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The aim was to determine whether probiotics-feeding can affect the expression and localization of avian beta defensins (AvBDs) and proinflammatory cytokines in response to Salmonella minnesota lipopolysaccharide (LPS) in the gastrointestinal tract. One-day-old male Chunky broiler chicks were fed with or without 0.4% probiotics for 7 days (P-group and non-P-group, respectively). Then, they were orally challenged with no LPS (0-LPS), 1 µg LPS (1-LPS), or 100 µg LPS (100-LPS) (n = 5, each), in experiment 1, and with no LPS and 1 µg LPS (n = 6, each) in experiment 2. Five hours after LPS challenge, the proventriculi and ceca were collected. A total of seven and eight AvBDs were identified in proventriculus and cecum, respectively. The density of ir-AvBD12 in the surface epithelium of proventriculus increased in the P-group in response to 1-LPS and 100-LPS stimulation. In experiment 1, the expression of two AvBDs in the proventriculus and six AvBDs in the cecum of 1-LPS chicks was higher in P-group than in the non-P-group. Results of experiment 2 showed similar tendency to experiment 1. These results suggest that probiotics-feeding may enhance the immunodefense system mediated by AvBDs but not by cytokine, against infection by Gram-negative bacteria.

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and lung as well as involvement of kidney, gastrointestinal system and heart. Aetiology and exact mechanism of disease is poorly understood. The association between antimicrobial peptides (AMPs) and other diseases such as idiopathic pulmonary fibrosis, diffuse panbronchiolitis, pulmoner alveolar proteinosis and psoriasis have been reported. A small number of studies have examined the role of AMPs on autoimmune diseases which has not been studied in scleroderma yet. We aimed to investigate AMP serum levels and their association with disease characteristics of SSc.MethodsForty-two patients (40 female, mean age 42 years) and 38 healthy subjects (32 female, mean age 38 years) were enrolled. For SSc patients, the following data were recorded: disease subset (limited/diffuse), autoantibodies (antinuclear, anti-centromere (ACA), and anti-SCL-70), blood tests, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), modified Rodnan skin score, presence and history of digital ulcers, kidney, gastrointestinal disease and lung involvement assessed by computed tomography and pulmonary function tests. Association between serum AMPs and disease characteristics were analysed.ResultsTwenty-nine of the patients had diffuse (69%) and 13 of the patients had limited (31%) systemic sclerosis. Average disease duration was 5.5 years. Pulmonary involvement was detected in 20 patients (47.6%). Serum concentration of alpha defensin was higher than healthy subjects (563 ± 415 vs 377 ± 269 ng/mL,p = 0.02). However, no difference was observed for beta-1 and beta-2 defensins in SSc patients and healthy controls. In sub-group analysis patients with interstitial lung disease had higher levels of alpha defensin than those without lung involvement (684 ± 473 vs 430 ± 299 ng/ml,p = 0.04). There was also correlation between alfa defensin serum concentrations and CRP (r = 0.34).ConclusionsAlpha defensin levels are increased in scleroderma patients and correlated with lung involvement indicating a role in the pathogenesis of disease.Trial registrationThis study is not a clinical trial study.

Chronic inflammatory responses can inflict permanent damage to host tissues. Specialized pro-resolving mediators downregulate inflammation but also can have other functions. The aim of this study was to examine whether oral epithelial cells express the receptors FPR2/ALX and DRV1/GPR32, which bind RvD1(n-3 DPA) , a recently described pro-resolving mediator derived from omega-3 docosapentaenoic acid (DPA), and whether RvD1(n-3 DPA) exposure induced significant responses in these cells. Gingival biopsies were stained using antibodies to FPR2/ALX and DRV1/GPR32. Expression of FPR2/ALX and DRV1/GPR32 was examined in primary oral epithelial cells by qRT-PCR, flow cytometry, and immunofluorescence. The effect of RvD1(n-3 DPA) on intracellular calcium mobilization and transcription of beta-defensins 1 and 2, and cathelicidin was evaluated by qRT-PCR. FPR2/ALX and DRV1/GPR32 were expressed by gingival keratinocytes in situ. In cultured oral epithelial cells, FPR2/ALX was detected on the cell surface, whereas FPR2/ALX and DRV1/GPR32 were detected intracellularly. Exposure to RvD1(n-3 DPA) induced intracellular calcium mobilization, FPR2/ALX internalization, DRV1/GPR32 translocation to the nucleus, and significantly increased expression of genes coding for beta-defensin 1, beta-defensin 2, and cathelicidin. This shows that the signal constituted by RvD1(n-3 DPA) is recognized by oral keratinocytes and that this can strengthen the antimicrobial and regulatory potential of the oral epithelium.

Candida glabrata is a common non-albicans Candida species found in patients with candidiasis and it sometimes develops antifungal resistance. Human beta-defensin-3 (hBD-3) is an antimicrobial peptide of immune system active against various types of microbes including Candida spp. This study investigated antifungal activity of hBD-3 and its synergistic effect with a first-line antifungal agent on C. glabrata clinical isolates. Candida spp. were characterised in patients with candidiasis. The antifungal activities of hBD-3 and fluconazole against C. glabrata were evaluated using Broth microdilution assay. The synergistic activity of these two agents was determined by checkerboard microdilution and time-killing assays. The cytotoxicity of hBD-3 was evaluated using LDH-cytotoxicity colorimetric assay. Of 307 episodes from 254 patients diagnosed with candidiasis, C. glabrata was found in 21 clinical isolates. Antifungal susceptibility tests of C. glabrata were performed, fluconazole demonstrated an inhibitory effect at concentrations of 0.25-8 μg/ml, but one antifungal resistant strain was identified (>64 μg/ml). hBD-3 showed an inhibitory effect against all selected strains at concentrations of 50-75 μg/ml and exhibited a synergistic effect with fluconazole at the fractional inhibitory concentration index (FICI) of 0.25-0.50. A concentration of 25 μg/ml of hBD-3 alone showed no cytotoxicity but synergistic activity was seen with fluconazole. In conclusion, hBD-3 has antifungal activity against C. glabrata and synergistic effects with fluconazole at concentrations that alone, have no cytotoxicity. hBD-3 could be used as an adjunctive therapy with first-line antifungal agents for patients with C. glabrata infection particularly those infected with fluconazole-resistant strains.

Aim To profile gingival tissue levels of human beta-defensin (hBD)-2 and hBD-3 in relation to gingival inflammation, Th17-related cytokine concentrations, Porphyromonas gingivalis counts, and gingipain and total protease activities. Materials and Methods Gingival tissue and subgingival plaque samples were collected from 21 periodontitis patients including 48 periodontal pocket sites with marginal, mild, or moderate to severe inflammation. hBD levels were determined by immunodetection, P. gingivalis counts with real-time polymerase chain reaction, protease activities with fluorogenic substrates, and cytokine concentrations with Luminex technique. Data were statistically analysed using Kruskal-Wallis and Mann-Whitney U tests and Spearman correlation coefficients. Results Subgingival plaque counts of P. gingivalis (p = .001) and gingipain activity (p

Objective: Type 2 diabetes mellitus (T2DM) is a well-defined risk factor of periodontitis and it can affect expression of human beta-defensins (hBDs) and cathelicidin (LL-37) as well. The aim of th...

Pseudomonas aeruginosa is a unique microorganism among the antibiotic-resistant microbial pathogens. Among the various therapeutic approaches, antimicrobial peptides are effective to combat this infection. The chimera C3 is a peptide derived from the human beta-defensins 2 and 3 that has previously displayed antibacterial activity against the Gram-negative and Gram-positive bacteria. In this research, the antibacterial activity and the effect of a new genetically designed chimera C3 (i.e. chimera C3-3) on the bacterial membrane was assessed by molecular dynamics (MD) simulation. To investigate the interactions of the peptide with the lipid bilayer model and their effects on each other, the characterizations e.g. Area Per Lipid (APL), density, deuterium order parameter, membrane thickness, Mean Square Displacement (MSD), Radial Distribution Function (RDF), hydrogen bonds (H-bond), root-mean-square-fluctuation (RMSF), bending modulus (KC) and free energy (ΔG) were compared between C3 and C3-3 peptides. Furthermore, the MD simulation of the pure membrane was also carried out. Finally, the comparison of the antibacterial effects showed that chimera C3-3 had the less antibacterial effect than the chimera C3 due to the N-terminal deletion of GII residues. Moreover, the results confirmed that the GII residue played the main role of an anchor in binding the membrane and deletion of anchor reduced the antibacterial activity.

Diabetic wound (DW) is considered as one of the serious complications associated with diabetes mellitus. Though some pharmacological approaches are available for managing DW, none of them has been reported to be very effective. Widely accepted options for its management include treatment of infection caused by various pathogens, wound debridement, reducing the period of the prolonged inflammatory phase, and supervision of the remodeling phase of wound healing. Satisfactory management of DW thus requires exploring new avenues for finding a potential therapeutic strategy. Literature shows that human beta defensins (HBDs) help in combating the insulin resistance by inhibiting the production of glucocorticoids, reducing chronic inflammation by acting through Toll-like receptor signaling pathway, and provoking cell migration, proliferation, angiogenesis, and stabilization of fibroblasts and keratinocytes, ultimately resulting in wound closure. In the present review, beneficial role of HBDs in the treatment of DW is discussed in detail.

Background Human beta defensin-3 (HβD-3) is an antimicrobial peptide present in saliva that protects tooth surfaces from microbial attack. These peptides are part of innate immunity so levels may be affected by different systemic diseases like anemia. Therefore, anemia may predispose an affected child to an increased risk of dental caries. The objectives of this study were to determine the association of early childhood caries (ECC) with HβD-3 levels and observe the association of HβD-3 levels with childhood anemia. Methods A total of 80 children admitted in a pediatric medical ward, age 48–71 months, of either sex were included in the study. The included children were categorized as cases (children with ECC n = 40) and controls (children without ECC n = 40). Children were further segregated into the anemic and non-anemic sub-groups based on the hospital record of hemoglobin level. The salivary concentration of HβD-3 was measured by Enzyme-Linked Immuno-sorbent assay (ELISA). IBM SPSS version 20 software was used for statistical analysis. Two sample t-test and one-way ANOVA were used to compare mean values while spearman was used for correlations at p Results The mean Salivary HβD-3 level in cases (8.87 ± 4.30) was significantly higher (p = 0.042) as compared to controls (7.23 ± 2.57). Salivary HβD-3 level in patients with caries and without anemia was highest (10.80 ± 4.50) whereas salivary HβD-3 level in the presence of caries and anemia was lowest (6.94 ± 3.13) amongst all groups. This difference was statistically significant (p = 0.001). Salivary HβD-3 level was found to be moderately correlated with cases (p = 0.002). An inverse correlation was found between salivary HβD-3 level and anemia (r = -0.479, p = 0.002). Conclusion Anemia may affect the innate immunity of children, and may result in a decreased level of salivary HβD3, thus increasing vulnerability to decay.

The β-defensins are important components of the vertebrate innate immune system. While mammalian β-defensins have wide-ranging antibacterial and immunomodulatory activities, those of amphibians remain largely uncharacterised. In this study, β-defensin cDNA was identified from the skin transcriptome of the Chinese spiny frog Quasipaa spinosa. This β-defensin (QS-BD) consists of a signal and a mature peptide. Sequence alignments with other amphibian β-defensins showed conservation of the functional mature peptide and that its closest relative is β-defensin from Zhangixalus puerensis. Synthetic QS-BD showed antibacterial activity against Vibrio vulnificus, Vibrio harveyi, Streptococcus iniae, and Aeromonas hydrophila. QS-BD showed bactericidal activity by destroying the cell membrane integrity, but did not hydrolyse genomic DNA. QS-BD treatment promoted respiratory bursts and upregulated the expression of interleukin-1β and tumour necrosis factor-α in the murine leukemic monocyte/macrophage cell line RAW264.7. This is the first demonstration of immunomodulatory activity by an amphibian β-defensin.

Human beta defensins (hBDs) may play an important role in the progression of lichen sclerosus (LS), due to their ability to induce excessive stimulation of extracellular matrix synthesis and fibroblast activation. The genetic ability of the individual to produce defensins, the presence of microbes influencing defensin production, and the sensitivity of microbes to defensins together regulate the formation of an ever-changing balance between defensin levels and microbiome composition. We investigated the potential differences in postmenopausal vaginal microbiome composition and vaginal hBD levels in LS patients compared to non-LS controls. LS patients exhibited significantly lower levels of hBD1 (p = 0.0003), and significantly higher levels of hBD2 (p = 0.0359) and hBD3 (p = 0.0002), compared to the control group. The microbiome of the LS patients was dominated by possibly harmful bacteria including Lactobacillus iners, Streptococcus anginosus or Gardnerella vaginalis known to initiate direct or indirect damage by increasing defensin level production. Our observations highlight that correcting the composition of the microbiome may be applicable in supplementary LS therapy by targeting the restoration of the beneficial flora that does not increase hBD2-3 production.

Objectives: Sepsis is a serious medical condition caused by the body’s systemic inflammatory response to infections. The antimicrobial peptides, human beta-defensins, play a key role in modulating host immune responses, and aberrant expression of human beta-defensins has been implicated in many infections and inflammatory diseases. However, little is known about the expression of human beta-defensin-3 in systemic infectious diseases. Methods: We investigated the gene expression and protein level of human beta-defensin-3 in peripheral whole blood from 107 participants—67 patients with sepsis and 40 healthy controls—and evaluated the feasibility of human beta-defensin-3 as an indicator for sepsis. Total RNA was extracted from peripheral blood samples, and relative mRNA expression of human beta-defensin-3 was determined by reverse transcription-quantitative polymerase chain reaction. Plasma concentration of human beta-defensin-3 was measured by enzyme-linked immunosorbent assay. Pearson’s correlation analysis was performed to assess the relationship between human beta-defensin-3 mRNA and protein levels. Receiver operating characteristic analysis was performed to evaluate the value of human beta-defensin-3 as a biomarker for sepsis. Results: Human beta-defensin-3 mRNA expression was significantly downregulated in sepsis patients compared to controls (p = 0.001). The mean fold change of mRNA expression (±standard error) was 0.82 ± 0.63 in sepsis patients and 1.39 ± 1.09 in controls. Plasma concentration of human beta-defensin-3 (pg/mL) was significantly lower in sepsis patients compared to healthy controls (p = 0.039). The mean protein concentration (±standard error) was 539.6 ± 39.4 in sepsis patients and 715.5 ± 53 in controls. There was a significant correlation between human beta-defensin-3 mRNA expression and the corresponding protein level in sepsis patients (r = 0.358, p = 0.04), but not in healthy controls (r = 0.124, p = 0.51). For discriminating sepsis patients from healthy controls, the area under the receiver operating characteristic curve was 0.722 (95% confidence interval: 0.597–0.847, p = 0.002) for human beta-defensin-3 mRNA and 0.689 (95% confidence interval: 0.557–0.827, p = 0.009) for human beta-defensin-3 protein. Conclusion: This is the first study to show the downregulation of human beta-defensin-3 gene expression and protein level in sepsis, which may contribute to the complex immunological imbalance in sepsis. The significant correlation between human beta-defensin-3 mRNA expression and protein concentration suggests that mRNA expression could be used to predict protein level. Our study also showed a potential role of human beta-defensin-3 as a blood-based biomarker for sepsis. More studies on the clinical significance of human beta-defensin-3 in sepsis could further support a biomarker development.

Beta-defensins consist in a group of cysteine-rich antimicrobial peptides (AMPs), widely found throughout vertebrate species, including teleost fish, with antimicrobial and immunomodulatory activities. However, although the European sea bass (Dicentrarchus labrax) is one of the most commercially important farmed fish species in the Mediterranean area, the characterization of its beta-defensins and its potential applications are still missing. In this study, we characterized two members of the beta-defensin family in this species. Phylogenetic and synteny analysis places sea bass peptides in the beta-defensin subfamilies 1 and 2, sharing similar features with the other members, including the six cysteines and the tertiary structure, that consists in three antiparallel beta-sheets, with beta-defensin 1 presenting an extra alpha-helix at the N-terminal. Further studies are necessary to uncover the functions of sea bass beta-defensins, particularly their antimicrobial and immunomodulatory properties, in order to develop novel prophylactic or therapeutic compounds to be used in aquaculture production.

The increasing prevalence of antibiotic-resistance and lack of effective antibiotics pose a serious threat to animal health and public health. Host antimicrobial peptides (AMPs) show broad-spectrum antimicrobial activity against various microbes with low potential for resistance development, compared to conventional antibiotics, indicating great potentials as therapeutic agents. Despite such promise, several limitations hinder the application of AMPs in the clinic, including high manufacturing cost, cytotoxicity, and stability in physiological conditions. New strategies are needed to solve those problems for their application. Avian beta-defensins (AvBD) are small, cationic, antimicrobial peptides. The potential application of AvBDs as antibiotic alternatives against antibiotic-resistant and zoonotic bacterial pathogens has been the subject of interest. In the first study, the biological functions of two AvBDs, AvBD-6 and AvBD-12, were determined under various experimental conditions. The results showed that AvBD-6 (+7) was more potent than AvBD-12 (+1) against E. coli, S. Typhimurium, and S. aureus as well as clinical isolates of extended-spectrum beta-lactamase (ESBL)-producing E. coli and K. pneumoniae. The antibacterial activity of AvBDs was greatly compromised under physiological salt concentrations. Both AvBDs demonstrated mild chemotactic property for chicken macrophages and AvBD-12, at relatively high concentrations, could also induce the migration of murine immature dendritic cells. The chemotactic property required the presence of chemokine receptor 2 (CCR2) on host cells and the conserved disulfide bridges of the peptides. The two AvBDs were nontoxic to CHO-K1, macrophages, or immature dendritic cells.

Sensitive receptors of the olfactory sensory system are located in the nasal cavity mucosa. The aim of this study was to evaluate the human beta-defensin-3 (hBD-3) gene expression in the surface epithelium of the nasal and sinonasal mucosa. Surgical samples from patients with nasal and sinonasal disease (n = 85) (sinus maxillaries mucosa, choana polyps, middle nasal passage polyps, sinus maxillaries polyps, inferior turbinate mucosa of hypertrophic rhinitis, inferior turbinate mucosa and the middle nasal passage mucosa as controls) were investigated. Total RNA was extracted and analysed by real-time RT-PCR for hBD-3 as well as beta-actin mRNA.hBD-3 gene expression was detected in all examined anatomical regions in 14.29-33.33% samples at low levels, but it was absent in the hypertrophic inferior turbinate mucosa (Fisher’s exact test, p < 0.05 compared to the middle nasal passage mucosa; p < 0.01, odds ratio (OR) 31.15, 95% confidence interval (CI) 1.53÷633.6 compared to the middle nasal passage polyps). The highest hBD-3 mRNA expression detection frequency was detected in the middle nasal passage polyps (53.84% cases) (p < 0.05, OR 7.00, CI 1.10÷44.63 compared to the sinus maxillaries mucosa). The highest levels of hBD-3 gene expression was detected in the middle nasal passage polyps also (Wilcoxon signed rank test, p < 0.05 compared to the hypertrophic inferior turbinate mucosa). Clinically, inflammatory polyps are found in the middle turbinate in patients with chronic rhinosinusitis but not in the inferior turbinate. In the context of chronic inflammation, apart from direct antimicrobial activity, high concentrations hBD-3 also potentially contributes to epithelial injury and fibrotic remodeling.

Staphylococcus epidermidis is an abundant skin commensal capable of activating cutaneous defense responses, such as induction of cytokines and antimicrobial peptides. To permanently colonize human skin and prevent inflammation S. epidermidis needs to control the induction of host defense mediators. We report here that S. epidermidis induces expression of the host regulator protein A20 in human keratinocytes, thereby controlling expression and release of interleukin-1 beta. siRNA-mediated knockdown of A20 expression strongly enhanced the induction of interleukin-1 beta gene expression and protein release in keratinocytes stimulated with S. epidermidis. Furthermore, siRNA-mediated knockdown of A20 resulted in enhanced gene expression and secretion of the antimicrobial peptide human beta-defensin-2 in keratinocytes facing S. epidermidis. Mechanistically, A20 negatively controlled S. epidermidis-induced activation of the transcription factor NF-kappaB. Together, these data indicate that S. epidermidis exploits A20 to attenuate cutaneous defense responses, which may help S. epidermidis to persist on human skin.

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.

Background Fecundity is important for farm blue fox (Vulpes lagopus), who with asthenospermia have be a problem in some of farms in China. A key symptom of asthenospermia is decreased sperm motility. The decreased secreting beta-defensin108 (vBD108) of blue fox is speculated be related to asthenospermia. To clarify this idea, the mRNA expression of vBD108 in testis and epididymis of blue foxes with asthenospermia were detected and compared to the healthy one. The antibody was prepared and analyzed by immunohistochemistry. Results The vBD108 in testis and epididymis was found both in blue fox with asthenospermia and healthy group by the method of immunohistochemistry. The expression of vBD108 mRNA in testes (P P Conclusions These results suggested that vBD108 deficiency may related to blue fox asthenospermia. Meanwhile, the study on the blue fox vBD108 provides a hopeful direction to explore the pathogenesis of blue fox asthenospermia in the future.

SARS-CoV-2 coronavirus infection induces heterogeneous symptoms, ranging from asymptomatic to lethal forms. Severe forms usually occur in the elderly and/or individuals with comorbidities. Children generally remain asymptomatic to primary infection, suggesting that they may have an effective local innate immune response. IFN-I and -III have non-redundant protective roles against SARS-CoV-2, although sometimes damaging the host. The expression and role of anti-viral peptides during SARS-CoV-2 infection have thus far been little studied. We aimed to identify the innate immune molecules present at the SARS-CoV-2 entry point. We analyzed the mRNA levels of type I (IFN-α and -β) and type III (IFN-λ1-3) interferons and selected antiviral peptides (i.e., β-defensins 1-3, α-defensins [HNP1-3, HD5] pentraxin-3, surfactant protein D, the cathelicidin LL-37 and interleukin-26) in nasopharyngeal swabs from 226 individuals of various ages, either infected with SARS-CoV-2 (symptomatic or asymptomatic) or negative for the virus. We observed that infection induced selective upregulation of IFN-λ1 expression in pediatric subjects (≤15 years), whereas IFN-α, IFN-β, IFN-λ2/λ3, and β-defensin 1-3 expression was unaffected. Conversely, infection triggered upregulation of IFN-α, IFN-β, IFN-λ2/λ3, and β-defensin 1-3 mRNA expression in adults (15-65 years) and the elderly (≥ 65 years), but without modulation of IFN-λ1. The expression of these innate molecules was not associated with gender or symptoms. Expression of the interferon-stimulated genes IFITM1 and IFITM3 was upregulated in SARS-CoV-2-positive subjects and reached similar levels in the three age groups. Finally, age-related differences in nasopharyngeal innate immunity were also observed in SARS-CoV-2-negative subjects. This study shows that the expression patterns of IFN-I/-III and certain anti-viral molecules in the nasopharyngeal mucosa of SARS-CoV-2-infected subjects differ with age and suggests that susceptibility to SARS-CoV-2 may be related to intrinsic differences in the nature of mucosal anti-viral innate immunity.

Host defense peptides (HDPs) exhibit a broad range of antimicrobial and immunomodulatory activities. In this sense, both functions are like different sides of the same coin. The direct antimicrobial side was discovered first, and widely studied for the development of anti-infective therapies. In contrast, the immunomodulatory side was recognized later and in the last 20 years the interest in this field has been continuously growing. Different to their antimicrobial activities, the immunomodulatory activities of host defense peptides are more effective in vivo. They offer a great opportunity for new therapeutic applications in the fields of anti-infective therapy, chronic inflammatory diseases treatment, novel vaccine adjuvants development and anticancer immunotherapy. These immune related functions of HDPs includes chemoattraction of leukocytes, modulation of inflammation, enhancement of antigen presentation and polarization of adaptive immune responses. Our attempt with this review is to make a careful evaluation of different aspects of the less explored, but attractive immunomodulatory side of the HDP functional coin.

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.

The family of human β-defensins consists of small cysteine-rich peptides, which are receiving significant attention due to their antimicrobial activity. The N-terminal cysteine motif of β-defensin is considered to contribute to its biological activity. Human β-defensin 118 (DEFB 118) is a particular anion β-defensin expressed predominantly in the male reproductive tract, but its physiological activity has not yet been revealed. In order to verify the potential role of the N-terminal domain of DEFB118 peptide in the regulation of infection, the truncated β-defensin core region of DEFB118 peptide was expressed with IMPACT-pTWIN1 system in Escherichia coli. Herein, the purified homogeneous DEFB118 peptide was identified by mass spectrometry and circular dichroism spectroscopy. The in vitro experiments revealed that DEFB118 peptide exhibited prominent LPS-binding potency (KD: 2.94 nM). Moreover, the DEFB118 core peptide significantly inhibited the mRNA level of LPS-induced inflammatory cytokines including IL-α, IL-1β, IL-6 and TNF-α in RAW264.7 cells, and correspondingly decreased secretion of IL-6 and TNF-α. We concluded that strong binding of DEFB118 to LPS might prevent LPS from binding to its receptor, and hence inhibited cytokines secretion. The results of this study may be a benefit to elucidate the immune protection of DEFB118 in the male reproductive tract.

1. Chicken salmonellosis is a common zoonotic infectious disease transmitted both vertically and horizontally. Avian beta-defensins (gallinacins) play an important role in the innate defence of the host and provide broad-spectrum immunity against multiple pathogens. 2. To detect the relationship between immune genes and salmonella carrier status and susceptibility to salmonellosis in chickens, polymorphisms with carrier-state susceptibility to salmonella and, hence, developing salmonellosis, were investigated in three avian beta-defensin genes

How herpesviruses circumvent mucosal defenses to promote infection of new hosts through the respiratory tract remains unknown due to a lack of host-specific model systems. We used the alphaherpesvirus equine herpesvirus type 1 (EHV1) and equine respiratory tissues to decipher this key event in general alphaherpesvirus pathogenesis. In contrast to several respiratory viruses and bacteria, EHV1 resisted potent antimicrobial equine β-defensins (eBDs) eBD2 and eBD3 by the action of glycoprotein M. Instead, eBD2 and -3 facilitated EHV1 particle aggregation and infection of rabbit kidney (RK13) cells. In addition, virion binding to and subsequent infection of respiratory epithelial cells were increased upon preincubation of these cells with eBD1, -2, and -3. Infected cells synthesized eBD2 and -3, promoting further host cell invasion by EHV1. Finally, eBD1, -2, and -3 recruited leukocytes, which are well-known EHV1 dissemination and latency vessels. The exploitation of host innate defenses by herpesviruses during the early phase of host colonization indicates that highly specialized strategies have developed during host-pathogen coevolution., β-Defensins protect the respiratory tract against the myriad of microbial pathogens entering the airways with each breath. However, this potentially hostile environment is known to serve as a portal of entry for herpesviruses. The lack of suitable respiratory model systems has precluded understanding of how herpesvirus virions overcome the abundant mucosal β-defensins during host invasion. We demonstrate how a central alphaherpesvirus, equine herpesvirus type 1 (EHV1), actually exploits β-defensins to invade its host and initiate viral spread. The equine β-defensins (eBDs) eBD1, -2, and -3 were produced and secreted along the upper respiratory tract. Despite the marked antimicrobial action of eBD2 and -3 against many bacterial and viral pathogens, EHV1 virions were resistant to eBDs through the action of the viral glycoprotein M envelope protein. Pretreatment of EHV1 virions with eBD2 and -3 increased the subsequent infection of rabbit kidney (RK13) cells, which was dependent on viral N-linked glycans. eBD2 and -3 also caused the aggregation of EHV1 virions on the cell surface of RK13 cells. Pretreatment of primary equine respiratory epithelial cells (EREC) with eBD1, -2, and -3 resulted in increased EHV1 virion binding to and infection of these cells. EHV1-infected EREC, in turn, showed an increased production of eBD2 and -3 compared to that seen in mock- and influenza virus-infected EREC. In addition, these eBDs attracted leukocytes, which are essential for EHV1 dissemination and which serve as latent infection reservoirs. These novel mechanisms provide new insights into herpesvirus respiratory tract infection and pathogenesis. IMPORTANCE How herpesviruses circumvent mucosal defenses to promote infection of new hosts through the respiratory tract remains unknown due to a lack of host-specific model systems. We used the alphaherpesvirus equine herpesvirus type 1 (EHV1) and equine respiratory tissues to decipher this key event in general alphaherpesvirus pathogenesis. In contrast to several respiratory viruses and bacteria, EHV1 resisted potent antimicrobial equine β-defensins (eBDs) eBD2 and eBD3 by the action of glycoprotein M. Instead, eBD2 and -3 facilitated EHV1 particle aggregation and infection of rabbit kidney (RK13) cells. In addition, virion binding to and subsequent infection of respiratory epithelial cells were increased upon preincubation of these cells with eBD1, -2, and -3. Infected cells synthesized eBD2 and -3, promoting further host cell invasion by EHV1. Finally, eBD1, -2, and -3 recruited leukocytes, which are well-known EHV1 dissemination and latency vessels. The exploitation of host innate defenses by herpesviruses during the early phase of host colonization indicates that highly specialized strategies have developed during host-pathogen coevolution.

Background: β-defensin 118 (DEFB118 ) is a novel host defence peptide (HDP) identified in human. To evaluate its potentials for future utilization, the DEFB118 gene was expressed in Escherichia coli ( E. coli ) and the recombinant protein was fully characterized. Methods: The DEFB118 protein was obtained by heterologous expression using E. coli Rosetta (DE3). Antibacterical activity of DEFB118 were determined by using various bacterial strains. IPEC-J cells challenged by E. coli K88 were used to determine its influences on inflammatory responses. Results: The E. coli transformants yielded more than 250 mg/mL D EFB118 protein after 4 h induction by 1.0 mM IPTG. The DEFB118 was estimated by SDS-PAGE to be 30 kDa, and MALDI-TOF analysis verified it is a human β-defensin 118. Importantly, the DEFB118 showed antimicrobial activities against both Gram-negative bacteria ( E. coli K88 and E. coli DH5α) and Gram-positive bacteria ( S. aureus and B. subtilis ), with a minimum inhibitory concentration (MIC) of 4 μg/mL. Hemolytic assays showed that DEFB118 had no detrimental impact on cell viability. Additionally, DEFB118 was found to elevate the viability of IPEC-J2 cells upon E. coli K88 challenge. Moreover, DEFB118 significantly decreased cell apoptosis in the late apoptosis phase and down-regulated the expression of inflammatory cytokines such as the IL-1β and TNF-a in the IPEC-J2 cells exposure to E. coli K88. Conclusions: These results suggested a novel function of the mammalian defensins, and the anti-bacterial and anti-inflammatory properties of DEFB118 may allow it a potential substitute for conventionally used antibiotics or drugs.

Defensins are short, rapidly evolving, cationic antimicrobial host defence peptides with a repertoire of functions, still incompletely realised, that extends beyond direct microbial killing. They are released or secreted at epithelial surfaces, and in some cases, from immune cells in response to infection and inflammation. Defensins have been described as endogenous alarmins, alerting the body to danger and responding to inflammatory signals by promoting both local innate and adaptive systemic immune responses. However, there is now increasing evidence that they exert variable control on the response to danger; creating a dichotomous response that can suppress inflammation in some circumstances but exacerbate the response to danger and damage in others and, at higher levels, lead to a cytotoxic effect. Focussing in this review on human β-defensins, we discuss the evidence for their functions as proinflammatory, immune activators amplifying the response to infection or damage signals and/or as mediators of resolution of damage, contributing to a return to homeostasis. Finally, we consider their involvement in the development of autoimmune diseases.

Anxiety disorders are the most prevalent psychiatric conditions within primary care, affecting up to 29% of people across their lifetime. Generalized Anxiety disorder (GAD) is frequently comorbid with Major Depressive Disorder (MDD), resulting in greater functional impairment. Gut microbiota have been shown to modulate brain chemistry and function, possibly also playing a role in the genesis of anxiety. Bacteria are also able to produce, or interact with the host metabolism of neuroactive substances, including classical neurotransmitters and trace amines, like octopamine, which although found in trace concentrations in the mammalian brain, can affect CNS function. Specifically, trace amines can affect catecholamine release, reuptake and biosynthesis, and modulate dopamine and serotonin metabolism. We investigated whether microbiota from patients with GAD with no signs of immune activation can alter behaviour in gnotobiotic mice and whether this is accompanied by changes in metabolites within the gastrointestinal tract. Germ-free NIH Swiss mice (n=35) were colonized with microbiota from either a GAD patient (n=18) with severe anxiety, comorbid depression, and low serum and fecal octopamine, or an age and sex-matched healthy control (HC) (n=17). Three weeks post- colonization, mouse behaviour was assessed by standard psychometric tests. Emotionality z-scores were calculated to provide a robust integrated behavioural assessment. Microbiota profiles were assessed by 16S rRNA based Illumina, fecal β-defensin-3 level was measured by ELISA. After sacrifice, mouse brain BDNF and GDNF expression was assessed by immunofluorescence, and gene expression in the hippocampus, amygdala, and olfactory bulbs was assessed by Nanostring. Stool and cecum metabolites were measured in all colonized mice by multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). There were no differences in fecal β-defensin levels between mice colonized with GAD microbiota as compared to mice colonized with HC microbiota. However, GAD mice exhibited greater anxiety and depressive-like behavior compared to HC mice in the digging and tail suspensions tests. Behavioural z-scoring across all six standard psychometric tests showed a significant increase in group emotionality score means of GAD-colonized mice compared to HC-colonized mice. Mice colonized with microbiota from a GAD patient had distinct bacterial profiles from mice colonized with HC microbiota. Compared to HC mice, GAD mice had lower levels of dopamine, octopamine and acetylcholine in cecum contents. Furthermore, GAD mice had higher expression of BDNF in the amygdala, lower expression of BDNF in the hippocampus, and lower expression of GDNF in the midbrain. GAD mice also had lower expression of CCR2 in the hippocampus, higher Cnlp/CAMP in the amygdala and olfactory bulb, and higher Nfkb1 in the olfactory bulb compared to HC mice. Our results suggest that microbiota from a selected patient with GAD has the ability to induce anxiety and depressive-like behavior, by mechanisms independent of immune system, likely by altered production of biogenic amines and neurotransmitters. Thesis Master of Science (MSc)

Background Leptospirosis is a zoonotic disease caused by Leptospira interrogans. Severe leptospirosis is often accompanied by kidney dysfunction caused by chronic infection. The kidney pathology involves bacterial invasion and inflammation caused by pro-inflammatory cytokines. Human beta defensins (hBDs) are antimicrobial peptides induced by microbial infection and/or pro-inflammatory cytokines. One function of hBDs is the recruitment of immune cells that leads to inflammation. However, the expression of hBDs by kidney epithelium in response to pathogenic Leptospira has never been investigated. Objective To determine the expression of hBDs in human kidney epithelium responses to Leptospira. Methods Human kidney cells were infected with Leptospira interrogans serovar Autumnalis in the presence or absence of anti-TLR2 neutralizing antibody (Ab) for 6 hours. TLR2, hBDs and pro-inflammatory cytokines mRNA expressions were analyzed by quantitative polymerase chain reaction (qPCR). Results Pathogenic Leptospira upregulated the expressions of pro-inflammatory cytokines and hBD2, but not TLR2, hBD1 and hBD3 in kidney cells. The expressions of hBD2 and pro-inflammatory cytokines were inhibited in the presence of anti-hTLR2 neutralizing Ab. Conclusions Our results provide the first evidence that pathogenic Leptospira induces hBD2 expression in kidney cells. The expressions of pro-inflammatory cytokines and hBD2 in the cells in response to pathogenic Leptospira are regulated by TLR2. Pro-inflammatory cytokines and hBD2 might be play role in recruitment of immune cells to the kidney and contribute to the development of inflammation-mediated tissue damage in the kidney. However, further study is needed to improve the understanding of the role of these molecules in immune response activation.