Human Beta-defensin Research Articles

Candida albicans Modulates Murine and Human Beta Defensin-1 during Vaginitis.

Vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC) are two forms of a disease caused by Candida spp. β-defensin (BD) is one of the most important families of antimicrobial peptides in the female genital tract and includes molecules that exert essential local functions as antimicrobial and PMN chemoattractant peptides. However, the information on their role during murine and human VVC and RVVC is limited. Thus, we analyzed the behavior and contribution of BD1 to the local response in a VVC mice model and the local cytokine profile and human BD1 and BD3 expression in cervicovaginal lavage from patients with VVC and RVVC. We demonstrated that, in patients with RVVC BD1, mRNA and protein expression were severely diminished and that the aspartate proteinase and lipase secreted by C. albicans are involved in that decrease. This study provides novel information about the pathogenesis of VVC and describes a highly efficient C. albicans escape strategy for perpetuating the infection; these results may contribute to the development of new or combined treatment approaches.

Susceptibility of Human Airway Tissue Models Derived From Different Anatomical Sites to Bordetella pertussis and Its Virulence Factor Adenylate Cyclase Toxin.

To study the interaction of human pathogens with their host target structures, human tissue models based on primary cells are considered suitable. Complex tissue models of the human airways have been used as infection models for various viral and bacterial pathogens. The Gram-negative bacterium Bordetella pertussis is of relevant clinical interest since whooping cough has developed into a resurgent infectious disease. In the present study, we created three-dimensional tissue models of the human ciliated nasal and tracheo-bronchial mucosa. We compared the innate immune response of these models towards the B. pertussis virulence factor adenylate cyclase toxin (CyaA) and its enzymatically inactive but fully pore-forming toxoid CyaA-AC-. Applying molecular biological, histological, and microbiological assays, we found that 1 µg/ml CyaA elevated the intracellular cAMP level but did not disturb the epithelial barrier integrity of nasal and tracheo-bronchial airway mucosa tissue models. Interestingly, CyaA significantly increased interleukin 6, interleukin 8, and human beta defensin 2 secretion in nasal tissue models, whereas tracheo-bronchial tissue models were not significantly affected compared to the controls. Subsequently, we investigated the interaction of B. pertussis with both differentiated primary nasal and tracheo-bronchial tissue models and demonstrated bacterial adherence and invasion without observing host cell type-specific significant differences. Even though the nasal and the tracheo-bronchial mucosa appear similar from a histological perspective, they are differentially susceptible to B. pertussis CyaA in vitro. Our finding that nasal tissue models showed an increased innate immune response towards the B. pertussis virulence factor CyaA compared to tracheo-bronchial tissue models may reflect the key role of the nasal airway mucosa as the first line of defense against airborne pathogens.

Cervicovaginal levels of human beta defensins during bacterial vaginosis.

To compare the cervicovaginal levels of human beta defensin (hBD)-1, 2 and 3 of women according to the status of Nugent-defined bacterial vaginosis (BV). A total of 634 women of reproductive age were included in the study. Participants were equally distributed in two groups: according to the classification of vaginal smears according to Nugent criteria in normal (scores 0 to 3) and BV (scores ≥7). Cervicovaginal fluid samples were used for measurements of hBDs1, 2 and 3 levels by enzyme-linked immunosorbent assay (ELISA). Levels of each hBD were compared between the two study groups using Mann-Whitney test, with p-value <0.05 considered as significant. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated for sociodemographic variables and hBD1-3 levels associated with BV a multivariable analysis. Correlation between Nugent score and measured levels of hBDs1-3 were calculated using Spearman's test. Cervicovaginal fluids from women with BV showed lower levels of hBD-1 [median 2,400.00 pg/mL (0-27,800.00); p<0.0001], hBD-2 [5,600.00 pg/mL (0-45,800.00); p<0.0001] and hBD-3 [1,600.00 pg/mL (0-81,700.00); p = 0.012] when compared to optimal microbiota [hBD-1: [median 3,400.00 pg/mL (0-35,600.00), hBD-2: 12,300.00 pg/mL (0-95,300.00) and hBD-3: 3,000.00 pg/mL (0-64,300.00), respectively]. Multivariable analysis showed that lower levels of hBD-1 (OR: 2.05; 95% CI: 1.46-2.87), hBD-2 (OR: 1.85; 95% CI: 1.32-2.60) and hBD-3 (OR: 1.90; 95% CI: 1.37-2.64) were independently associated BV. Significant negative correlations were observed between Nugent scores and cervicovaginal levels of hBD-1 (Spearman's rho = -0.2118; p = 0.0001) and hBD-2 (*Spearman's rho = -0.2117; p = 0.0001). Bacterial vaginosis is associated with lower cervicovaginal levels of hBDs1-3 in reproductive-aged women.

Exposure to per- and polyfluoroalkyl substances associates with an altered lipid composition of breast milk.

The composition of human breast milk is highly variable inter- and intra-individually. Environmental factors are suspected to contribute to such compositional variation, however, their impact on breast milk composition is currently poorly understood. We sought to (1) define the impact of maternal exposure to per- and polyfluoroalkyl substances (PFAS) on lipid composition of human breast milk, and (2) to study the combined impact of maternal PFAS exposure and breast milk lipid composition on the growth of the infants.In a mother-infant study (n=44) we measured the levels of PFAS and lipids in maternal serum and conducted lipidomics analysis of breast milk collect 2-4days after the delivery and at 3months of infant age, by using ultra high performance liquid chromatography combined with quadrupole-time-of-flight mass spectrometry. Gastrointestinal biomarkers fecal calprotectin and human beta defensin 2 were measured in the stool samples at the age of 3, 6, 9, and 12months. Maternal diet was studied by a validated food frequency questionnaire. PFAS levels were inversely associated with total lipid levels in the breast milk collected after the delivery. In the high exposure group, the ratio of acylated saturated and polyunsaturated fatty acids in triacylglycerols was increased. Moreover, high exposure to PFAS associated with the altered phospholipid composition, which was indicative of unfavorable increase in the size of milk fat globules. These changes in the milk lipid composition were further associated with slower infant growth and with elevated intestinal inflammatory markers. Our data suggest that the maternal exposure to PFAS impacts the nutritional quality of the breast milk, which, in turn, may have detrimental impact on the health and growth of the children later in life.

Experimental and simulation studies reveal mechanism of action of human defensin derivatives

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.

STEM-20. THE ROLE OF HUMAN BETA DEFENSINS IN THE CLONOGENICITY OF GLIOBLASTOMA MULTIFORME

Abstract INTRODUCTION Glioblastoma (GBM) is the most common primary malignant brain tumor with a median overall survival of 12-15months. GBM aggressiveness and poor response to treatment are often attributed to a small population of stem-like cells referred to as glioma stem cells (GSCs). Human Beta-Defensins (HBDs), a family of small molecules initially thought to function as anti-microbials, have been implicated in various cancers with functions that are cancer type specific, including proinflammatory and immunosuppressive roles. GOAL: This study aimed to elucidate HBDs expression in GSCs and differentiated gioma cells (DGSs). METHODS We identified HBD2 and HBD3 in glioma and GSCs and DGSs by immunofluorescence (IF) and immunohistochemistry (IHC). We also assessed the role HBD2/3 play in GBM oncogenesis by investigating their effects on the self-renewal capacity of GSCs. RESULTS HBD2 and HBD3 are found in both bulk tumor and GSCs by IHC and IF. Expression of HBD2 and HBD3 mRNA levels are elevated in GBM patient tissue in comparison to lower grade gliomas by 16.2 &amp; 1.9 fold (respectively; p &amp;lt; 0.0001). Additionally, transcriptional expression of HBD2 and HBD3 are increased by 0.68 and 1.15 fold respectively in GSCs versus autologous DGCs (p &amp;lt; 0.05; p&amp;lt; 0.005 respectively), suggesting a role for HBD 2/3 in oncogenesis. Interestingly however, HBD2 and HBD3 pretreatment of GSC cell lines showed decreased self-renewal capacity by 34.2 and 66.4% (p &amp;lt; 0.001), determined by the reduced number of large neurospheres ( &amp;gt;250mm). CONCLUSIONS Our results demonstrate the presence of HBD2/3 in GBM samples by IHC and IF with increased HBD2/3 mRNA expression in GBM samples. Interestingly DGCs contain less HBD2/3 than their GCS counterparts, and clonogenicity assays demonstrate a decrease in oncogenesis, suggesting that HBD’s role in proliferation and clonogenicity of DGCs and GSCs may be context dependent, leading to additional questions and future studies.

Nano- and Macroscale Imaging of Cholesterol Linoleate and Human Beta Defensin 2-Induced Changes in Pseudomonas aeruginosa Biofilms.

The biofilm production of Pseudomonas aeruginosa (PA) is central to establishing chronic infection in the airways in cystic fibrosis. Epithelial cells secrete an array of innate immune factors, including antimicrobial proteins and lipids, such as human beta defensin 2 (HBD2) and cholesteryl lineolate (CL), respectively, to combat colonization by pathogens. We have recently shown that HBD2 inhibits biofilm production by PA, possibly linked to interference with the transport of biofilm precursors. Considering that both HBD2 and CL are increased in airway fluids during infection, we hypothesized that CL synergizes with HBD2 in biofilm inhibition. CL was formulated in phospholipid-based liposomes (CL-PL). As measured by atomic force microscopy of single bacteria, CL-PL alone and in combination with HBD2 significantly increased bacterial surface roughness. Additionally, extracellular structures emanated from untreated bacterial cells, but not from cells treated with CL-PL and HBD2 alone and in combination. Crystal violet staining of the biofilm revealed that CL-PL combined with HBD2 effected a significant decrease of biofilm mass and increased the number of larger biofilm particles consistent with altered cohesion of formed biofilms. These data suggest that CL and HBD2 affect PA biofilm formation at the single cell and community-wide level and that the community-wide effects of CL are enhanced by HBD2. This research may inform future novel treatments for recalcitrant infections in the airways of CF patients.

Inactivation of HIV-1 in Polarized Infant Tonsil Epithelial Cells by Human Beta-Defensins 2 and 3 Tagged with the Protein Transduction Domain of HIV-1 Tat.

Mother-to-child transmission (MTCT) of HIV-1 may occur during pregnancy, labor, and breastfeeding; however, the molecular mechanism of MTCT of virus remains poorly understood. Infant tonsil mucosal epithelium may sequester HIV-1, serving as a transient reservoir, and may play a critical role in MTCT. Innate immune proteins human beta-defensins 2 (hBD-2) and -3 may inactivate intravesicular virions. To establish delivery of hBD-2 and -3 into vesicles containing HIV-1, we tagged hBDs with the protein transduction domain (PTD) of HIV-1 Tat, which facilitates an efficient translocation of proteins across cell membranes. Our new findings showed that hBD-2 and -3 proteins tagged with PTD efficiently penetrated polarized tonsil epithelial cells by endocytosis and direct penetration. PTD-initiated internalization of hBD-2 and -3 proteins into epithelial cells led to their subsequent penetration of multivesicular bodies (MVB) and vacuoles containing HIV-1. Furthermore, PTD played a role in the fusion of vesicles containing HIV-1 with lysosomes, where virus was inactivated. PTD-initiated internalization of hBD-2 and -3 proteins into ex vivo tonsil tissue explants reduced the spread of virus from epithelial cells to CD4+ T lymphocytes, CD68+ macrophages, and CD1c+ dendritic cells, suggesting that this approach may serve as an antiviral strategy for inactivating intraepithelial HIV-1 and reducing viral MTCT.

139 Local dermatitis upon tapestripping and vaseline treatment of healthy human skin is attenuated by coal tar: insights into AHR-targeting therapies for inflammatory skin diseases

Coal tar therapy, an old dermatological therapy, exerts its therapeutic effect through activation of the aryl hydrocarbon receptor (AHR) and AHR-targeting molecules show therapeutic efficacy in phase 2/3 trials for chronic inflammatory skin diseases. The anti-inflammatory mechanisms of AHR activation in the skin are partially understood, but mostly studied in animal models. Here we aimed to investigate the therapeutic effects of AHR activation by coal tar application on epidermal responses after skin barrier disruption and inflammatory cues in human skin. Healthy volunteers (n=9) were treated with pix lithanthracis (5% in vaseline lanette) or vehicle (vaseline lanette) after complete removal of the stratum corneum by tape stripping. Non-invasive biophysical measurements (TEWL, hydration, erythema), stratum corneum tapes (NMF and protein profiling) and biopies were taken during 4 days of treatment. Application of vaseline on tapestripped skin led to an acute inflammatory response characterized by increased keratinocyte proliferation and subsequent acanthosis, a dense dermal CD45+ and MPO+ immune cell infiltrate indicative of (neutrophilic) granulocytes and increased levels of human beta defensin-2 (hBD2). Most strikingly, coal tar treated sites clearly showed less immune cell infiltrate, less pronounced acanthosis and hBD2 levels in the stratum corneum were significantly lower than upon vaseline treatment. No differential effects were found between vaseline and coal tar treatment for the other parameters we investigated. This in vivo human acute dermatitis model indicates that activation of AHR signaling reduces the influx of inflammatory cells potentially resulting in less epidermal activation. The lack in positive effects on skin barrier parameters by coal tar may be due to the relative short study period. Further investigations on the inflammatory signaling molecules driving the acute inflammation can provide new insights in the coal tar and AHR-mediated therapeutic effects.

Efficacy of Bifidobacterium animalis subsp. lactis, BB-12® on infant colic - a randomised, double-blinded, placebo-controlled study.

To evaluate the administration of Bifidobacterium animalis subsp. lactis, BB-12® (BB-12) on infant colic in breastfed infants, a double-blind, placebo-controlled randomised study was conducted in Chengdu, China from April 2016 to October 2017 with 192 full-term infants less than 3 months of age and meeting the ROME III criteria for infant colic. After a 1-week run-in the infants were randomly assigned to receive daily BB-12 (1×109 cfu/day) or placebo for 3 weeks. Crying/fussing time were recorded using a 24 h structured diary. The primary endpoint was the proportion of infants achieving a reduction in crying and fussing time of ≥50% from baseline. Parent's/caregiver's health related quality of life was measured using a modified PedsQL™ 2.0 Family Impact Module and immunological biomarkers were evaluated from faecal samples at baseline and after the 21-day intervention. The percentage of infants achieving a reduction in the daily crying/fussing time ≥50% after the 21-day intervention was significantly higher in the infants supplemented with BB-12 (P<0.001). The mean number of crying episodes was significantly reduced in the BB-12 group compared to the placebo group (10.0±3.0 to 5.0±1.87 vs 10.5±2.6 to 7.5±2.8, respectively) (P<0.001) and the mean daily sleep duration was markedly increased from baseline to end of intervention in the BB-12 group compared to the infants in the placebo group (60.7±104.0 vs 31.9±102.7 min/day, respectively) (P<0.001). The faecal levels of human beta defensin 2, cathelicidin, slgA, calprotectin and butyrate were statistically higher in the BB-12 group compared to the placebo group after the 21-day intervention. At the end of the intervention the parent's/caregiver's physical, emotional and social functioning scores were significantly higher for the BB-12 group compared to the placebo group (all P<0.05). Supplementation of BB-12 is effective in reducing crying and fussing in infants diagnosed with infant colic.

Human beta-defensins 2 and 4 are dysregulated in patients with coronavirus disease 19

Antimicrobial peptides (AMPs) have recently been proposed as significant immunological factors involved in pathogenesis of coronavirus disease 19 (COVID-19). Human β-defensins (hBDs) are among these AMPs, but the evidence is not well detailed. Therefore, this case-control study analyzed levels of hBD1, hBD2, hBD3 and hBD4 in serum of 103 patients with severe COVID-19 and 105 healthy controls. Most patients were older than 45 years (80.6%), and more than 50% suffered from chronic diseases (cardiovascular and diabetes). Results revealed that median levels of hBD1 and hBD3 did not show significant differences between patients and controls. On the contrary, HBD2 levels were significantly decreased in patients compared to controls (1036 vs. 1289 ng/L; p < 0.001), while HBD4 levels were significantly increased (4.04 vs. 2.43 ng/L; p < 0.001). Receiver operating characteristic curve analysis demonstrated the predictive significance of hBD2 (area under the curve [AUC] = 0.795; 95% confidence interval [CI] = 0.729–0.861; p < 0.001) and hBD4 (AUC = 0.816; 95% CI = 0.756–0.876; p < 0.001) in discriminating between COVID-19 patients and controls. Logistic regression analysis (adjusted for age, gender and body mass index) confirmed the significance of hBD2 (odds ratio [OR] = 0.996; corrected p = 0.004) and hBD4 (OR = 4.948; corrected p < 0.001) in susceptibility to COVID-19. In conclusion, the study indicated that hBD2 showed low levels in serum of patients infected with severe COVID-19, while hBD4 showed elevated levels. These differences in HBDs were not influenced by age, gender, body mass index, or chronic disease.

Hybrid derivative of cathelicidin and human beta defensin-2 against Gram-positive bacteria: A novel approach for the treatment of bacterial keratitis

Bacterial keratitis (BK) is a major cause of corneal blindness globally. This study aimed to develop a novel class of antimicrobial therapy, based on human-derived hybrid host defense peptides (HyHDPs), for treating BK. HyHDPs were rationally designed through combination of functional amino acids in parent HDPs, including LL-37 and human beta-defensin (HBD)-1 to -3. Minimal inhibitory concentrations (MICs) and time-kill kinetics assay were performed to determine the concentration- and time-dependent antimicrobial activity and cytotoxicity was evaluated against human corneal epithelial cells and erythrocytes. In vivo safety and efficacy of the most promising peptide was examined in the corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively. A second-generation HyHDP (CaD23), based on rational hybridization of the middle residues of LL-37 and C-terminal of HBD-2, was developed and was shown to demonstrate good efficacy against methicillin-sensitive and methicillin-resistant S. aureus [MIC = 12.5–25.0 μg/ml (5.2–10.4 μM)] and S. epidermidis [MIC = 12.5 μg/ml (5.2 μM)], and moderate efficacy against P. aeruginosa [MIC = 25-50 μg/ml (10.4–20.8 μM)]. CaD23 (at 25 μg/ml or 2× MIC) killed all the bacteria within 30 min, which was 8 times faster than amikacin (25 μg/ml or 20× MIC). After 10 consecutive passages, S. aureus (ATCC SA29213) did not develop any antimicrobial resistance (AMR) against CaD23 whereas it developed significant AMR (i.e. a 32-fold increase in MIC) against amikacin, a commonly used treatment for BK. Pre-clinical murine studies showed that CaD23 (0.5 mg/ml) achieved a median reduction of S. aureus bioburden by 94% (or 1.2 log10 CFU/ml) while not impeding corneal epithelial wound healing. In conclusion, rational hybridization of human-derived HDPs has led to generation of a potentially efficacious and safe topical antimicrobial agent for treating Gram-positive BK, with no/minimal risk of developing AMR.

Computational vaccinology guided design of multi-epitopes subunit vaccine designing against Hantaan virus and its validation through immune simulations.

The Hantaan virus belongs to Bunyaviridae family, an emerging virus that is responsible for hemorrhagic fevers. The virus is distributed worldwide and as of now there is no successful antiviral drug or vaccine developed to protect against the viral infections. Immunization or vaccination is an alternative approach for the protection against viral infections. A cost effective and thermodynamically stable vaccine should be developed to prevent a future possible pandemic. In this study a vaccine candidate was designed against the Hantaan virus, multiple immunoinformatics and reverse vaccinology tools were utilized for the prediction of both B and T cell epitopes for Nuceloprotein, RNA dependent RNA polymerase L and Envelope protein of the Hantaan virus. The individual epitopes were modeled for docking with respective HLAs and a multi-epitopes subunit vaccine candidate was constructued by joining together carefully evaluated B and T cell epitopes with suitable linkers. The vaccine model was evaluated for several physiochameical parameters i.e. Molecular weight, instability index and aliphatic index among the others, followed by 3D modeling of the vaccine for docking with TLR-4. Based on previous studies, Human beta-defensin was liked at the N-terminus of the vaccine sequence as an adjuvant to enhance immunogenicity. The docked complexes of vaccine-TLR-4 were then evaluated for residual interactions. Moreover, to validate final vaccine construct, immune simulations was carried out by C-IMMSIM server. A natural immune reponse was predicted by the immune simulation analysis. In-silico cloning was carried out using E. coli as host resulting in 0.93 CAI value, which suggests that the vaccine construct will attain maximal expression in E. coli host. The vaccine designed in this study needs experimental verification to confirm the immunogenicity and efficacy of the vaccine and ultimately used against Hantaan virus associated infections.

Silver Nanoparticle-Coated Polyhydroxyalkanoate Based Electrospun Fibers for Wound Dressing Applications

Wound dressings are high performance and high value products which can improve the regeneration of damaged skin. In these products, bioresorption and biocompatibility play a key role. The aim of this study is to provide progress in this area via nanofabrication and antimicrobial natural materials. Polyhydroxyalkanoates (PHAs) are a bio-based family of polymers that possess high biocompatibility and skin regenerative properties. In this study, a blend of poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxyoctanoate-co-3-hydroxy decanoate) (P(3HO-co-3HD)) was electrospun into P(3HB))/P(3HO-co-3HD) nanofibers to obtain materials with a high surface area and good handling performance. The nanofibers were then modified with silver nanoparticles (AgNPs) via the dip-coating method. The silver-containing nanofiber meshes showed good cytocompatibility and interesting immunomodulatory properties in vitro, together with the capability of stimulating the human beta defensin 2 and cytokeratin expression in human keratinocytes (HaCaT cells), which makes them promising materials for wound dressing applications.

Human beta defensin levels and vaginal microbiome composition in post-menopausal women diagnosed with lichen sclerosus

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.

Binding free energy calculation of human beta defensin 3 with negatively charged lipid bilayer using free energy perturbation method

Human β defensin type 3 (hBD-3) is a cationic peptide having strong antimicrobial activities even at high salt concentrations. The conserved sequence is believed to contribute to its unique antibacterial activities. To design novel drugs based on hBD-3, predicting the binding free energy contribution of each residue on hBD-3 with bacterial membrane is important. Firstly, the stable binding structure of hBD-3 dimer in analog form bound on POPG lipid bilayer was predicted using NAMD simulations, which was confirmed by RMSD, buried surface area, hydrogen bonds, distance map, and insertion depth map calculations. Then, free energy perturbation (FEP) method was applied to calculate the binding free energy of each residue by mutating it into Alanine. It was found that the positively charged residues on the tail region of hBD-3 contribute significantly to its binding with membrane. The result emphasized the importance of electrostatic interactions to hBD-3's binding with bacterial membrane.

Correlations between initial human-beta-defensin-1 level and quality of life of patients during anti-tuberculosis therapy

Background. The quality of life of patients with tuberculosis is an important component of the treatment effectiveness.&#x0D; Objective. To find the correlations between initial human-beta-defensin-1 (HBD-1) level and quality of life of patients during anti-tuberculosis therapy.&#x0D; Materials and methods. 100 patients with pulmonary tuberculosis were included in the study. The patients were diagnosed, treated and monitored according to current state protocols and World Health Organization guidelines. Additionally, the level if HBD-1 was measured in blood plasma by ELISA at the treatment onset. The patients were interviewed using SF-25 scale at the treatment inset, after 30 days and after 60 days. The parameters of physical functioning, role-physical functioning, bodily pain, general health, vitality, social functioning, emotional-role functioning, and mental health were assessed.&#x0D; Results. We found correlations between the initial level of HBD-1 and quality of life parameters: physical functioning (-0.43), role-physical functioning (-0.34), bodily pain (-0.23), general health (-0.42), social functioning (-0.42), emotional-role functioning (-0.36); p&lt;0.05. The obtained negative correlations indicate that a high initial level of HBD-1 is a predictor of lower quality of life during treatment.&#x0D; Conclusions. An increase in the level of HBD-1 at the treatment onset can be considered a predictor of a decrease in the quality of life during treatment in patients with pulmonary tuberculosis.

Effect of Early Feeding on Intestinal Permeability and Inflammation Markers in Infants with Genetic Susceptibility to Type 1 Diabetes: A Randomized Clinical Trial

To assess whether weaning to an extensively hydrolyzed formula (EHF) decreases gut permeability and/or markers of intestinal inflammation in infants with HLA-conferred diabetes susceptibility, when compared with conventional formula. By analyzing 1468 expecting biological parent pairs for HLA-conferred susceptibility for type 1 diabetes, 465 couples (32%) potentially eligible for the study were identified. After further parental consent, 332 babies to be born were randomized at 35th gestational week. HLA genotyping was performed at birth in 309 infants. Out of 87 eligible children, 73 infants participated in the intervention study: 33 in the EHF group and 40 in the control group. Clinical visits took place at 3, 6, 9, and 12months of age. The infants were provided either EHF or conventional formula whenever breastfeeding was not available or additional feeding was required over the first 9months of life. The main outcome was the lactulose to mannitol ratio (L/M ratio) at 9months. The secondary outcomes were L/M ratio at 3, 6, and 12months of age, and fecal calprotectin and human beta-defensin 2 (HBD-2) levels at each visit. Compared with controls, the median L/M ratio was lower in the EHF group at 9months (.006 vs .028; P=.005). Otherwise, the levels of intestinal permeability, fecal calprotectin, and HBD-2 were comparable between the two groups, although slight differences in the age-related dynamics of these markers were observed. It is possible to decrease intestinal permeability in infancy through weaning to an extensively hydrolyzed formula. This may reduce the early exposure to dietary antigens. Clinicaltrials.gov: NCT01735123.

Role of antimicrobial peptides system in inflammatory periodontal diseases non-specific oral cavity protection.

The system of antimicrobial peptides (AMP) is one of the most ancient mechanisms of the macroorganism resistance to infectious pathogens invasion. The aim of the study was to determine the role of the antimicrobial peptides system and periodontal pathogenic markers in the development and progression of inflammatory periodontal diseases. Gingival pocket washes (91 samples in total) for the research were received from patients with inflammatory periodontal diseases (chronic periodontitis and gingivitis) and intact periodontium. Using ELISA, the content of antimicrobial peptides was determined: human alpha-defensin (HNP 1-3), beta-defensin (HBD 1-3) and cathelicidin (LL-37). Periodontal pathogenic markers were isolated during RT-PCR. The study revealed differences in AMP concentrations by groups: level of HBD 2 in patients with chronic periodontitis was 1,36 times higher than those in the group of patients with chronic gingivitis (p=0,023) and 2,39 times higher than those in the control group (p<0,001), the content of HNP 1-3 in the group of patients with chronic periodontitis was reduced by 1,23 times compared with the indicators of the group of patients with gingivitis (p=0,045) and by 1,97 times compared with the indicators of the control group (p<0,001). The frequency of detection of periodontal pathogenic bacteria genes was 88,0% in patients with periodontitis, 76,92% in patients with gingivitis and 33,3% in the group with intact periodontium. HBD 2 content moderately correlated with the definition of P. gingivalis (r=0,612; p=0,022), T. forsythensis (r= 0,434; p=0,015), A. actinomycetemcomitans (r=0,483; p=0,006), a moderate negative correlation was detected between the content of HNP 1-3 and the release of periodontal pathogens in associations (P. gingivalis with T. forsythensis and T. denticola) (r=-0,388; p=0,031) in the group of patients with chronic periodontitis. Thus, the revealed relationships and correlations indicate shifts in the processes of reparative regeneration of the oral cavity and the regulation of local immunity in response to microbial invasion.

Human Beta-Defensin 2 and 3 Inhibit HIV-1 Replication in Macrophages.

Human beta-defensins (hBDs) are broad-spectrum antimicrobial peptides, secreted by epithelial cells of the skin and mucosae, and astrocytes, which we and others have shown to inhibit HIV-1 in primary CD4+ T cells. Although loss of CD4+ T cells contributes to mucosal immune dysfunction, macrophages are a major source of persistence and spread of HIV and also contribute to the development of various HIV-associated complications. We hypothesized that, besides T cells, hBDs could protect macrophages from HIV. Our data in primary human monocyte-derived macrophages (MDM) in vitro show that hBD2 and hBD3 inhibit HIV replication in a dose-dependent manner. We determined that hBD2 neither alters surface expression of HIV receptors nor induces expression of anti-HIV cytokines or beta-chemokines in MDM. Studies using a G-protein signaling antagonist in a single-cycle reporter virus system showed that hBD2 suppresses HIV at an early post-entry stage via G-protein coupled receptor (GPCR)-mediated signaling. We find that MDM express the shared chemokine-hBD receptors CCR2 and CCR6, albeit at variable levels among donors. However, cell surface expression analyses show that neither of these receptors is necessary for hBD2-mediated HIV inhibition, suggesting that hBD2 can signal via additional receptor(s). Our data also illustrate that hBD2 treatment was associated with increased expression of APOBEC3A and 3G antiretroviral restriction factors in MDM. These findings suggest that hBD2 inhibits HIV in MDM via more than one CCR thus adding to the potential of using β-defensins in preventive and therapeutic approaches.