Human Β-defensin Research Articles

Extracellular Vesicles of Commensal Skin Microbiota Alleviate Cutaneous Inflammation in Atopic Dermatitis Mouse Model by Re-Establishing Skin Homeostasis

Atopic dermatitis (AD) is a chronic inflammatory cutaneous disorder in which the skin is affected by microbial dysbiosis. The role of commensal skin microbiota in AD is of great interest. Extracellular vesicles (EVs) are important regulators of skin homeostasis and pathology. The mechanism of preventing AD pathogenesis through commensal skin microbiota-derived EVs remains poorly understood. In this study, we investigated the role of commensal skin bacterium Staphylococcus epidermidis-derived EVs (SE-EVs). We showed that SE-EVs significantly decreased the expression of proinflammatory genes (TNFα, IL1β, IL6, IL8, and iNOS) through lipoteichoic acid and increased the proliferation and migration of calcipotriene (MC903)-treated HaCaT cells. Furthermore, SE-EVs increased the expression of human β-defensins 2 and 3 in MC903-treated HaCaT cells through toll-like receptor 2, enhancing resistance to S.aureus growth. In addition, topical SE-EV application remarkably attenuated inflammatory cell infiltration (CD4+ T cells and Gr1+ cells), T helper 2 cytokine gene expression (Il4, Il13, and Tlsp), and IgE levels in MC903-induced AD-like dermatitis mice. Intriguingly, SE-EVs induced IL-17A+ CD8+ T-cell accumulation in the epidermis, which may represent heterologous protection. Taken together, our findings showed that SE-EVs reduced AD-like skin inflammation in mice and may potentially be a bioactive nanocarrier for the treatment of AD.

A Healthy Vaginal Microbiota Remains Stable during Oral Probiotic Supplementation: A Randomised Controlled Trial.

The primary objective of this randomised, placebo-controlled, triple-blind study was to assess whether orally consumed Lactobacillus acidophilus La-14 (La-14) and Lacticaseibacillus rhamnosus HN001 (HN001) colonise a healthy human vagina. Furthermore, potential effects on vaginal microbiota and immune markers were explored. Fifty women devoid of vaginal complaints (Nugent score 0-3 and vaginal pH ≤ 4.5) were randomised into a 2-week intervention with either La-14 and HN001 as the verum product or a comparable placebo. Vaginal swab samples were collected at baseline, after one and two weeks of intervention, and after a one-week follow-up, for assessing colonisation of the supplemented lactobacilli, vaginal microbiota, and six specific immune markers. Colonisation of L. acidophilus and L. rhamnosus was not observed above the assay detection limit (5.29 and 5.11 log 10 genomes/swab for L. acidophilus and L. rhamnosus, respectively). Vaginal microbiotas remained stable and predominated by lactobacilli throughout the intervention, and vaginal pH remained optimal (at least 90% of participants in both groups had pH 4.0 or 4.5 throughout the study). Immune markers elafin and human β-defensin 3 (HBD-3) were significantly decreased in the verum group (p = 0.022 and p = 0.028, respectively) but did not correlate with any microbiota changes. Adverse events raised no safety concerns, and no undesired changes in the vaginal microbiota or immune markers were detected.

Salivary Human β-Defensin 1-3 and Human α-Defensin-1 Levels in Relation to the Extent of Periodontal Disease and Tooth Loss in the Elderly.

The oral innate immune response may diminish with aging. In the present study, the aim was to examine human β-defensin (hBD) 1-3 and human neutrophil peptide (HNP)-1 levels in the saliva of an elderly population to establish the extent of periodontal disease and tooth loss. A total of 175 individuals aged ≥ 65 years were divided into five groups based on the number of teeth with a pocket depth ≥ 4 mm as follows: 17 pocket-free individuals (Control), 55 individuals having 1-6 pocket teeth (PerioA), 33 individuals having 7-13 pocket teeth (PerioB), 29 individuals having at least 14 pocket teeth (PerioC), and 41 edentulous individuals. Their salivary defensin levels were measured with ELISA kits. The salivary HNP-1 levels were significantly higher in the Perio groups (PerioB: p < 0.001 and PerioC: p < 0.001) in comparison to the Control. The associations between salivary HNP-1 levels and the number of pocket teeth remained significant after adjustments for age, gender, level of education, and number of teeth. The salivary HNP and hBD levels differed in terms of their correlation to the extent of periodontal disease and tooth loss in the elderly.

High-Throughput Screening for Epigenetic Compounds That Induce Human β-Defensin 1 Synthesis

Antimicrobial host defense peptides (HDPs) are critically important for innate immunity. Small-molecule compounds with the ability to induce HDP synthesis are being actively explored for antimicrobial therapy. To facilitate the discovery of the compounds that specifically activate human β-defensin 1 (DEFB1) gene transcription, we established a cell-based high-throughput screening assay that employs HT-29/DEFB1-luc, a stable reporter cell line expressing the luciferase gene driven by a 3-Kb DEFB1 gene promoter. A screening of a library of 148 small-molecule epigenetic compounds led to the identification of 28 hits, with a minimum strictly standardized mean difference of 3.0. Fourteen compounds were further selected and confirmed to be capable of inducing DEFB1 mRNA expression in human HT-29 colonic epithelial cells. Desirably, the human cathelicidin antimicrobial peptide (CAMP) gene was also induced by these epigenetic compounds. Benzamide-containing histone deacetylase inhibitors (HDACi) were among the most potent HDP inducers identified in this study. Additionally, several major genes involved in intestinal barrier function, such as claudin-1, claudin-2, tight junction protein 1, and mucin 2, were differentially regulated by HDP inducers. These findings suggest the potential for the development of benzamide-based HDACi as host-directed antimicrobials for infectious disease control and prevention.

Host Cationic Antimicrobial Molecules Inhibit S. aureus Exotoxin Production.

Innate immune molecules, including antimicrobial peptides (for example, defensins) and lysozyme, function to delay or prevent bacterial infections. These molecules are commonly found on mucosal and skin surfaces. Staphylococcus aureus is a common pathogen and causes millions of infections annually. It is well known that innate immune molecules, such as defensins and lysozyme, either poorly inhibit or do not inhibit the growth of S. aureus. Our current studies show that the α-defensin human neutrophil α-defensin-1 (HNP-1) and lysozyme inhibit exotoxin production, both hemolysins and superantigens, which are required for S. aureus infection. HNP-1 inhibited exotoxin production at concentrations as low as 0.001 μg/mL. Lysozyme inhibited exotoxin production at 0.05 to 0.5 μg/mL. Both HNP-1 and lysozyme functioned through at least one two-component system (SrrA/B). The β-defensin human β-defensin 1 (HBD-1) inhibited hemolysin but not superantigen production. The cation chelator S100A8/A9 (calprotectin), compared to EDTA, was tested for the ability to inhibit exotoxin production. EDTA at high concentrations inhibited exotoxin production; these were the same concentrations that interfered with staphylococcal growth. S100A8/A9 at the highest concentration tested (10 μg/mL) had no effect on S. aureus growth but enhanced exotoxin production. Lower concentrations had no effect on growth or exotoxin production. Lysostaphin is regularly used to lyse S. aureus. The lytic concentrations of lysostaphin were the only concentrations that also inhibited growth and exotoxin production. Our studies demonstrate that a major activity of innate defensin peptides and lysozyme is inhibition of staphylococcal exotoxin production but not inhibition of growth. IMPORTANCE Staphylococcus aureus causes large numbers of both relatively benign and serious human infections, which are mediated in large part by the organisms' secreted exotoxins. Since 1921, it has been known that lysozyme and, as shown later in the 1900s, other innate immune peptides, including human neutrophil α-defensin-1 (HNP-1) and human β-defensin 1 (HBD-1), are either not antistaphylococcal or are only weakly inhibitory to growth. Our study confirms those findings but, importantly, shows that at subgrowth inhibitory concentrations, these positively charged innate immune peptides inhibit exotoxin production, including both hemolysins and the superantigen toxic shock syndrome toxin-1. The data show that the principal activity of innate immune peptides in the host is likely to be inhibition of exotoxin production required for staphylococcal mucosal or skin colonization rather than growth inhibition.

Differential invasiveness & expression of antimicrobial peptides in Shigella serotypes.

The study of Shigella pathogenesis at present is severely hampered by the lack of a relevant animal model that replicates human bacillary dysentery. Different Shigella serogroups cause varying severity of clinical illness. Ex vivo colonization of Shigella flexneri, S. dysenteriae and S. sonnei were characterized in human paediatric colonic pinch biopsies in the in vitro organ culture (IVOC) model to study the invasiveness of Shigella by gentamicin protection assay (GPA). Furthermore, the expression of antimicrobial peptides (AMPs) in response to different serotypes of Shigella was also studied in IVOC model. IVOC explants were inoculated with 109 colony forming units of different serotypes of Shigella and recovery of bacteria studied. Histopathological analysis was carried out to study inflammatory immune responses. GPA was done to elucidate the invasiveness of different serotypes of Shigella. Secretions of AMPs were measured by enzyme-linked immunosorbent assay (ELISA). Western blotting was performed to check the expression of AMPs and nuclear factor kappa B in IVOC explants. After 24 h post-infection, the colon biopsies showed intense inflammatory reaction. In both IVOC and GPA, S. dysenteriae 1 was the most invasive as compared to S. flexneri and S. sonnei. S. sonnei was the least invasive. ELISA demonstrated that S. sonnei dampened the HBD (human β-defensin)-2 responses whereas there was augmentation by S. dysenteriae and there was a modest but non-significant increase by S. flexneri. A modest increase in HBD-3 by S. sonnei and S. flexneri was observed but was not found to be significant. However, western blotting data showed upregulation of all AMPs by all serotypes. Western blotting is more sensitive than ELISA. In the present study, differences in invasiveness and AMP production induced by different serotypes of Shigella were found. Human intestinal IVOC represents a model system to investigate early interaction between pathogenic bacteria and the human gut.

The role of TGF-β and antimicrobial peptides in basal cell carcinoma: a systematic review.

Basal cell carcinoma (BCC) is the most common skin cancer in humans, occurring in more than 50% of Caucasians during their lifetime, with a frequency rate that is continually increasing. We present a systematic review summarizing the role of transforming growth factor β (TGF-β), cathelicidin, and human β-defensins (HBDs) in the pathogenesis of BCC. The major online databases including PubMed, Scopus, Embase, Web of Science, Cochrane Library, and Google Scholar were searched to extract studies regarding the levels of TGF-β, HBD, and cathelicidin in BCC. A total of 14 studies met the inclusion criteria and were included in this systematic review. There were 6 studies that included initially established levels of TGF-β in BCCs. A total of 87 BCCs were analysed, and a common result was that the TGF-β levels increase in the BCCs compared to the control groups. Analogously, 2 studies contained numerical data on HBD levels but with a different in methodology. The level of cathelicidin was established in 108 BCCs and was significantly higher in the BCC group than in the control group. The presented review shows evidence that proteins like TGF-β, HBD, and cathelicidin play a role in developing BCC. Protein levels or their expression are elevated in patients with BCC. Furthermore, a critical review of the literature was presented and discussed, highlighting its shortcomings.

Лікування хворих на істинну та мікробну екзему з урахуванням порушень імунної системи

The purpose of the study is to analyze the effect of drugs with immunomodulatory effect on the changed indicators of the immune system of patients with idiopathic eczema (IE) and microbial eczema (ME). Materials and methods. 43 patients with chronic eczema (19 with idiopathic and 24 with microbial) aged 18–63 were examined. Eczema severity was assessed using the EASI. The immunological study carried out before and after treatment included the determination of the subpopulation composition of blood lymphocytes (CD3+, CD4+ and CD8+) by flow cytofluorimetry, interleukins (IL)- 4, –10, –17 and interferon (IFN) γ, lactoferrin (LF) and human β-defensin 2 (hBD2) by immunoenzymatic method using appropriate commercial test systems. The innate immune system was assessed by the phagocytic index (PhI), phagocytic number (PhN) and phagocytosis completion index (PhCI). Patients with IE against the background of traditional therapy additionally received cholecalciferol (vitamin D3), ME – glucosaminylmuramylpentapeptide (GMPP), than cholecalciferol. The duration of HMPP administration and the dose of vitamin D3 depended on the severity of the dermatosis. Results and conclusions. The developed therapy contributed to regression of clinical manifestations, reduction of EASI. With IE and ME, clinical remission was achieved in 31.6% and 29.2%, significant improvement in 57.9% and 54.1%, and improvement in 10.5% and 16.7% of patients, respectively. Under the influence of immunomodulatory agents, it was possible to normalize most indicators of immunity, except for some parameters of innate immunity (PhCI and LF in ME), the level of IL (IL-17 in IE).

Human β-defensin 2 ameliorates acute GVHD by limiting ileal neutrophil infiltration and restraining T cell receptor signaling

Acute graft-versus-host disease (aGVHD), which is driven by allogeneic T cells, has a high mortality rate and limited treatment options. Human β-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide. In addition to its antimicrobial effects, hBD-2 has immunomodulatory functions thought to be mediated by CCR2 and CCR6 in myeloid cells. In this study, we analyzed the effect of recombinant hBD-2 on aGVHD development. We found that intestinal β-defensin expression was inadequately induced in response to inflammation in two independent cohorts of patients with aGVHD and in a murine aGVHD model. Treatment of mice with hBD-2 reduced GVHD severity and mortality and modulated the intestinal microbiota composition, resulting in reduced neutrophil infiltration in the ileum. Furthermore, hBD-2 treatment decreased proliferation and proinflammatory cytokine production by allogeneic T cells in vivo while preserving the beneficial graft-versus-leukemia effect. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation, and metabolism in a CCR2- and CCR6-independent manner by reducing proximal T cell receptor signaling. Furthermore, hBD-2 treatment diminished alloreactive T cell infiltration and the expression of genes involved in T cell receptor signaling in the ilea of mice with aGVHD. Together, we found that both human and murine aGVHD were characterized by a lack of intestinal β-defensin induction and that recombinant hBD-2 represents a potential therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Serum, saliva, and gingival tissue human β-defensin levels in relation to retinoic acid use.

Retinoic acid is an active derivative of vitamin A and regulates the differentiation, proliferation, and antimicrobial peptide expression profiles of human cells. The aim of the present study was to analyze the effect of systemic retinoic acid use on serum, saliva, and gingival tissue levels of human β-defensin (hBD)-1, hBD-2, and hBD-3. A total of 69 participants (34 systemic retinoic acid users and 35 healthy controls) were enrolled in this study. Plaque index, probing pocket depth, bleeding on probing (BOP), and clinical attachment loss were measured. Saliva and serum hBD-1, hBD-2, and hBD-3 levels were quantified by enzyme-linked immunosorbent assay. Gingival tissue hBD-1, hBD-2, and hBD-3 levels were determined by immunohistochemistry. A univariate general linear model was used in adjusted comparisons of hBD1, hBD-2, and hBD-3. P values of < 0.05 were considered statistically significant. Reduced salivary levels of hBD-2 (P = 0.042), but not hBD-1 or hBD-3, were detected in systemic retinoic acid users compared to non-user controls. There was a significant difference in the adjusted (for BOP%) salivary hBD-2 concentrations between retinoic acid and control groups (P = 0.031). No difference was observed in serum or tissue levels of hBD-1, hBD-2, or hBD-3 between the two study groups. Systemic retinoic acid use was associated with suppressed salivary hBD-2 level, which was independent of gingival inflammation.

Fecal HBD-2 and Claudin-3 may be potential biomarkers to predict the deterioration of necrotizing enterocolitis: A prospective study.

Necrotizing enterocolitis (NEC) is a critical gastrointestinal disease. We aim to explore the value of fecal human β-defensin 2 (HBD-2), Claudin-3, high-mobility group box-1 protein (HMGB-1), and resistin-like molecule β (Relmβ) as well as some laboratory metrics to predict the deterioration of NEC. Infants diagnosed with NEC at Stage II were enrolled in our study. Those who progressed to Stage III were included in the Stage III group and the rest were included in the Stage II group. Clinical data and laboratory metrics of the infants were collected. Fecal samples of HBD2, HMGB-1, Claudin-3, and Relmβ collected during their enrollment were determined by using enzyme-linked immunosorbent assay (ELISA) kits. Student's t-test, the Mann-Whitney U test, the chi-square test, receiver operating characteristic (ROC), and logistic regression analysis were performed. Sixty infants diagnosed with NEC at Stage II were enrolled in our study, with 27 in the Stage III group (n = 27) and 33 in the Stage II group (n = 33). Although many of these NEC cases were late preterm and term infants, the infants in the Stage III group had a lower gestational age (P < 0.05). The incidence of gestational diabetes mellitus, peritonitis, intestinal adhesion, and sepsis was higher and more infants in the Stage III group underwent surgeries (P < 0.05). The levels of HBD-2 and Claudin-3 were higher and neutrophil count was lower in the Stage III group than in the Stage II Group, and the area under the curve (AUC) was 0.754, 0,755, and 0.666, respectively (P < 0.05). HBD-2 ≥ 1649.02 ng/g and Claudin-3 ≥ 2488.71 pg/g were included in the multivariate stepwise logistic regression analysis (P < 0.05), and the AUC of the model was 0.805 (95% CI: 0.688-0.922). Fecal HBD-2 and Claudin-3 may be potential biomarkers to predict the deterioration of NEC from Stage II to Stage III.

Lactoplantibacillus plantarum KG Lysates Inhibit the Internalization of Staphylococcus aureus by Human Keratinocytes through the Induction of Human Beta-Defensin 3

Human β-defensin 3 (hBD3) is an antimicrobial peptide and a chemoattractant molecule expressed in human keratinocytes. Lysates prepared from Lactoplantibacillus plantarum KG (KG lysates), a fermented lactobacillus strain with Ginseng extracts, significantly induced hBD3 in human keratinocytes. hBD3 induced by KG lysates affected the internalization of Staphylococcus aureus by human keratinocytes. The intracellular pathway for the induction of hBD3 by KG lysates was examined using inhibitors. Briefly, KG lysates stimulated the late signaling pathway including JAK1/2, which might be induced by the EGFR- or IFN-γ-mediated pathway, and affected hBD3 induction. Through this study, we confirmed that KG lysates have the ability to control bacterial infections, especially S. aureus, in human keratinocytes. Thus, KG lysates can be provided in various formulations for the development of food and pharmaceutical materials to control bacterial infection.

Human β-Defensin 3 Inhibits Porphyromonas Gingivalis Lipopolysaccharide-Induced Oxidative and Inflammatory Responses of Microglia by Suppression of Cathepsins B and L.

Recently, the effects of antibacterial peptides are suggested to have therapeutic potential in Alzheimer's disease. Furthermore, systemic treatment of Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) induced Alzheimer's disease-like neuropathological changes in middle-aged mice. Then, we examined whether human β-defensins (hBDs), antimicrobial peptides produced by the oral mucosa and salivary glands, can suppress Pg LPS-induced oxidative and inflammatory responses by microglia. hBD3 (1 μM) significantly suppressed Pg LPS-induced production of nitric oxide and interleukin-6 (IL-6) by MG6 cells, a mouse microglial cell line. hBD3 (1 μM) also significantly inhibited Pg LPS-induced expression of IL-6 by HMC3 cells, a human microglial cell line. In contrast, neither hBD1, hBD2 nor hBD4 failed to inhibit their productions. Furthermore, hBD3 suppressed Pg LPS-induced p65 nuclear translocation through the IκBα degradation. Pg LPS-induced expression of IL-6 was significantly suppressed by E64d, a cysteine protease inhibitor, and CA-074Me, a known specific inhibitor for cathepsin B, but not by pepstatin A, an aspartic protease inhibitor. Interestingly, hBD3 significantly inhibited enzymatic activities of recombinant human cathepsins B and L, lysosomal cysteine proteases, and their intracellular activities in MG6 cells. Therefore, hBD3 suppressed oxidative and inflammatory responses of microglia through the inhibition of cathepsins B and L, which enzymatic activities are necessary for the NF-κB activation.

Investigation of human β-defensins 1, 2 and 3 in human saliva by molecular dynamics.

Human β-defensins present in saliva have a broad spectrum of antimicrobial activities that work against infections in oral cavity. To provide a better understanding of these molecules' properties and functions at the molecular level, we have investigated and compared the important structural properties of human β-defensin-1, -2 and -3 using molecular dynamics simulations. Our results have shown that human β-defensin-3 has a more flexible structure in water than the other two because of its high hydrophilicity, low β-sheet content and high repulsive forces between its charged residues. Moreover, we found that the location of the salt bridges is important in protein's stability in water. Molecular dynamics simulations of human β-defensins 1, 2 and 3 revealed that the hbd-3 is more flexible in water than hbd-1 and hbd-2.

Evaluation of human β‑defensins in the cerebrospinal fluid of suspected meningitis.

Human β-defensins (HBDs) are an important class of antimicrobial peptides that have immunomodulatory functions; however, the role of HBDs have not been well explored in the pathogenesis of meningitis. A cross-sectional study was performed to explore the levels of HBD1, HBD2, HBD3, and HBD4 in the cerebrospinal fluid (CSF) of 176 suspected meningitis cases. CSF samples were first subjected to PCR analysis using a set of universal primers targeting a portion of the eubacteria 16S rRNA gene. The analysis demonstrated that 66 samples (37.5%) were PCR-positive, whilst 110 samples (62.5%) were PCR-negative. DNA sequence analysis of the PCR-positive products identified two broad categories of bacteria, Gram-negative (68.2%) and Gram-positive (31.8%). A total of 88 PCR-negative CSF samples showed abnormal leukocyte counts, glucose concentrations, and/or protein concentrations, and were considered abnormal (ABN). The remaining 22 CSF samples were considered normal (NOR). HBD1, HBD2, and HBD4 levels did not exhibit significant differences between PCR-positive, ABN, and NOR CSF samples. However, HBD3 levels were significantly higher in the ABN CSF samples than in the NOR CSF samples (P=0.005). HBD3 levels were also elevated in the PCR-positive CSF samples compared with the NOR CSF samples, but the difference was not significant (P=0.151). HBD2, HBD3, and HBD4 were correlated with leukocyte counts, glucose concentration, and protein concentration. In conclusion, HBD3 levels were significantly elevated in the CSF of suspected meningitis cases regardless of the cause of meningitis. The CSF levels of certain HBDs were affected by specific diagnostic laboratory parameters for meningitis, including leukocyte counts, glucose concentration, and protein concentration.

Effects of Fluticasone Propionate on Klebsiella pneumoniae and Gram-Negative Bacteria Associated with Chronic Airway Disease.

Inhaled corticosteroids (ICS) are commonly prescribed first-line treatments for asthma and chronic obstructive pulmonary disease (COPD). Recent evidence has shown that ICS use is associated with changes in the airway microbiome, which may impact clinical outcomes such as potential increased risk for pneumonia in COPD. Although the immunomodulatory effects of corticosteroids are well appreciated, whether ICS could directly influence the behavior of respiratory tract bacteria has been unknown. In this pilot study we explored the effects of fluticasone proprionate, a commonly prescribed inhaled corticosteroid, on respiratory bacteria with an expanded focus on Klebsiella pneumoniae, a species previously implicated in fluticasone-associated pneumonia in COPD. We observed significant effects of fluticasone proprionate on growth responses of K. pneumoniae, as well as other bacterial species isolated from asthmatic patients. Fluticasone-exposed K. pneumoniae displayed altered expression of several bacterial genes and reduced the metabolic activity of bronchial epithelial cells and their expression of human β-defensin 2. Targeted assays identified a fluticasone metabolite from fluticasone-exposed K. pneumoniae cells, suggesting this species may be capable of metabolizing fluticasone proprionate. Collectively, these observations support the hypothesis that specific members of the airway microbiota possess the functional repertoire to respond to or potentially utilize corticosteroids in their microenvironment. These findings lay a foundation for novel research directions into the potential direct effects of ICS, often prescribed long term to patients, on the broader airway microbial community and on the behavior of specific microbial species implicated in asthma and COPD outcomes. IMPORTANCE Inhaled corticosteroids are widely prescribed for many respiratory diseases, including asthma and COPD. While they benefit many patients, corticosteroids can also have negative effects. Some patients do not improve with treatment and even experience adverse side effects. Recent studies have shown that inhaled corticosteroids can change the make-up of bacteria in the human respiratory tract. However, whether these medications can directly impact the behavior of such bacteria has been unknown. Here, we explored the effects of fluticasone propionate, a commonly prescribed inhaled corticosteroid, on Klebsiella pneumoniae and other airway bacteria of interest, including primary species isolated from adult asthma patients. We provide evidence of growth responses to direct fluticasone exposure in culture and further examined fluticasone's effects on K. pneumoniae, including gene expression changes and effects of fluticasone-exposed bacteria on airway cells. These findings indicate that members of the human airway bacterial community possess the functional ability to respond to corticosteroids, which may have implications for the heterogeneity of treatment response observed clinically.

Uncoupling melanogenesis from proliferation in epidermal melanocytes responding to stimulation with psoriasis-related proinflammatory cytokines

BackgroundFew studies have addressed the impact of the psoriasis-related proinflammatory cytokines on the proliferation and melanogenesis of melanocytes (MCs) in lesional psoriatic skin. ObjectiveWe investigated the effects of TNFα, IL17A, and IL8 on the proliferation and melanin synthesis of MCs. MethodsSkin specimens were biopsied from patients with psoriasis vulgaris at the active stage, or from the tail skin of Dct-LacZ mice with imiquimod (IMQ)-induced psoriasiform dermatitis. Cultured keratinocytes (KCs), MCs, and human skin explants were used in this study. The numbers of MCs were measured via β-galactosidase staining, EdU incorporation and HMB45 immunohistochemical staining. The expression of human β-defensin 3 (hBD3) in KCs was silenced by siRNA, the conditioned medium (CM) from siRNA-transfected KCs was used to treat MCs, then followed by αMSH stimulation. The melanogenesis-related genes were examined by using qRT-PCR and western blotting. ResultsThe increased number of MCs and decreased melanin content were highly relevant to the enhanced expression of IL8 and BD3 both in human psoriatic skin and in IMQ-treated mouse tail skin. IL8 expression in KCs and CXCR2 expression in MCs was significantly increased by IL17A and TNFα, the αMSH-induced upregulations of microphthalmia-associated transcription factor (MITF) and tyrosinase in MCs were abrogated by the CM from hBD3-unsilenced KCs, but not from hBD3-silenced KCs. ConclusionOur results suggest the roles of IL8-CXCR2 activation in promoting MC proliferation and of BD3 upregulation in reducing melanogenesis. These findings have been implicated in the underlying mechanism that active psoriasis prefers hypopigmentation despite chronic inflammation.

Improving Fmoc Solid Phase Synthesis of Human Beta Defensin 3.

Human β-defensin 3, HBD-3, is a 45-residue antimicrobial and immunomodulatory peptide that plays multiple roles in the host defense system. In addition to interacting with cell membranes, HBD-3 is also a ligand for melanocortin receptors, cytokine receptors and voltage-gated potassium channels. Structural and functional studies of HBD-3 have been hampered by inefficient synthetic and recombinant expression methods. Herein, we report an optimized Fmoc solid-phase synthesis of this peptide using an orthogonal disulfide bonds formation strategy. Our results suggest that utilization of an optimized resin, coupling reagents and pseudoproline dipeptide building blocks decrease chain aggregation and largely improve the amount of the target peptide in the final crude material, making the synthesis more efficient. We also present an alternative synthesis of HBD-3 in which a replacement of a native disulfide bridge with a diselenide bond improved the oxidative folding. Our work enables further biological and pharmacological characterization of HBD-3, hence advancing our understanding of its therapeutic potential.

Design of a novel multi-epitope vaccine candidate against hepatitis C virus using structural and nonstructural proteins: An immunoinformatics approach.

Hepatitis C virus (HCV) infects the liver and causes chronic infection. Several mutations in the viral genome have been associated with drug resistance development. Currently, there is no approved vaccine against the HCV. The employment of computational biology is the primary and crucial step for vaccine design or antiviral therapy which can substantially reduce the duration and cost of studies. Therefore, in this study, we designed a multi-epitope vaccine using various immunoinformatics tools to elicit the efficient human immune responses against the HCV. Initially, various potential (antigenic, immunogenic, non-toxic and non-allergenic) epitope segments were extracted from viral structural and non-structural protein sequences using multiple screening methods. The selected epitopes were linked to each other properly. Then, toll-like receptors (TLRs) 3 and 4 agonists (50S ribosomal protein L7/L12 and human β-defensin 2, respectively) were added to the N-terminus of the final vaccine sequence to increase its immunogenicity. The 3D structure of the vaccine was modeled. Molecular dynamics simulations studies verified the high stability of final free vaccines and in complex with TLR3 and TLR4. These constructs were also antigenic, non-allergenic, nontoxic and immunogenic. Although the designed vaccine traits were promising as a potential candidate against the HCV infection, experimental studies and clinical trials are required to verify the protective traits and safety of the designed vaccine.

Lacticaseibacillus rhamnosus Lcr35 Stimulates Epithelial Vaginal Defenses upon Gardnerella vaginalis Infection.

Dysbiosis of the vaginal microbiome as a result of overgrowth of anaerobic bacteria, such as Gardnerella vaginalis, and low levels of "healthy" lactobacilli leads to bacterial vaginosis (BV), usually associated with a low-grade inflammatory process. Despite appropriate antibiotic treatment, G. vaginalis-associated BV is characterized by significant recurrence. The use of probiotics could be an interesting alternative therapy due to their ability to rebalance vaginal microbiota. In this study, we investigated the effects of a well-characterized probiotic strain, Lacticaseibacillus rhamnosus Lcr35, on epithelial vaginal and dendritic cell (DC) immune responses after G. vaginalis infection. In an in vitro coculture model with human monocyte-derived dendritic cells and a vaginal epithelial cell (VEC) monolayer, the Lcr35 strain induced DC activation, as evidenced by the induction of maturation and synthesis of interleukin-8 (IL-8) and CCL-20 chemokines upon apical challenge of the VECs by G. vaginalis. Analysis of the vaginal epithelial response showed that the presence of Lcr35 significantly increased the production of the proinflammatory cytokines IL-8 and IL-1β and human β-defensin 2 (HBD-2), whereas the concentration of secretory leukocyte protease inhibitor (SLPI) was decreased in G. vaginalis-infected vaginal epithelial cells. Treatment with recombinant SLPI was associated with upregulation of Lcr35-stimulated IL-8 and HBD-2 production. These results suggest that inhibition of SLPI by Lcr35 in vaginal epithelial cells contributes to the host defense response against G. vaginalis infection.