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Human defensins are cysteine-rich peptides (Cys-rich peptides) of the innate immune system. Defensins contain an ancestral structural motif (i.e., γ-core motif) associated with the antimicrobial activity of natural Cys-rich peptides. In this study, low concentrations of human α- and ß-defensins showed microbicidal activity that was not associated with cell membrane permeabilization. The cell death pathway was similar to that previously described for human lactoferrin, also an immunoprotein containing a γ-core motif. The common features were (1) cell death not related to plasma membrane (PM) disruption, (2) the inhibition of microbicidal activity via extracellular potassium, (3) the influence of cellular respiration on microbicidal activity, and (4) the influence of intracellular pH on bactericidal activity. In addition, in yeast, we also observed (1) partial K+-efflux mediated via Tok1p K+-channels, (2) the essential role of mitochondrial ATP synthase in cell death, (3) the increment of intracellular ATP, (4) plasma membrane depolarization, and (5) the inhibition of external acidification mediated via PM Pma1p H+-ATPase. Similar features were also observed with BM2, an antifungal peptide that inhibits Pma1p H+-ATPase, showing that the above coincident characteristics were a consequence of PM H+-ATPase inhibition. These findings suggest, for the first time, that human defensins inhibit PM H+-ATPases at physiological concentrations, and that the subsequent cytosolic acidification is responsible for the in vitro microbicidal activity. This mechanism of action is shared with human lactoferrin and probably other antimicrobial peptides containing γ-core motifs.

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Atopic dermatitis (AD) is a frequent inflammatory condition that usually begins during early childhood, but it increasingly starts to debut, even in the elderly. Based on immunoglobulin E (IgE) levels and clinical features, two subsets of this disease have been recognized: intrinsic and extrinsic. When speaking about AD, most specialists think about filaggrin (FLG) mutations resulting in epidermal barrier defects, which is the case in most atopic patients, but some have a normal barrier, as seen by imaging, and still have specific clinical lesions along with metal allergies. Specific molecules (IL-10, IFN-γ, and HBD-3) have been shown to greatly impact the interactions between internal and external factors in this peculiar form of AD. A less-known protein, suprabasin, has been highlighted as a promising explanation for nickel anomalies in intrinsic AD.

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There is an unmet clinical need for a non-invasive and cost-effective test for oral squamous cell carcinoma (OSCC) that informs clinicians when a biopsy is warranted. Human beta-defensin 3 (hBD-3), an epithelial cell-derived anti-microbial peptide, is pro-tumorigenic and overexpressed in early-stage OSCC compared to hBD-2. We validate this expression dichotomy in carcinoma in situ and OSCC lesions using immunofluorescence microscopy and flow cytometry. The proportion of hBD-3/hBD-2 levels in non-invasively collected lesional cells compared to contralateral normal cells, obtained by ELISA, generates the beta-defensin index (BDI). Proof-of-principle and blinded discovery studies demonstrate that BDI discriminates OSCC from benign lesions. A multi-center validation study shows sensitivity and specificity values of 98.2% (95% confidence interval [CI] 90.3-99.9) and 82.6% (95% CI 68.6-92.2), respectively. A proof-of-principle study shows that BDI is adaptable to a point-of-care assay using microfluidics. We propose that BDI may fulfill a major unmet need in low-socioeconomic countries where pathology services are lacking.

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The use of immune compounds as antimicrobial adjuvants is a classic idea recovering timeliness in the current antibiotic resistance scenario. However, the activity of certain antimicrobial peptides against ESKAPE Gram-negatives has not been sufficiently investigated. The objective of this study was to determine the activities of human defensins HNP-1 and hBD-3 alone or combined with permeabilizing/peptidoglycan-targeting agents against clinical ESKAPE Gram-negatives [Acinetobacter baumannii (AB), Enterobacter cloacae (EC), Klebsiella pneumoniae (KP), and acute/chronic Pseudomonas aeruginosa (PA)]. Lethal concentrations (LCs) of HNP-1 and hBD-3 were determined in four collections of multidrug resistant EC, AB, KP, and PA clinical strains (10-36 isolates depending on the collection). These defensins act through membrane permeabilization plus peptidoglycan building blockade, enabling that alterations in peptidoglycan recycling may increase their activity, which is why different recycling-defective mutants were also included. Combinations with physiological lysozyme and subinhibitory colistin for bactericidal activities determination, and with meropenem for minimum inhibitory concentrations (MICs), were also assessed. HNP-1 showed undetectable activity (LC > 32 mg/L for all strains). hBD-3 showed appreciable activities: LC ranges 2-16, 8-8, 8->32, and 8->32 mg/L for AB, EC, KP, and PA, being PA strains from cystic fibrosis significantly more resistant than acute origin ones. None of the peptidoglycan recycling-defective mutants showed greater susceptibility to HNP-1/hBD-3. Combination with colistin or lysozyme did not change their bactericidal power, and virtually neither did meropenem + hBD-3 compared to meropenem MICs. This is the first study comparatively analyzing the HNP-1/hBD-3 activities against the ESKAPE Gram-negatives, and demonstrates interesting bactericidal capacities of hBD-3 mostly against AB and EC. IMPORTANCE: In the current scenario of critical need for new antimicrobials against multidrug-resistant bacteria, all options must be considered, including classic ideas such as the use of purified immune compounds. However, information regarding the activity of certain human defensins against ESKAPE Gram-negatives was incomplete. This is the first study comparatively assessing the in vitro activity of two membrane-permeabilizing/peptidoglycan construction-blocking defensins (HNP-1 and hBD-3) against relevant clinical collections of ESKAPE Gram-negatives, alone or in combination with permeabilizers, additional peptidoglycan-targeting attacks, or the blockade of its recycling. Our data suggest that hBD-3 has a notable bactericidal activity against multidrug-resistant Acinetobacter baumannii and Enterobacter cloacae strains that should be considered as potential adjuvant option. Our results suggest for the first time an increased resistance of Pseudomonas aeruginosa strains from chronic infection compared to acute origin ones, and provide new clues about the predominant mode of action of hBD-3 against Gram-negatives (permeabilization rather than peptidoglycan-targeting).

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Asthma, a common pediatric pulmonary disease, significantly affects children's healthy development. This study aimed to investigate the functions of human ß defensin-3 (HBD-3) in asthma progression. For this purpose, blood samples from asthmatic and healthy children were collected. Moreover, the airway smooth muscle cells (ASMCs) were treated with platelet-derived growth factor BB (PDGF-BB) to develop an in vitro asthma model, then evaluated cell viability and migration via CCK-8 and transwell assays. The mRNA levels of interferon γ (INF-γ), interleukin 4 (IL-4), interleukin 10 (IL-10), alpha-smooth muscle actin (α-SMA), HBD-3, and the protein levels of phosphatidylinositol 3-kinase (PI3K) along with protein kinase B (AKT) were detected. Similarly, the N6-methyladenosine (m6A) content in the ASMCs and m6A levels of HBD-3 were also measured. Results indicated an upregulated HBD-3 in the asthmatic children. The ASMCs were found to be stimulated by PDGF-BB, in addition to the promotion of cell viability and migration. The INF-γ, IL-4, and α-SMA levels were reduced, while IL-10 was elevated in PDGF-BB-stimulated ASMCs. Silencing HBD-3 in PDGF-BB stimulated ASMCs was found to exert the opposite effect by inhibiting cell viability and migration, enhancing the levels of INF-γ, IL-4, and α-SMA, while the IL-10 levels were found to decline. PDGF-BB stimulation of ASMCs resulted in activation of the PI3K/AKT signaling pathway, which was blocked post HBD-3 silencing, while the role of si-hBD in PDGF-BB stimulated ASMCs was neutralized post-treatment with IGF-1. Finally, it was found that METTL3 overexpression prominently upregulated the m6A levels of HBD-3 and decreased the mRNA expression and stability of HBD-3 in the PDGF-BB-stimulated ASMCs. The study concluded that METTL3-mediated HBD-3 participates in the progression of asthma through the PI3K/AKT signaling pathway.

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It is still a challenge to prevent the formation of bacterial biofilms on the surfaces of oral implants. A chemical peptide with binding and antibacterial properties may be a promising agent if used to modify titanium (Ti) surfaces to inhibit biofilm formation. In this study, peptides were designed by linking the antimicrobial sequence derived from human ß-defensin-3 (hBD-3) to the Ti-binding peptide-1 (TBP-1) sequence by using a triple glycine (G) linker. The antimicrobial activity and biocompatibility characteristics of the chemical-peptide-modified Ti surface were then evaluated and the potential antibacterial mechanism was investigated. This study demonstrated that the chemical-peptide-modified surface exhibited satisfactory bactericidal activities against Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis. In addition to its potent bacteria-killing efficacy, the surface-immobilised chemical peptide also demonstrated excellent biocompatibility to L929 cells. Moreover, the disruption of the integrity of the bacterial membrane partially revealed the antibacterial mechanism of the peptide. This study demonstrated the potential of chemical-peptide-modified Ti surfaces for preventing the occurrence of peri-implant diseases, thereby providing a promising approach to improving the survival rate of oral implants.

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Sepsis is a life-threatening condition characterized by a dysregulated host response to infection. Early and accurate diagnosis of sepsis is crucial for timely intervention and improved patient outcomes. In recent years, there has been growing interest in identifying reliable biomarkers to aid in the diagnosis of sepsis. This study aims to evaluate the levels of two potential biomarkers, high-mobility group box 1 (HMGB1) and human ß-defensin 3 (HBD-3), and compare their diagnostic efficacy in sepsis. We aimed to assess HMGB-1 and HBD-3 levels in sepsis and assess the combined diagnostic validity of HMGB-1 and HBD-3. In this case-control study, the plasma concentration of HMGB-1 and HBD-3 was measured using an enzyme-linked immunosorbent assay (ELISA). Two groups, totaling 144 people, were formed; 66 patients treated in the ICU for sepsis were included in the patient group. 78 Blood donors from the Salmaniya Medical Complex Blood Bank who had no prior infection or inflammatory disease made up the Control group. The statistical computations were performed using the STATA 8® statistical software tool (StataCorp LP, College Station, TX, USA). In patients' mean HMGB-1 levels were 2.1442 ng/ml, compared to 0.62141 ng/ml in the control group. The mean HBD-3 level was 1068.453 ng/ml in sepsis patients versus 589.935 ng/ml in controls. A significant difference between the two groups has been observed in both biomarkers (P < 0.05). The sensitivity of HMGB-1 was 75.8% and 41.3%, respectively. The sensitivity and specificity of HBD-3 were 63.6% and 93.5%, respectively. The levels of HMGB-1 and HBD-3 between healthy and septic subjects varied significantly. HMGB-1 and HBD-3 levels in the blood tested together might accurately identify sepsis. These findings contribute to the growing body of evidence supporting the utility of biomarkers in sepsis diagnosis, and may ultimately aid in the development of more effective diagnostic strategies for sepsis management.

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Antibiotic resistance often confers a fitness cost to the resistant cell and thus raises key questions of how resistance is maintained in the absence of antibiotics and, if lost, whether cells are genetically primed for re-evolving resistance. To address these questions, we have examined vancomycin-intermediate Staphylococcus aureus (VISA) strains that arise during vancomycin therapy. VISA strains harbor a broad spectrum of mutations, and they are known to be unstable both in patients and in the laboratory. Here, we show that loss of resistance in VISA strains is correlated with a fitness increase and is attributed to adaptive mutations, leaving the initial VISA-adaptive mutations intact. Importantly, upon a second exposure to vancomycin, such revertants evolve significantly faster to become VISA, and they reach higher resistance levels than vancomycin-naive cells. Further, we find that sub-lethal concentrations of vancomycin stabilize the VISA phenotype, as do the human ß-defensin 3 (hBD-3) and the bacteriocin nisin that both, like vancomycin, bind to the peptidoglycan building block, lipid II. Thus, factors binding lipid II may stabilize VISA both in vivo and in vitro, and in case resistance is lost, mutations remain that predispose to resistance development. These findings may explain why VISA infections often are re-occurring and suggest that previous vancomycin adaptation should be considered a risk factor when deciding on antimicrobial chemotherapy.

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Background: Leprosy, a chronic infectious peripheral neuropathy, is caused by Mycobacterium leprae. This bacterium produces triacylated lipopeptides that can induce the immune system via the Toll-like receptor 2/1 (TLR 2/1) complex. Activation of TLR 2/1 produces proinflammatory cytokines and antimicrobial peptides (AMPs), including human beta-defensin-3 (HBD-3) and cathelicidin. Purpose: To analyze differences in gene expression of HBD-3 and cathelicidin in the skin of leprosy patients, household contacts, and healthy individuals. Patients and Methods: An analytic observational study was conducted at the Outpatient Clinic of Dermatology and Venereology of Dr Mohammad Hoesin General Hospital, Palembang, Indonesia, from January 2021 to June 2022. In each group of 18 subjects, 72 samples were collected, including skin lesion in leprosy patients, normal skin in leprosy patients, household contacts, and healthy individuals. A comparison of HBD-3 and cathelicidin gene expression between the four groups was analyzed using Pearson Chi Square, Kruskal-Wallis, and Mann-Whitney Test. Results: The median value of HBD-3 gene expression on skin lesion in leprosy patients was 260.61 (0.19-3734.10); normal skin in leprosy patients was 1.91 (0.01-151.17); household contacts skin was 7.93 (0.27-121.10); and healthy individuals' skin was 1.00 (1.00-1.00) is highly significant difference (p < 0.0001). The median value of cathelicidin gene expression on skin lesion in leprosy patients was 38.72 (0.28-1852.17); normal skin in leprosy patients was 0.48 (0.01-15.83); household contacts skin was 9.8 (0.04-128.0); and healthy individual skin was 1.00 (1.00-1.00), also highly significant difference (p < 0.0001). Conclusion: Gene expression of HBD-3 and cathelicidin increased in skin lesions of leprosy patients and household contacts.

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Enormous efforts in recent past two decades to eradicate the pathogen that has been prevalent in half of the world's population have been problematic. The biofilm formed by Helicobacter pylori provides resistance towards innate immune cells, various combinatorial antibiotics, and human antimicrobial peptides, despite the fact that these all are potent enough to eradicate it in vitro. Biofilm provides the opportunity to secrete various virulence factors that strengthen the interaction between host and pathogen helping in evading the innate immune system and ultimately leading to persistence. To our knowledge, this review is the first of its kind to explain briefly the journey of H. pylori starting with the chemotaxis, the mechanism for selecting the site for colonization, the stress faced by the pathogen, and various adaptations to evade these stress conditions by forming biofilm and the morphological changes acquired by the pathogen in mature biofilm. Furthermore, we have explained the human GI tract antimicrobial peptides and the reason behind the failure of these AMPs, and how encapsulation of Pexiganan-A(MSI-78A) in a chitosan microsphere increases the efficiency of eradication.

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West Nile virus (WNV) is an emerging flavivirus transmitted through mosquito bites and responsible for a wide range of clinical manifestations. Following their inoculation within the skin, flaviviruses replicate in keratinocytes of the epidermis, inducing an innate immune response including the production of antimicrobial peptides (AMPs). Among them, the cathelicidin LL-37 and the human beta-defensin (hBD)-3 are known for their antimicrobial and immunomodulatory properties. We assessed their role during WNV infection of human primary keratinocytes. LL-37 reduced the viral load in the supernatant of infected keratinocytes and of the titer of a viral inoculum incubated in the presence of the peptide, suggesting a direct antiviral effect of this AMP. Conversely, WNV replication was not inhibited by hBD-3. The two peptides then demonstrated immunomodulatory properties whether in the context of keratinocyte stimulation by poly(I:C) or infection by WNV, but not alone. This study demonstrates the immunostimulatory properties of these two skin AMPs at the initial site of WNV replication and the ability of LL-37 to directly inactivate West Nile viral infectious particles. The results provide new information on the multiple functions of these two peptides and underline the potential of AMPs as new antiviral strategies in the fight against flaviviral infections.

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The multi-bacterial environment of the oral cavity makes it hard for periodontal regeneration. As a class of antimicrobial peptide, beta defensin has been found to show broad-spectrum antibacterial ability. In addition, connective tissue growth factor (CTGF) is demonstrated to play a great role in multi-physiological events such as angiogenesis, wound healing and, more importantly, fibrogenesis. In this study, human ß defensin 3 (hBD3) and CTGF were co-transfected into bone marrow derived mesenchymal stem cells (BMSCs) for preparing cell sheets. The transfection efficiency was detected through fluorescence of eGFP and western blot assay. Our results showed that the hBD3 and CTGF proteins were highly and stably expressed in the BMSCs after transfection. The results of RT-PCR and induced differentiation indicated that hBD3 promoted osteogenic differentiation of BMSCs, while CTGF significantly increased fibrogenic differentiation even in the presence of hBD3. The BMSCs acquired stronger capacity in terms of promoting M2 polarization of RAW 264.7 macrophages fulfilled by the transfection and secretion of hBD3 and CTGF. To further evaluate the periodontal remodeling performance of cell sheets, a coralline hydroxyapatite (CHA)-chitosan based hydrogel-human tooth system was designed to simulate the natural periodontal environment. The results showed that dense extracellular matrix, oriented fiber arrangement, and abundant collagen deposition appeared in the area of BMSCs sheets after subcutaneous transplantation. Altogether, our data showed that the lentivirus transfected BMSCs sheets had a promising application prospect for periodontal repair.

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BACKGROUND: Nanomaterials of biomedicine and tissue engineering have been proposed for the treatment of periodontitis in recent years. This study aimed to investigate the effects of gold nanoparticles (AuNPs) combined with human ß-defensin 3 (hBD3) on the repair of the alveolar bones of experimental periodontitis in rats. METHODS: A model of experimental periodontitis was established by ligation of the maxillary second molars with silk thread in rats, which were treated with or without AuNPs combined with hBD3. Micro-computerized tomography (micro-CT) scanning, enzyme-linked immunosorbent assay, and histological and immunohistochemical staining, including alkaline phosphatase (ALP), osteoprotegerin (OPG), tartrate-resistant acid phosphatase (TRAP), and receptor activator of NF-κB ligand (RANKL), were used to analyze the samples. RESULTS: Micro-CT demonstrated that the alveolar bone resorption was significantly reduced after the treatment with AuNPs combined with hBD3. Levels of TNF-α and IL-6 were decreased markedly compared with the ligation group. H&E and Masson staining showed that AuNPs combined with hBD3 group had less inflammatory cell infiltration, collagen fibrosis and fracture, but higher calcification in the new bone tissue. Moreover, the administration of AuNPs combined with hBD3 increased the expression levels of ALP and OPG (related to bone formation) while decreasing the expression levels of TRAP and RANKL (related to bone resorption) expression. CONCLUSIONS: AuNPs combined with hBD3 had a protective effect on the progression of experimental periodontitis in rats and played a certain role in suppressing osteoclastogenesis and alleviating the inflammatory destruction of periodontitis along with the promotion of bone repair.

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Mannheimia haemolytica-induced bovine respiratory disease causes loss of millions of dollars to Canadian cattle industry. Current antimicrobials are proving to be ineffective and leave residues in meat. Antimicrobial peptides (AMPs) may be effective against M. haemolytica while minimizing the risk of drug residues. Cationic AMPs can kill bacteria through interactions with the anionic bacterial membrane. Human ß-Defensin 3 (HBD3) and microcin J25 (MccJ25) are AMPs with potent activity against many Gram-negative bacteria. We tested the microbicidal activity of wild-type HBD3, three HBD3 peptide analogues (28 amino acid, 20AA, and 10AA) derived from the sequence of natural HBD3, and MccJ25 in vitro against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis. Since AMPs can act as chemoattractant we tested the chemotactic effect of HBD3, 28AA, 20AA, and 10AA peptides on bovine neutrophils in Boyden chamber. Minimum bactericidal concentration (MBC) assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogues with an MBC of 50 µg/mL. The 10AA analogue had MBC 6.3 µg/mL which is likely a result of lower final inoculum size. MccJ25 didn't have significant bactericidal effect below an MBC < 100 µg/mL. Bovine neutrophils showed chemotaxis towards HBD3 and 20AA peptides (P < 0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). The data show that these peptides are effective against M. haemolytica and are chemotactic for neutrophils in vitro.

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BACKGROUND: The global problem of antibiotic resistance requires the search for and development of new methods of treatment. One of the promising strategies is the use of low doses of antimicrobial peptides, in particular, human defensins HNP-1, hBD-1, and hBD-3, in combination with antibacterial drugs already used in clinical practice. This approach may be used to increase the effectiveness of conventional antibiotics. However, this requires thorough study of the effectiveness of defensins in combination with antibiotics against a large number of bacterial strains with known phenotypes of antibiotic resistance. The aim of this work was to study the antibacterial effect of HNP-1, hBD-1 and hBD-3 in combination with rifampicin or amikacin against clinical isolates of Staphylococcus aureus (n = 27) and Escherichia coli (n = 24) collected from hospitalized patients. METHODS: The standard checkerboard assay was used to determine minimum inhibitory concentrations (MICs) of antimicrobials. The combined microbicidal effects of two substances (defensin + conventional antibiotic) were assessed by the fractional inhibitory concentration index (FICI). RESULTS: The highest anti-staphylococcal activity (including methicillin-resistant strains) among defensins was demonstrated by hBD-3 that had MIC of 1 (0.5-4) mg/L (hereinafter, MIC values are presented as median and interquartile range). The MIC of HNP-1 against S. aureus was 4 (2-8) mg/L; the MIC of hBD-1 was 8 (4-8) mg/L. Against E. coli, the most effective was also found to be hBD-3 that had MIC of 4 (4-8) mg/L; the MIC of HNP-1 was 12 (4-32) mg/L. The combinations of HNP-1 + rifampicin and hBD-3 + rifampicin demonstrated synergistic effects against S. aureus. Against E. coli, combinations of HNP-1 + amikacin and hBD-3 + amikacin also showed synergy of action.

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The antimicrobial peptide human Beta defensin 3 (hBD3) is an essential part of the innate immune system and is involved in protection against respiratory pathogens by specifically permeabilizing bacterial membranes. The Gram-positive bacterium Streptococcus pneumoniae causes serious diseases including pneumonia, meningitis, and septicemia, despite being frequently exposed to human defense molecules, including hBD3 during colonization and infection. Thus, the question arises how pneumococci adapt to stress caused by antimicrobial peptides. We addressed this subject by analyzing the proteome of S. pneumoniae after treatment with hBD3 and compared our data with the proteomic changes induced by LL-37, another crucial antimicrobial peptide present in the human respiratory tract. As antimicrobial peptides usually cause membrane perturbations, the response to the membrane active cationic detergent cetyltrimethylammonium bromide (CTAB) was examined to assess the specificity of the pneumococcal response to antimicrobial peptides. In brief, hBD3 and LL-37 induce a similar response in pneumococci and especially, changes in proteins with annotated transporter and virulence function have been identified. However, LL-37 causes changes in the abundance of cell surface modification proteins that cannot be observed after treatment with hBD3. Interestingly, CTAB induces unique proteomic changes in S. pneumoniae. Though, the detergent seems to activate a two-component system that is also activated in response to antimicrobial peptide stress (TCS 05). Overall, our data represent a novel resource on pneumococcal adaptation to specific cell surface stresses on a functional level. This knowledge can potentially be used to develop strategies to circumvent pneumococcal resistance to antimicrobial peptides.

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Antimicrobial peptides are considered to be one of the candidate antimicrobial agents for antibiotic-resistant bacterial infection in the future. The effects of antimicrobial peptide hBD3-CBD on Pseudomonas aeruginosa PA14 and PA14 ΔexsA were analyzed by the bactericidal effects, hemolysis assays, pyocyanin pigment productions, and virulence factor expressions (exoU, exoS, hcnA, and lasB). Pyocyanin production and virulence factor expressions are important features of the type III secretion system in Pseudomonas aeruginosa. HBD3-CBD killed PA14 and PA14 ΔexsA with similar efficiency; it lowered the hemolysis levels of PA14 and PA14 ΔexsA and reduced the pyocyanin production, biofilm formation, and exoU, exoS, and lasB expressions in PA14. Compared with PA14, PA14 ΔexsA showed a lower hemolysis effect, pyocyanin production, exoU, and lasB expressions. The effects of hBD3-CBD on the PA14 toxin secretion were similar to the changes in the type III secretion system mutant isolate PA14 ΔexsA. Our results demonstrated that the type III secretion system was involved in the biological functions on PA 14 from hBD3-CBD.

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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 < .001), as well as interleukin (IL)-1ß (p = .012), IL-10 (p = .024), IL-17A (p = .002), IL-17F (p = .006), and IL-23 (p = .036) concentrations were elevated in severely inflamed sites, whereas no change was observed in hBD-2 and hBD-3 levels. Negative correlations were found between protease activity and hBD-2 (p = .033) and hBD-3(p = .003) levels. CONCLUSIONS: Shift in gingival inflammation from marginal to mild stage is related to elevations in subgingival plaque P. gingivalis counts and gingipain activity, but not to tissue hBD levels. Negative correlations between hBDs and total protease activity suggest the degradation of these antimicrobial peptides in progressed inflammation.

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@#Introduction: Dental caries in children is a major problem of mouth disease throughout the world, so too is there currently an increase in health problems in children due to obesity. Human Beta defensing(HBD) has been found in saliva and from several studies stated that HBD aside from being a broad-spectrum antimicrobial can act as an immunomodulator. The purpose of this study is to reveal whether there is a relationship between obesity and HBD-3 salivary concentration in caries patients and caries-free patients. Methods: This cross-sectional observational study was involved 62 children with caries and caries-free, aged 9-11 years, students at Qommarudin Islamic Boarding School, Gresik, East Java Indonesia. dental caries examination, carried out in accordance with World Health Organization (WHO) diagnostic criteria. Body mass index (BMI) was measured from the height and weight of individuals, HBD3 concentrations were tested with an ELISA kit from Bioassay Technology Laboratory (China) from saliva samples. Evaluate the results with the Kruskal Wallis test, followed by the Mann-Whitney test. The level of significance used in this statistical test was 0.05. Results: there was a relationship between BMI level and HBD-3 concentration in the caries group (p <0.05, p = 0.009) with a moderate level of association. but there was no significant relationship in the caries-free group (p> 0.05, p = 0.189). Conclusion: There was an association between BMI and HBD-3 salivary concentration in caries patients but there was no relationship in the caries-free group.

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Human ß-defensin 3 (HBD3) is an antimicrobial peptide up-regulated in the oral tissues of individuals with head and neck squamous cell carcinomas (HNSCC) and oral squamous cell carcinomas (SCC) and present in high concentrations in their saliva. In this study, we determined if HBD3 contributes to HNSCC pathogenesis by inducing programmed death-ligand 1 (PD-L1) expression on HNSCC cell lines. For this, SCC cell lines SCC4, SCC15, SCC19, SCC25, and SCC99 (5.0 × 104 viable cells) were used. Cells were incubated with IFNγ (0.6 µM) and HBD3 (0.2, 2.0, or 20.0 µM) for 24 h. Cells alone served as controls. Cells were then treated with anti-human APC-CD274 (PD-L1) and Live/Dead Fixable Green Dead Cell Stain. Cells treated with an isotype antibody and cells alone served as controls. All cell suspensions were analyzed in a LSR II Violet Flow Cytometer. Cytometric data was analyzed using FlowJo software. Treatment with IFNγ (0.6 µM) increased the number of cells expressing PD-L1 (p < 0.05) with respect to controls. Treatment with HBD3 (20.0 µM) also increased the number of cells expressing PD-L1 (p < 0.05) with respect to controls. However, treatment with IFNγ (0.6 µM) was not significantly different from treatment with HBD3 (20.0 µM) and the numbers of cells expressing PD-L1 were similar (p = 1). Thus, HBD3 increases the number of cells expressing PD-L1. This is a novel concept, but the role HBD3 contributes to HNSCC pathogenesis by inducing PD-L1 expression in tumors will have to be determined.