional markers expressed by these populations are linked to the pathogenesis of psoriasis. Objectives We evaluated the expression of functional markers on circulating intermediate monocytes. Our goal was to correlate specific populations and their markers with the clinical severity of psoriasis. Methods A cohort of 43 psoriatic patients was subjected to analysis. The proportion of intermediate monocytes with CD86 expression was evaluated by flow cytometry. Serum beta defensin-2 levels were measured by ELISA. Immunofluorescent staining was performed in order to identify the presence of CD14+CD16+ cells that co-expressed CD86 in affected skin tissues. Results Upregulated expression of CD86 on the intermediate subset (but not the number of intermediate monocytes) correlated with clinical severity as measured by PASI scores and serum beta defensin-2 levels. Immunostaining also showed the presence of CD86+CD14+CD16+ cells in the epidermis and dermis of psoriatic plaques, which was associated with increased epidermal proliferation. Conclusion These results suggest that the expression of CD86 on circulating intermediate monocytes could be used as an index in clinical practice and provide novel insights into how these cells join a complex immune network under the pathological conditions of psoriasis.

Infectious bronchitis virus (IBV) is an enveloped RNA virus that causes deformities in eggshells. The aim of this study was to investigate the innate immune response to IBV, and to determine whether prostaglandin (PG) E2, which is synthesized during inflammation, is involved in the innate immune response in the uterine mucosa. The effects of intra-oviductal inoculation with attenuated IBV (aIBV) on the expression of viral RNA recognition receptors and innate antiviral factors were examined by real-time PCR and immunohistochemistry, and on PGE2 levels by ELISA. Then, the effects of PGE2 on the expression of innate antiviral factors in cultured uterine mucosal cells were examined. The results showed that the expression of RNA virus pattern recognition receptors (TLR3, 7, and MDA5), antimicrobial peptides (avian β-defensins, including AvBD1, 2, 4-6 and cathelicidins, including CATH1 and 3), and interferons (IFNα, β, γ, λ) were upregulated, and the expression of cyclooxygenase 2 (PG synthase) and the level of PGE2 were increased in the uterine mucosa following aIBV inoculation. The number of AvBD2-positive cells in the mucosa also increased in response to aIBV. In cultured mucosal cells (mainly epithelial), the expression of AvBD4, 10-13 and IFNα, β, and λ was upregulated following incubation with 500 nM PGE2. These results suggest that the expression of viral RNA-recognition receptors, AvBDs, CATHs, and IFNs and PGE2 are induced by the IBV antigen, and that the expression of a different set of AvBDs is also induced by PGE2 in the cultured uterine mucosal cells. These antiviral factors may play a role in the protection of the uterine mucosa from IBV infection.

The present study evaluated whether two different antimicrobial peptides (AMP): porcine beta defensins-1 (PBD1) and −2 (PBD2) at three concentrations (1.5 μM, 3 μM and 5 μM) could be a suitable alternative to antibiotics in liquid-stored boar semen. Two separate experiments were conducted with liquid-stored boar semen preserved at 17 °C for 9–10 days. In the first one, we evaluated the impact of adding three concentrations of each AMP on the bacterial growth and sperm quality of boar semen stored for 10 days. In the second experiment, the ability of these AMPs to control bacterial growth was determined over a 9-day period, following artificial inoculation with Escherichia coli at 107 and 108 CFU mL−1. In both experiments, sperm viability was assessed through flow cytometry, sperm motility was determined with Computer Assisted Sperm Analysis (CASA) and the inhibitory effect on microbial growth was evaluated by bacteria culture on Luria Bertani agar. PBD1 and PBD2 were found to significantly (P

Background β-defensins are an excellent antimicrobial peptide against microbial infection in which dendritic cells (DCs) play a crucial role by improving the innate and adaptive immune defense. However, it is unclear whether BDs affect DC maturation. This work aimed to study the effects of mouse β-defensin-14 (MBD-14) on DC maturation. Methods Via in vitro using mouse bone marrow DCs, the maturation of DCs was evaluated by cell morphological staining, flow cytometry, endocytosis assay, and allogeneic mixed lymphocyte reaction, respectively. And it was also assessed by in vivo establishing a mouse air-pouch model for flow cytometric determination, cytokine analysis, and histological staining. Additionally, CLI-095, an inhibitor of Toll-like receptor-4 (TLR-4), was used to determine whether TLR-4 is possibly involved in DC maturation. Results It was found MBD-14 promoted DCs to form more filopodia and lamellipodia, increased the expression of DC maturation markers (CD40 and MHC-II), decreased their endocytic capacity, and enhanced T-cell proliferation. The analyses of the air-pouch exudates were consistent with the in vitro results of MBD-14 activating DCs. And when CLI-095 was applied, DC maturation was inhibited partly. Conclusions This work demonstrates that MBD-14 can promote the maturation of DCs in which TLR-4 is possibly involved.

Human β-defensins (HBD), an antimicrobial peptide encoded by the (DEFB) gene. In humans they play a significant role in Helicobacter pylori immunological responses to. Four variants in β-defensins 1 and 2 genes (rs1800972 C/T, rs1047031 C/T, rs2740090 C/T and rs2740091 G/A) were included in a H. pylori patients in Iraq (80 Recurrent H. pylori infection and 80 Treated H. pylori patients). To determine these variants, we used polymerase chain reaction with tetra-primers amplification and refractory mutation system. Logistic regression of rs2740090 showed the allele and genotypes has a significant association with Recurrent H. pylori patients' susceptibility. Regarding rs1800972, T allele and CT genotype were significantly associated with an increased possibility of recovery from H. pylori infections, but the highest frequencies was found in Treated H. pylori patients for alleles in rs2740091. For SNP rs1047031, alleles and genotypes no significant difference was found. Tetra-locus haplotype (rs1800972, rs1047031, rs2740090 and rs2740091) revealed that (C-G-C-C and C-G-C-T) significantly elevated the haplotype frequency in Treated H. pylori patients. Moreover, the rs2740090 variant was associated with a Recurrent H. pylori patients' susceptibility. The overall result can lead to final conclusion that some of genetic polymorphisms in immune gene can play a role in drug susceptibility and thus we can predict the right dose and drug for treatment of person with specific genotype.

Dogs with allergies are prone to skin infections and treatments/preventatives to boost innate immune-defenses are beneficial. The aim of this study was to evaluate the effects of Boldo and Meadowsweet extracts on the expression of β-defensins (cBD), cathelicidin (cCath), and pro-inflammatory cytokines in canine keratinocyte. This study had two phases. Phase I evaluated mRNA expression of cBD103 and cCath, and secretion of cCath, IL-8 and TNF-α by keratinocytes harvested from healthy (n=5) and atopic (n=5) age-matched beagles exposed to Boldo (2% to 0.2%) and Meadowsweet (1% to 0.2%) extracts. Phase II focused on atopic keratinocytes (n=14) exposed to 0.2% Boldo, 0.2% Meadowsweet, and a mixture of 0.1% of both extracts. Phase I: cBD103 mRNA (all concentrations) and TNF-α secretion (2% Boldo) were increased in atopic compared with healthy keratinocytes. In atopic keratinocytes, cBD103 was increased after exposure to 1.5% and 0.2% Boldo. In healthy keratinocytes, 1% and 0.2% Meadowsweet, and 2% Boldo increased and decreased IL-8 secretion, respectively. In atopic keratinocytes, IL-8 increased after exposure to 1% and 0.4% Meadowsweet extract. Phase II: cBD103 mRNA increased after exposure to 0.2% Meadowsweet and to 0.1% mixture. cCath was increased after 0.2% Boldo, but decreased after 0.2% Meadowsweet or the 0.1% mixture. TNF-α secretion was decreased after 0.2% Boldo. It is concluded that low concentrations of both extracts and their combination may have some effects on cCath and cBD103 without stimulating an inflammatory response. However, more studies are needed to clarify the effects of these extracts on the local immunity.

Antimicrobial peptides are small peptides that play important roles in a host's innate immune response. As an important antimicrobial peptide, β-defensin widely distribute in mammals, insects and plants with broad-spectrum antimicrobial activity. In this study, the β-defensin gene of the channel catfish, Ictalurus punctatus, was cloned, sequenced, and subjected to a bioinformatic analysis. The β-defensin gene of the channel catfish contains three exons and two introns, and encodes a precursor peptide consisting of two domains: a signal peptide of 24 amino acid residues and a mature peptide of 43 amino acid residues. The mature peptide is estimated to have a molecular mass of 7.1kDa and a theoretical isoelectric point of 8.21. Channel catfish β-defensin (ccBD) has six conserved cysteine residues, forming three disulfide bridges at C1-C5, C2-C4, and C3-C6, and a β-sheet in the predicted three-dimensional structure. A phylogenetic analysis suggests that ccBD belongs to the type 1 β-defensins. Real-time quantitative PCR showed that channel catfish β-defensin transcripts are constitutively expressed in various tissues in healthy fish, with highest expression in the skin. The expression of ccBD in vivo increased significantly in the head kidney (2.9-fold), gill (2.2-fold), and skin (6.6-fold) at 48h after bacterial (Edwardsiella ictaluri) challenge. In vitro, lipopolysaccharide (LPS), a bacterial mimic, induced significant changes in ccBD expression in leukocytes from the spleen (3.4-fold) and head kidney (3.9-fold) 24h after stimulation. Chemically synthesized ccBD displayed marked inhibitory activity against a broad range of bacteria. These results suggest that ccBD is involved in the innate antibacterial defenses of the channel catfish.

Beta_defensin have been solely found in vertebrates until β-defensin-like peptides were described as transcript isoforms in two species of Panulirus genus. They were considered as putative antimicrobials since their biological activity have not been demonstrated. Here we purified and characterized a defensin-like peptide from the hemocytes of spiny lobster P. argus, hereafter named panusin. Structurally, panusin presents a cysteine-stabilized α/β motif, and is prone to form homodimers. Biological activity of panusin showed broad-spectrum antimicrobial activity, characterized for being strikingly salt-resistant. Panusin did not showed hemolytic activity but was demonstrated its binding capacity to different lipid membrane models, indicating amphipathicity of β-sheet core as driving force for its antimicrobial activity. Panusin is considered a new kind of arthropod defensin which share structural and biological features with beta-defensin from vertebrates. The presence of beta-defensin like peptides in crustacean might suggest the emergence of the evolutionary relationship of β-defensins from vertebrates., This work was partially supported by IFS Grant reference No F/5024-1 and F/5024-2, and by the VLIR-UOS Grant reference ZEIN2013Z134

Our previous results indicate that HBD2 and HBD3 are chemotactic for a broad spectrum of leukocytes in a CCR6- and CCR2-dependent manner. In this study we report that pre-stimulation of primary human macrophages or THP-1 cells with HBD2 or HBD3 results in a synergistic, enhanced expression of pro-inflammatory cytokines and chemokines induced by TLR ligand re-stimulation. Experiments using specific inhibitors of the ATP-gated channel receptor P2X7 or its functional ligand ATP, suggest that the enhanced expression of pro-inflammatory cytokines and chemokines seems to be mediated by P2X7R. Furthermore, our data provide evidence that beta-defensins do not directly interact with P2X7R but rather induce the release of intracellular ATP. Interference with ATP release abrogated the synergistic effect mediated by HBD2 and HBD3 pre-stimulation in THP-1 cells. However, extracellular ATP alone seems not to be sufficient to elicit the enhanced synergistic effect on cytokine and chemokine expression observed by pre-stimulation of primary human macrophages or THP-1 cells with HBD2 or HBD3. Collectively, our findings provide new insights into the molecular mechanisms how HBD2 and HBD3 interact with cells of myeloid origin and demonstrate their immuno-modulating functions during innate immune responses.

Made available in DSpace on 2018-12-11T17:02:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-09-01 Objective Controversies exist regarding the relationship between the concentrations of antimicrobial peptides (AMPs) and presence of dental caries in children. Thus, the aim of this study was to examine levels of AMPs in saliva of caries-free (CF), early childhood caries (ECC) and severe early childhood caries (S-ECC) children to determine if the levels of these salivary peptides individually or in combinations were related to caries severity and mutans streptococci levels. Design 36 to 60 month-old children were selected to participate in this study. Children were grouped into CF group (n = 29), ECC group (n = 25) and S-ECC group (n = 29). Saliva was collected from children for microbiological analysis by culture. Salivary concentrations of cathelicidin LL-37, human β-defensin 2 (hBD-2), human β-defensin 3 (hBD-3) and histatin-5 (HTN-5) were determined by ELISA. Results Salivary concentrations of AMPs did not differ among CF, ECC and S-ECC groups. Data showed positive correlations between mutans streptococci levels and salivary hBD-2 or HTN-5. Positive correlations were found between hBD-2, hBD-3, LL-37 and HTN-5. Combinations among AMPs, mainly LL-37, were positively associated with caries levels. Conclusions Salivary concentrations of AMPs individually were not associated with the severity of early childhood caries. The stimulus of caries appears to trigger a biological response, however, with a combination of these peptides. UNESP-Univ. Estadual Paulista Department of Pediatric Dentistry and Public Health Araçatuba Dental School, Rua José Bonifácio, 1193 Forsyth Institute Department of Microbiology Harvard University Harvard School of Dental Medicine UNESP-Univ. Estadual Paulista Department of Pediatric Dentistry and Public Health Araçatuba Dental School, Rua José Bonifácio, 1193

Influenza is a disease of the respiratory system caused by single stranded RNA viruses with varying genotypes. Immunopathogenesis to influenza viruses differs based on virus strain, dose, and mouse strain used in laboratory models. Although effective mucosal immune defenses are important in early host defense against influenza, information on the kinetics of these immune defense mechanisms during the course of influenza infection is limited. We investigated changes to antimicrobial peptides and primary innate immune cells at early time points after infection and compared these variables between two prominent H1N1 influenza A virus (IAV) strains, A/CA/04/2009 and A/PR/08/1934 in C57BL/6 mice. Alveolar and parenchymal macrophage ratios were altered after IAV infection and pro-inflammatory cytokine production in macrophages was induced after IAV infection. Genes encoding antimicrobial peptides, β-defensin (Defb4), bactericidal-permeability increasing protein (Bpifa1), and cathelicidin antimicrobial peptide (Camp), were differentially regulated after IAV infection and the kinetics of Defb4 expression differed in response to each virus strain. Beta-defensin reduced infectivity of A/CA/04/2009 virus but not A/PR/08/1934. Beta defensins also changed the innate immune cell profile wherein mice pre-treated with β-defensin had increased alveolar macrophages and CD103+ dendritic cells, and reduced CD11b+ dendritic cells and neutrophils. In addition to highlighting that immune responses may vary based on influenza virus strain used, our data suggest an important role for antimicrobial peptides in host defense against influenza virus.

Bacterial resistance against antibiotics has become an increasing challenge in the treatment of cutaneous infections. Consequences can be severe, especially in infected wounds following previous local radiotherapy. Certain endogenous peptide antibiotics, the host defence peptides (HDPs), exhibit broad-spectrum antimicrobial activity and promote wound healing. Their use as supplements to conventional antibiotics is a current topic of discussion; however, knowledge of their quantities in healthy and compromised tissue is a prerequisite for such discussion. To date, no data concerning HDP quantities in irradiated skin are available.Expression profiles of the genes encoding HDPs, namely human beta-defensin-1 (DEFB1, hBD-1), beta-defensin-2 (DEFB4A, hBD-2), beta-defensin-3 (DEFB103, hBD-3) and S100A7, were assessed in samples of non-irradiated and irradiated neck.A reduction in the expression of all of the examined genes was observed in irradiated skin when compared with non-irradiated skin (statistically significant in the case of S100A7, P = 0.013). Immunohistochemistry revealed differences in HDP distribution with respect to the epithelial layers.The study demonstrates a significant reduction in HDP gene expression in neck skin as a result of radiotherapy. These findings might represent a starting point for novel treatments of cutaneous infections in irradiated patients, such as topical supplementation of synthetic HDP.

β-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal β-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant β-defensins remain unknown. In addition, the regulation of the immune system by mammalian β-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized β-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 μg/ml was able to reduce the internalization of S. aureus into bMECs (∼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (∼5-fold) and membrane abundance (∼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1β (∼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (∼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of β-defensins from plants as immunomodulators of the mammalian innate immune response.

Defensins are moonlighting peptides which are broadly distributed throughout all the living kingdoms. They play a multitude of important roles in human health and disease, possessing several immunoregulatory functions and manifesting broad antimicrobial activities against viruses, bacteria, and fungi. Based on their patterns of intramolecular disulfide bridges, these small cysteine-rich cationic proteins are divided into three major types, α-, β-, and θ-defensins, with the α- and β-defensins being further subdivided into a number of subtypes. The various roles played by the defensins in the innate (especially mucosal) and adoptive immunities place these polypeptides at the frontiers of the defense against the microbial invasions. Current work analyzes the antimicrobial activities of human and animal defensins in light of their intrinsic disorder propensities.

Atopic dermatitis is a chronic skin condition with complex etiology. It is characterized by skin barrier defects and T helper type 2 (Th2)-polarized inflammation. Although mutations in the filaggrin gene are known to be prominent genetic risk factors for the development of atopic dermatitis, the interdependency between these and an altered cytokine milieu is not fully understood. In this study, we evaluated the direct effects of filaggrin deficiency on the cornified envelope, tight junction proteins, and innate immune response, and report the effects of Th2 cytokines in normal and filaggrin-deficient skin equivalents. Supplementation with IL-4 and IL-13 led to distinct histologic changes and significantly increased skin surface pH, both of which were enhanced in filaggrin knockdown skin equivalents. We detected a compensatory up-regulation of involucrin and occludin in filaggrin-deficient skin that was dramatically disturbed when simultaneous inflammation occurred. Furthermore, we found that a lack of filaggrin triggered an up-regulation of human ?-defensin 2 via an unknown mechanism, which was abolished by Th2 cytokine supplementation. Taken together, these results indicate that defects in the epidermal barrier, skin permeability, and cutaneous innate immune response are not primarily linked to filaggrin deficiency but are rather secondarily induced by Th2 inflammation.

Glucocorticoids (GCs) have been extensively used as the mainstream treatment for chronic inflammatory disorders. The persistent use of steroids in the past decades and the association with secondary infections warrants for detailed investigation into their effects on the innate immune system and the therapeutic outcome. In this study, we analyse the effect of GCs on antimicrobial polypeptide (AMP) expression. We hypothesize that GC related side effects, including secondary infections are a result of compromised innate immune responses. Here, we show that treatment with dexamethasone (Dex) inhibits basal mRNA expression of the following AMPs; human cathelicidin, human beta defensin 1, lysozyme and secretory leukocyte peptidase 1 in the THP-1 monocytic cell-line (THP-1 monocytes). Furthermore, pre-treatment with Dex inhibits vitamin D3 induced cathelicidin expression in THP-1 monocytes, primary monocytes and in the human bronchial epithelial cell line BCi NS 1.1. We also demonstrate that treatment with the glucocorticoid receptor (GR) inhibitor RU486 counteracts Dex mediated down-regulation of basal and vitamin D3 induced cathelicidin expression in THP-1 monocytes. Moreover, we confirmed the anti-inflammatory effect of Dex. Pre-treatment with Dex inhibits dsRNA mimic poly IC induction of the inflammatory chemokine IP10 (CXCL10) and cytokine IL1B mRNA expression in THP-1 monocytes. These results suggest that GCs inhibit innate immune responses, in addition to exerting beneficial anti-inflammatory effects.

Traditionally, calcitriol has been considered a calcium and phosphate regulating hormone, but has recently been shown to play a pivotal role in innate immunity. Many barrier and immune cells have membrane and intracellular receptors that recognize different microbial antigens. Activation of these receptors induces synthesis of 1α-hydroxylase, which acts on 25 hydroxyvitamin D to generate intracellular calcitriol. Calcitriol activates its receptor and enhances the synthesis of important human antibiotics like cathelicidin and β2-defensin while inhibiting hepcidin. These pluripotent peptides have an important role in innate immunity, and their regulation is abnormal in hypovitaminosis D. The literature on their secretion mechanisms, levels in different organic fluids, mechanism of action, and relationship with vitamin D is reviewed here.

Experimental models of bacterial and viral infections in cattle have suggested vitamin D has a role in innate immunity of cattle. The intracrine vitamin D pathway of bovine macrophages, however, has only been shown to activate a nitric oxide-mediated defense mechanism, as opposed to cathelicidin and β-defensin antimicrobial peptides in human macrophages. In this study we have investigated the actions of 1,25-dihydroxyvitamin D3 (1,25D) on a cluster of eleven bovine β-defensin genes on the basis of RNAseq data indicating they were targets of 1,25D in cattle. Treatment of bovine monocyte cultures with 1,25D (10 nM, 18 h) in the absence and presence of LPS stimulation increased the expression of bovine β-defensin 3 (BNBD3), BNBD4, BNBD6, BNBD7, and BNBD10 genes 5 to 10-fold compared to control (P0.05). Treatment of lipopolysaccharide (LPS)-stimulated monocytes with 0-100 ng/mL 25-hydroxyvitamin D3 also increased BNBD3, BNBD4, BNBD7, and BNBD10 in a dose-dependent manner. Treatment of monocytes with the protein translation inhibitor, cycloheximide, however, blocked upregulation of the β-defensins in response to 1,25D suggesting the β-defensins in cattle are not direct targets of the vitamin D receptor. Furthermore, preliminary investigation of vitamin D's contribution to β-defensin expression in vivo revealed that intramammary 1,25D treatment of lactating cows increased BNBD7 expression in mammary macrophages. In conclusion, our data demonstrate that multiple β-defensin genes are upregulated by 1,25D in cattle, providing further indication that vitamin D contributes to bovine innate immunity.

Background Bacterial colonisation with Moraxella catarrhalis may partly sustain chronic inflammation in the lower airways of patients with chronic obstructive pulmonary disease (COPD). In addition, this bacterium causes infectious exacerbations of COPD, which often necessitate treatment with antibiotics. Antimicrobial peptides are the body's own antibiotic substances with bactericidal and bacteriostatic, as well as immunomodulatory function. In particular, human beta-defensin 3 (hBD-3) exerts an antimicrobial effect against an extraordinarily broad spectrum of pathogens. We therefore investigated the role of hBD-3 in infections of pulmonary epithelial cells with M. catarrhalis. Methods The antimicrobial activity of hBD-3 vs. M. catarrhalis was evaluated in an antimicrobial susceptibility assay. We analyzed hBD-3 secretion of M. catarrhalis-infected pulmonary epithelial cells using ELISA. The role of M. catarrhalis-specific virulence factors, toll-like receptors (TLR) 2 and 4, MAPK pathways, and transcription factors AP-1 and NF-κB in the induction and regulation of hBD-3 expression were explored with specific inhibitors, small interference RNA, Western Blot, and chromatin immunoprecipitation (ChIP) assays. Results HBD-3 exhibited a strong bactericidal effect against M. catarrhalis. M. catarrhalis induced hBD-3 expression in pulmonary epithelial cells, which was dependent on M. catarrhalis membranous lipoolygosaccharide (LOS), while the surface proteins UspA1 and UspA2 were not involved. Gene silencing of TLR2, but not TLR4, led to a reduced hBD-3 secretion after stimulation with M. catarrhalis or M. catarrhalis LOS. Inhibition of MAPKs ERK1/2 and JNK, but not p38, reduced hBD-3 secretion. HBD-3 expression was mediated through the recruitment of AP-1 to the hBD-3 gene promoter and was independent of NF-κB. Conclusion The immune response of pulmonary epithelial cells towards M. catarrhalis involves secretion of hBD-3, which has a bactericidal effect against this pathogen. Binding of M. catarrhalis virulence factor LOS to TLR2 causes an ERK1/2- and JNK-dependent induction of AP-1-related transcription of the hBD-3 gene, resulting in the production and secretion of hBD-3.

The gut epithelium is critically involved in maintaining intestinal immune homeostasis. Acting as a physical barrier, it separates the intestinal microflora from cells of the immune system. In addition to its barrier function, the intestinal epithelium expresses defensins, natural, endogenous antimicrobial peptides. In humans, specialized epithelial cells, termed Paneth cells, located primarily in the small intestine express two defensins, Human Defensin-5 (HD-5) and Human Defensin-6 (HD-6). Previously, we have shown that HD-5 potently kills bacteria and induces secretion of interleukin-8 by intestinal epithelial cells. We show that HD-6 specifically and synergistically enhances the HD-5-induced IL-8 secretion, but does not alter its anti-bacterial activity. Further, we find that HD-5 decreases the trans-epithelial electrical resistance of intestinal epithelial cells and that HD-6 negates this effect of HD-5.

In this study we identified mechanisms at the colonic mucosa by which MUC2 mucin regulated the production of β-defensin in a proinflammatory milieu but functionally protected susceptible bacteria from its antimicrobial effects. The regulator role of MUC2 on production of β-defensin 2 in combination with the proinflammatory cytokine interleukin-1β (IL-1β) was confirmed using purified human colonic MUC2 mucin and colonic goblet cells short hairpin RNA (shRNA) silenced for MUC2. In vivo, Muc2(-/-) mice showed impaired β-defensin mRNA expression and peptide localization in the colon as compared with Muc2(+/-) and Muc2(+/+) littermates. Importantly, purified MUC2 mucin abrogated the antimicrobial activity of β-defensin 2 against nonpathogenic and enteropathogenic Escherichia coli. Sodium metaperiodate oxidation of MUC2 removed the capacity of MUC2 to stimulate β-defensin production and MUC2's inhibition of defensin antimicrobial activity. This study highlights that a defective MUC2 mucin barrier, typical in inflammatory bowel diseases, may lead to deficient stimulation of β-defensin 2 and an unbalanced microbiota that favor the growth of β-defensin-resistant microbes such as Clostridium difficile.

Antibiofilm activity of several human defensin analogs that have the ability to kill planktonic bacteria, against pre-established biofilms of Escherichia coli MG1655 and Staphylococcus aureus NCTC 8530 were examined. Linear and linear fatty acylated analogs did not show any activity while disulfide constrained analogs disrupted pre-established S. aureus biofilms. Chimeric analogs of human β-defensin 1 and θ-defensin, hBTD-1 and [d]hBTD-1 were highly active against S. aureus biofilms. Among the analogs tested, only the d-enantiomer [d]hBTD-1 showed activity against E. coli biofilm. Our study provides insights into the structural requirements for the eradication of pre-established biofilms in defensin analogs.

β-defensins (BD) are the largest family of vertebrate defensins with potent antimicrobial, chemotactic and immune-regulatory activities. Four BD genes (BD1-4) have been cloned previously in rainbow trout but none have been reported in other salmonids. In this study seven BD genes (BD1a-b, 2-4, 5a-b) are characterised in Atlantic salmon and additional BD genes (BD1b and BD5) in rainbow trout. Bioinformatic analysis revealed up to seven BD genes in the genomes of other salmonids that belong to five subfamilies (BD1-5) due to whole genome duplications. BD1-2 and BD4-5 are also present in basal teleosts but only BD1 and/or BD5 are present in advanced teleosts due to loss of one chromosomal locus. BD3 is salmonid specific. Fish BD have a unique three-coding exon structure. Fish BD are highly divergent between subfamilies but conserved within each subfamily. Atlantic salmon BD genes are differentially expressed in tissues, often with low level expression in systemic immune organs (head kidney and spleen) yet with at least one BD gene highly expressed in mucosal tissues, heart, blood and liver. This suggests an important role of these BD genes in innate immunity in mucosa, liver and blood in Atlantic salmon.

At present, expressing antimicrobial peptides in bacterial models is considered a routine lab bench work. However, low expression yields of these types of proteins are usually obtained. In this work, the antimicrobial peptide human β-defensin 2 (HBD2) was obtained in low expression yields in Escherichia coli BL21(DE3). To improve the expression yields of HBD2, some variables such as cell density, temperature, and length of induction, as well as the inducer concentration, were investigated using a 24-factorial design of experiments (DoE). This approach allowed us to identify the identification of critical variables (main effects and interactions among factors) affecting bacterial HBD2 expression. After the evaluation of 19 different combination, the best condition to express HBD2 had a pre-induction temperature of 37 °C, a cell density of 1.0 U (600 nm), an induction temperature of 20 °C and a 0.1 mM of gene expression inducer (IPTG) over four hours. Under such conditions, the expression yield of the HBD2 peptide was one order of magnitude higher than the peptide expression performed initially.

Defensins play a vital role in the early stage of infection before adaptive immune responses are generated. Thus far, only limited detailed genomic data for avian defensin genes have been described. Here, using bacterial artificial chromosome libraries, we found that a 95 kb and 177 kb sequences in the golden pheasant (Chrysolophus pictus, Galliformes) and hwamei (Garrulax canorus, Passeriformes) corresponds to 16 and 30 defensin genes, respectively. Fluorescence in situ hybridization assigned defensin gene clusters in the golden pheasant and hwamei to chromosomes 2q and 3q, respectively. In combination with the previous chicken (Gallus gallus, Galliformes) and zebra finch (Taeniopygia guttata, Passeriformes) defensin clusters, the comparative genomic analysis revealed that lineage-specific duplications and deletions have given rise to clearly different genomic structures. On the basis of genomic characteristics, we further found that transposable elements act as agents of evolution, causing direct and indirect copy number variations in defensin genes via duplications. Tissue expression analysis showed that the Passeriformes-specific duplicated AvBD1 and AvBD3 genes are mainly expressed in the upper and lower respiratory tract, tongue, and spleen. Our analyses indicate that the duplication-and-deletion of defensin genes conformed to the birth-and-death evolutionary process and that transposable elements induced the duplication of defensin genes. Moreover, the respiratory system-specific expression pattern of Passeriformes-specific expanded AvBD1 and AvBD3 genes suggests their important role in maintaining the singing trait of songbirds. The understanding of the genomic structure, expression, and evolution of defensin genes can provide a crucial immunological foundation to study and prevent avian diseases.

Defensins are small effector molecules of the innate immune system that are present in almost all organisms including plants and animals. These peptides possess antimicrobial activity against a broad range of microbes including bacteria, fungi and viruses and act as endogenous antibiotics. α-Defensins are a subfamily of the defensin family and their expression is limited to specific tissues. Equine DEFA1 is an enteric α-defensin exclusively secreted by Paneth cells and shows an activity against a broad spectrum of microbes, including typical pathogens of the horse such as Rhodococcus equi, various streptococci strains, Salmonella choleraesuis, and Pasteurella multocida. Here, we report the three-dimensional structure of DEFA1 solved by NMR-spectroscopy and demonstrate its specific function of aggregating various phospholipids.

Chemokines and their receptors are vital for the trafficking of immune cells. In an orchestrated fashion, up- and down-regulation of chemokines and their receptors contribute to both immune system homeostasis as well as inflammation. The CC chemokine, CCL20 and its cognate receptor, CCR6, are described as one of the few chemokine-receptor pairs that show exclusivity. In our review, we analyze observations which indicate that CCR6 does not have CCL20 as an exclusive ligand as once appreciated. For example, attempts to study the pair, utilizing mainly CCR6-deficient mice, are confounded by a family of non-chemokine ligands known as β-defensins that can bind to CCR6 and potentially can activate the cell. Therefore, a review of the activities of other potential binding partners of CCR6 is essential for interpretation of the current literature on this matter and for an understanding of their involvement in basic immunology and pathology.

Human β-defensins play a pivotal role in the innate immune response. Although expressed by and acting at epithelial surfaces, little is known about their specific interaction with epithelial structures. Here, we identify the transmembrane protein CD98 as a cell surface receptor involved in the internalization of human β-defensin 3 (hBD3) in human epithelial A549 cells. CD98 and hBD3 extensively colocalize on the basolateral domain of A549. While verifying their direct binding by fluorescence resonance energy transfer and surface plasmon resonance, we mapped the interaction to CD98 residues 304-414, i.e. to the region known to interact with the proteins of intestinal bacteria during colonic invasion. Treatment of A549 cells with hBD3 dramatically reduces CD98 expression and conversely, knockdown of CD98 expression impairs hBD3 cell surface binding and internalization. Competition for bacterial binding to CD98 and downregulation of CD98 expression may represent novel mechanisms for the antibacterial activity of hBD3 Human β-defensins play a pivotal role in the innate immune response. Although expressed by and acting at epithelial surfaces, little is known about their specific interaction with epithelial structures. Here, we identify the transmembrane protein CD98 as a cell surface receptor involved in the internalization of human β-defensin 3 (hBD3) in human epithelial A549 cells. CD98 and hBD3 extensively colocalize on the basolateral domain of A549. While verifying their direct binding by fluorescence resonance energy transfer and surface plasmon resonance, we mapped the interaction to CD98 residues 304-414, i.e. to the region known to interact with the proteins of intestinal bacteria during colonic invasion. Treatment of A549 cells with hBD3 dramatically reduces CD98 expression and conversely, knockdown of CD98 expression impairs hBD3 cell surface binding and internalization. Competition for bacterial binding to CD98 and downregulation of CD98 expression may represent novel mechanisms for the antibacterial activity of hBD3.

SummaryDefensins are short cationic, amphiphilic, cysteine-rich peptides that constitute the front-line immune defense against various pathogens. In addition to exerting direct antibacterial activities, defensins inactivate several classes of unrelated bacterial exotoxins. To date, no coherent mechanism has been proposed to explain defensins’ enigmatic efficiency toward various toxins. In this study, we showed that binding of neutrophil α-defensin HNP1 to affected bacterial toxins caused their local unfolding, potentiated their thermal melting and precipitation, exposed new regions for proteolysis, and increased susceptibility to collisional quenchers without causing similar effects on tested mammalian structural and enzymatic proteins. Enteric α-defensin HD5 and β-defensin hBD2 shared similar toxin-unfolding effects with HNP1, albeit to different degrees. We propose that protein susceptibility to inactivation by defensins is contingent to their thermolability and conformational plasticity and that defensin-induced unfolding is a key element in the general mechanism of toxin inactivation by human defensins.

Production of the human cathelicidin antimicrobial peptide gene (hCAP18/LL-37), is regulated by 1α,25-dihydroxyvitamin D3 (1,25D3) and is critical in the killing of pathogens by innate immune cells. In addition, secreted LL-37 binds extracellular receptors and modulates the recruitment and activity of both innate and adaptive immune cells. Evidence suggests that during infections activated immune cells locally produce increased levels of 1,25D3 thus increasing production of hCAP18/LL-37. The relative expression levels of hCAP18/LL-37 among different immune cell types are not well characterized. The aim of this study was to determine the relative levels of hCAP18/LL-37 in human peripheral blood immune cells and determine to what extent 1,25D3 increased its expression in peripheral blood-derived cells. We show for the first time, a hierarchy of expression of hCAP18 in freshly isolated cells with low levels in lymphocytes, intermediate levels in monocytes and the highest levels found in neutrophils. In peripheral blood-derived cells, the highest levels of hCAP18 following treatment with 1,25D3 were in macrophages, while comparatively lower levels were found in GM-CSF-derived dendritic cells and osteoclasts. We also tested whether treatment with parathyroid hormone in combination with 1,25D3 would enhance hCAP18 induction as has been reported in skin cells, but we did not find enhancement in any immune cells tested. Our results indicate that hCAP18 is expressed at different levels according to cell type and lineage. Furthermore, potent induction of hCAP18 by 1,25D3 in macrophages and dendritic cells may modulate functions of both innate and adaptive immune cells at sites of infection.

Antimicrobial peptides (AMPs), an important part of the innate immune system, are crucial for defense against invading microorganisms. Whereas AMPs have been extensively studied in adult skin, little is known about the impact of AMPs in the developing human skin. We therefore compared the expression and regulation of AMPs in fetal, neonatal, and adult keratinocytes (KCs) in vitro. The constitutive expression of human β-defensin-2 (HBD-2), HBD-3, S100 protein family members, and cathelicidin was significantly higher in KCs from fetal skin than in KCs from postnatal skin. The capacity to further increase AMP production was comparable between prenatal and postnatal KCs. Analysis of skin equivalents (SEs) revealed a strong constitutive expression of S100 proteins in fetal but not in neonatal and adult SEs. The elevated AMP levels correlated with reduced H3K27me3 (tri-methyl-lysine 27 on histone H3) levels and increased expression of the histone demethylase JMJD3. Knockdown of JMJD3 in fetal KCs elevated H3K27me3 levels and significantly downregulated the expression of HBD-3, S100A7, S100A8, S100A9, and cathelicidin. Our data indicate a crucial contribution of histone modifications in the regulation of AMP expression in the skin during ontogeny. The elevated AMP expression in prenatal skin might represent an essential defense strategy of the unborn.

Defensins are components of the innate immune system that promote the directional migration and activation of dendritic cells, thereby modulating the adaptive immune response. Because matrix glycosaminoglycan (GAG) is known to be important for these functions, we characterized the structural features of human β-defensin 6 (hBD6) and GAG interaction using a combination of structural and in silico analyses. Our results showed that GAG model compounds, a pentasaccharide (fondaparinux, FX) and an octasaccharide heparin derivative (dp8) bind to the α-helix and in the loops between the β2 and β3 strands, inducing the formation of a ternary complex with a 2:1 hBD6:FX stoichiometry. Competition experiments indicated an overlap of GAG and chemokine receptor CCR2 binding sites. An NMR-derived model of the ternary complex revealed that FX interacts with hBD6 along the dimerization interface, primarily contacting the α-helices and β2-β3 loops from each monomer. We further demonstrated that high-pressure NMR spectroscopy could capture an intermediate stage of hBD6-FX interaction, exhibiting features of a cooperative binding mechanism. Collectively, these data suggest a “sandwich-like” model in which two hBD6 molecules bind a single FX chain and provide novel structural insights into how defensin orchestrates leukocyte recruitment through GAG binding and G protein-coupled receptor activation. Despite the similarity to chemokines and hBD2, our data indicate different properties for the hBD6-GAG complex. This work adds significant information to the currently limited data available for the molecular structures and dynamics of defensin carbohydrate binding.

The capability to express an innate antimicrobial action through the production of beta-defensins in the skin of chick embryos has been studied by immunocytochemistry. The immunolabeling of periderm granules present in the initial embryonic epidermis of the chick is obtained with a lizard anti-defensin antibody (Ac-BD-15) that recognizes a homologous peptide in the chick beta-defensin 9. Similar granules, termed honeycomb granules, are also stored in some basophilic granulocytes of adult and hatchling chicks, but did not show immunoreactivity to the antibody. It is unclear whether this morphology indicates different organelles, content or lack of available antigens due to organelle packaging. The remarkable ultrastructural similarity between periderm granules and the honeycomb granules present in basophil granulocytes further indicates that embryonic keratinocytes can synthesize antimicrobial peptides together with corneous proteins. These peptides may participate in the formation of an epidermal microbial barrier during ontogenesis or protect the embryo in case of damage to the amniotic-periderm barrier. These transient organelles, apart from the early keratinization of the epidermis that forms the initial barrier before hatching may also contribute to the formation of an efficient anti-microbial barrier against the penetration of microbes during embryonic life until hatching when the adaptive immunity system is still immature.

Chronic rejection predominantly manifested as bronchiolitis obliterans syndrome (BOS), still remains a major problem affecting long-term outcomes in human lung transplantation (LTx). Donor specific antibodies (DSA) and infiltration of neutrophils in the graft have been associated with the development of BOS. This study determines the role of defensins, produced by neutrophils, and its interaction with α-1-antitrypsin (AAT) towards induction of airway inflammation and fibrosis which are characteristic hallmarks of BOS. Bronchoalveolar lavage (BAL) and serum from LTx recipients, BOS+ (n=28), BOS- (n=26) and normal healthy controls (n=24) were analyzed. Our results show that BOS+ LTx recipients had higher α-defensins (HNP1-3) and β-defensin2 HBD2 concentration in BAL and serum compared to BOS-DSA-recipients and normal controls (p=0.03). BOS+ patients had significantly lower serum AAT along with higher circulating concentration of HNP-AAT complexes in BAL (p=0.05). Stimulation of primary small airway epithelial cells (SAECs) with HNPs induced expression of HBD2, adhesion molecules (ICAM and VCAM), cytokines (IL-6, IL-1β, IL-13, IL-8 and MCP-1) and growth-factor (VEGF and EGF). In contrast, anti-inflammatory cytokine, IL-10 expression decreased 2-fold (p=0.002). HNPs mediated SAEC activation was completely abrogated by AAT. In conclusion, our results demonstrates that neutrophil secretory product, α-defensins, stimulate β-defensin production by SAECs causing upregulation of pro-inflammatory and pro-fibrotic signaling molecules. Hence, chronic stimulation of airway epithelial cells by defensins can lead to inflammation and fibrosis the central events in the development of BOS following LTx.

Defensins are an effector component of the innate immune system with broad antimicrobial activity. Humans express two types of defensins, α- and β-defensins, which have antiviral activity against both enveloped and non-enveloped viruses. The diversity of defensin-sensitive viral species reflects a multitude of antiviral mechanisms. These include direct defensin targeting of viral envelopes, glycoproteins, and capsids in addition to inhibition of viral fusion and post-entry neutralization. Binding and modulation of host cell surface receptors and disruption of intracellular signaling by defensins can also inhibit viral replication. In addition, defensins can function as chemokines to augment and alter adaptive immune responses, revealing an indirect antiviral mechanism. Nonetheless, many questions regarding the antiviral activities of defensins remain. Although significant mechanistic data are known for α-defensins, molecular details for β-defensin inhibition are mostly lacking. Importantly, the role of defensin antiviral activity in vivo has not been addressed due to the lack of a complete defensin knockout model. Overall, the antiviral activity of defensins is well established as are the variety of mechanisms by which defensins achieve this inhibition; however, additional research is needed to fully understand the role of defensins in viral pathogenesis.

Here, we report the characterization of three avian β-defensins (AvBDs) from the goose, named anser_AvBD1, AvBD3, and AvBD6, respectively. All of anser_AvBDs exhibited broad antibacterial activity. In addition, the antibacterial activity of all of the AvBDs against Staphylococcus aureus and Proteus mirabilis decreased significantly in the presence of 100mM NaCl (P

Reports of human infections with highly pathogenic H5N1 avian influenza viruses in many countries in Asia and Africa with varying case fatality rates highlight the pandemic potential of these viruses. In order to contain a rapidly spreading influenza virus in a pandemic scenario, a vaccine which can induce rapid and robust immune responses, preferably in a single dose, is necessary. Murine beta-defensin 2 (Mbd2), a small molecular weight protein expressed by epithelial cells, has been shown to enhance antigen-specific immune responses by recruiting and activating professional antigen presenting cells to the site of vaccination. This study assessed the potential of Mbd2 to enhance the immunogenicity and protective efficacy of a human adenovirus (HAd)-based vaccine expressing the hemagglutinin (HA) and nucleoprotein (NP) [HAd-HA-NP] of an H5N1 influenza virus. A single inoculation of mice with both HAd-HA-NP and a HAd vector expressing Murine β-defensin 2 (HAd-Mbd2) resulted in significantly higher levels of both humoral and cell-mediated immune responses compared to the groups vaccinated only with HAd-HA-NP. These responses were evident even at day 7 post-immunization. Furthermore, the HAd-HA-NP+HAd-Mbd2-immunized group receiving the lowest vector dose (2 × 10(7)+1 × 10(7)) was completely protected against an rgH5N1 virus challenge on day 7 post-vaccination. These results highlight the potential of Mbd2 as a genetic adjuvant in inducing rapid and robust immune responses to a HAd-based vaccine.

β-defensin-4 is a member of antimicrobial peptides (APs) of the immunity system. This molecule has antimicrobial activity but it seems to be involved in articular inflammatory processes too, as it happens during osteoarthritic disease (OA). Considering the possible relation existing between (OA) and temporomandibular disorders (TMD), the aim of our study was to evaluate immunohistochemically the presence of β- defensin-4 in pathological temporomandibular joint (TMJ) discs affected by internal derangement without reduction (ADDwoR).Eighteen TMJ-displaced disc specimens were considered in this study and were analysed by immunohistochemical evaluation. They were compared with a control sample of sixteen healthy discs and two scores, intensity of staining (IS) and extent score (ES) were estimated.Findings of our analysis showed a significant difference between control and study sample (P 0.001). IS and ES of control sample and pathological sample were 1 and 4 respectively.Our results confirmed the presence of β-defensin-4 in human TMJ discs affected by ADDwoR, hypothesing a possible role of this molecule in articular bone disruption.

The resistance of turtle skin to infections is likely due to the production of a thick corneous layer but also of soluble antimicrobial molecules released in the stratum corneum. The present study has determined the localization of beta-defensin peptides previously identified by biomolecular analysis in turtle skin. After the production of a specific antibody for one peptide indicated as Ts-BD-1, light and ultrastructural localization indicates that the peptide is mainly present in some variably dense primary granules of granulocytes. These cells are sparse in the connective tissues and within blood vessels present in the dermis and subdermal inter-muscular connective tissue. A weak labeling is also present in the epidermis, especially localized in sparse secretory granules discharged among corneocytes of the stratum corneum. This localization suggests that antimicrobial peptides are common constituents of the narrow extracellular spaces located among superficial corneocytes where bacteria or their remnants are frequently detected. The study suggests that turtle beta-defensins are mainly contained in granulocytes involved in the immunological surveillance of the skin and that they become active after skin wounding.

Human β-defensin 1 (hBD-1) is an antimicrobial peptide expressed by epithelia and hematopoietic cells. We demonstrated recently that hBD-1 shows activity against enteric commensals and Candida species only after its disulfide bonds have been reduced by thioredoxin (TRX) or a reducing environment. Here we show that besides TRX, glutaredoxin (GRX) is also able to reduce hBD-1, although with far less efficacy. Moreover, living intestinal and lymphoid cells can effectively catalyze reduction of extracellular hBD-1. By chemical inhibition of the TRX system or specific knockdown of TRX, we demonstrate that cell-mediated reduction is largely dependent on TRX. Quantitative PCR in intestinal tissues of healthy controls and inflammatory bowel disease patients revealed altered expression of some, although not all, redox enzymes, especially in ulcerative colitis. Reduced hBD-1 and TRX localize to extracellular colonic mucus, suggesting that secreted or membrane-bound TRX converts hBD-1 to a potent antimicrobial peptide in vivo.

Background: Human milk contains hyaluronan (HA). Results: Milk HA concentration is highest immediately after delivery. Treatment of epithelium with physiologic levels of milk-derived HA increases intracellular expression of β-defensin (in vitro and in vivo) and resistance to Salmonella. Conclusion: Milk HA enhances functional antimicrobial defense mechanisms of the intestinal epithelium. Significance: Milk HA may be a mediator of maternal protection of newborns. Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.