hBD-2 Gene Research Articles

Features of innate immunity in children with hypertrophy of the palatine tonsils

Hypertrophy of pharyngeal lymphoid tissue is among the most common problems in pediatric otorhinolaryngology, in particular, hypertrophy of the palatine tonsils. This disorder is characterized by increased size of a single or both palatine tonsils combined with various clinical symptoms. The principles of treatment in children with this pathology remain debatable, since the long-term effects of bilateral tonsillotomy are still not fully understood. The aim of the study was to assess the expression levels of genes encoding the innate immunity molecules (TLR4, HBD1, HBD2, IL1β) in the mucous membranes of palatine tonsils in children with palatine tonsillitis before and after treatment.We conducted a study of 78 patients divided into three independent groups. The 1st group (comparison) included 20 somatically healthy children. The 2nd group included 28 children with grade 2 palatine tonsil hypertrophy who underwent local treatment. The 3rd group included 30 children with grade 3 hypertrophy of the palatine tonsils, who, by clinical indications, underwent bilateral tonsillotomy. Determination and evaluation of innate immunity indices was carried out both before starting the treatment, and one month later, using the real-time PCR method.In children with hypertrophy of palatine tonsils, the initial values of TLR4 gene expression and antimicrobial peptides differed from those of healthy children. A decreased expression of HBD1 and HBD2 genes, which provide immediate protection against pathogens, was revealed. The values of TLR4 gene expression differed in groups of children with varying degrees of palatine tonsillar hypertrophy. In patients with bilateral tonsillotomy, an increased expression of TLR4 gene and a decreased expression of antimicrobial peptide genes (HBD1, HBD2) were revealed, which may indicate a readiness for development of tonsillar inflammation in response to pathogens. One month after surgical treatment, the indices of innate immunity were comparable with those of healthy children thus confirming the validity of surgical treatment. In the 2nd group of patients, the TLR4 gene expression one month after conservative treatment remained reduced, the expression of β-defensin HBD1 gene increased and exceeded the indicators of the group of healthy children, the expression level of the IL-1β gene was reduced.The revealed imbalance between the TLR4, HBD1 and HBD2 expression levels confirms an important role of innate immunity mechanisms in pathogenesis of palatine tonsillitis. The assessment of innate immunity indices may be used as an additional criterion in administration of therapy for hypertrophy of palatine tonsils and evaluation of its efficiency.

<i>HBD1</i> and <i>LL37</i> gene expression in children with atopic dermatitis

Atopic dermatitis (AD) is a multifactorial genetically determined inflammatory skin disease characterized by itching, chronic course, age-related features of localization and lesion morphology. Atopic dermatitis is caused by complex interactions between genetic, immunological, and environmental factors. The barrier function of the skin is impaired in atopic dermatitis. Antimicrobial peptides, e.g., LL-37, b-defensins are involved in maintaining the skin barrier function (especially, intercellular contacts). An imbalance of antimicrobial peptides may cause different disorders, including allergic pathologies. The aim of this study is to investigate gene expression profile of the HBD1 and LL37 encoding antimicrobial peptides in the samples of skin and blood mononuclear cells obtained from the children with moderate and severe atopic dermatitis before and after treatment. By means of real-time polymerase chain reaction, the levels of HBD1 and LL37 gene expression were evaluated in the samples. Statistical analysis showed significantly increased (p 0.017) expression levels of both HBD1 (H-test = 24.76; 2, n = 72; p = 0.00001), and LL37 genes (H-test = 15.69; 2, n = 72; p = 0.00039) in blood cells of AD patients compared to the control group, as well as decreased (p 0.05) levels of HBD1 expression in the affected skin compared to the control group. Our data on the cathelicidin gene in the skin do not differ from the literature data, since its expression is reduced in AD. In our series, an increase of the gene expression was revealed in PBMCs. The HBD1 peptide is expressed in both monocytes and macrophages, representing a link between innate and adaptive immunity. In our study, the expression of the HBD1 gene was increased only in blood, thus suggesting activation of innate immunity components at the systemic level in response to inflammation. Of importance, understanding the role of immunological markers in AD will help to develop novel prognostic approaches in management of the patients with atopic disorders. Therefore, one should understand pathogenetic mechanisms of allergic diseases.

A Transcriptome-Wide Association Study of Cognitive Impairment in Subjects With Metabolic Syndrome Adjusted for Adherence to Mediterranean Diet

ObjectivesMild cognitive impairment has been associated with several genetic and lifestyle factors including diet. However, the number of transcriptome-wide association studies (TWAS) in this field is still very scarce. Our aim was to carry out a TWAS adjusted for adherence to Mediterranean diet (MedDiet) to identify the differentially expressed genes associated with a global cognitive function score, tacking diet into account.MethodsA set of cognitive functions related to cognitive decline was assessed in 103 subjects (aged 64.8 years) with metabolic syndrome. A principal component analysis (PCA) on 5 variables including semantic and verbal fluency, the trail making test (TMT) A and B and the digits total test, was performed to reduce multidimensionality. Only one component with eigenvalues >1 was identified. This latent variable had strong direct correlations with semantic and verbal fluency and the digits test, and negative with TMT-A and TMT-B. The standardized score was used as the global cognitive variable representing attention and executive functions. RNA was isolated from leukocytes and after quality control, using the Affymetrix human 20 array, a TWAS was undertaken according to the quality control procedures.ResultsIn the TWAS adjusted for sex, age, batch effect, leukocyte types, body mass index, smoking, medications and adherence to MedDiet, we identified several top-ranked differentially expressed genes associated with the global cognitive score, including: HBD3 (Methyl-cpg Binding Domain protein 3) gene (p = 8.9 × 10–6) as the hit, previously associated with neuropathy; GABRP (Gamma-Aminobutyric Acid Type A receptor Pi), involved in the synaptic transmission; and LAG3 (Lymphocyte activating 3), related to neuroinflammation, among others. Moreover, MedDiet was significantly related to the expression of LAG3 gene.ConclusionsIn this TWAS, a composite score for cognitive function is associated with differential expression of several genes related with neurotransmission and neuroinflammation in leukocytes, some of them being additionally modulated by adherence to MedDiet.Funding SourcesGeneralitat Valenciana (grant PROMETEO/2021/021). Grant PID2019-108858RB-I00 is funded by AEI 10.13039/501,100,011,033 and, by “ERDF A way of making Europe”.

β-defensin 1 Gene Polymorphisms are Associated with Kidney Disease in Northwestern Mexicans with Type 2 Diabetes

ABSTRACT Diabetic kidney disease (DKD) is one of the more limiting complications to the quality of life of diabetes mellitus patients. Studies including cultured cells, animal models, and case-control studies highlight the role of human β-defensin-1 (hBD-1) in diabetes. This study assessed the association of hBD-1 gene (DEFB1) functional variations −52 G/A (rs1799946), −44 C/G (rs1800972) and −20 G/A (rs11362) with type 2 diabetes mellitus (T2DM) in order to investigate its effects on genetic susceptibility and progression to DKD in a Mexican population. A total of 214 T2DM patients with and without DKD (n = 102 and n = 112, respectively) and 117 healthy subjects participated in this case–control study. Genotyping was made by PCR-RFLPs. Clinical and biochemical parameters of all patients were measured. There was no statistically significant difference in genotype or allele frequencies between patients and healthy individuals. Nevertheless, compared with patients without DKD, DKD patients have a reduced prevalence of AA genotype of −52 G/A (OR = 0.307, 95% CI = 0.104–0.905, p =.026), as well as a higher frequency of GA genotype of −20 G/A variant (OR = 1.875, 95%CI = 1.031–3.409, p = .038). Our results suggest that rs1799946 and rs11362 could be useful variants to stratify T2DM Mexican patients in order to prescribe closer follow-up to prevent or retard DKD. Further tests in different ethnic groups are encouraged.

HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii

Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA-protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9-mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.

Human beta-defensin-1 rs2738047 polymorphism is associated with shisha smoking risk among Saudi population

Human β-defensin (HBD), a member of the antimicrobial peptides, is essential for respiratory epithelial cells’ microbial defense, and is affected by cigarette smoking (CS). Its expression is upregulated by stimulation from microbes or inflammation. Genetic polymorphisms in the HBD-1 gene have been implicated in the development of various smoking-related diseases, including chronic obstructive pulmonary disease and asthma. Thus, we sought to analyze possible associations between HBD-1 single-nucleotide polymorphism (SNP) in HBD-1 gene and CS in ethnic Saudi Arabian subjects. Variants rs1047031 (C/T), rs1799946 (C/T), rs2738047 (C/T), and rs11362 (C/T) were investigated by genotyping 575 blood specimens from males and females, smokers/non-smokers: 288/287. The CT and CT+TT genotypes of rs1799946 presented an ~5-fold increased correlation with CS among the female smokers, compared with the female controls (OR = 5.473, P = 0.02003; and OR = 5.211, P = 0.02028, respectively), an observation similar to rs11362 SNP in female smokers, but with protective effects in TT genotype, compared with the CC reference allele (OR = 0.143, P = 0.04368). In shisha smokers, the heterozygous CT and the CT/TT genotype of rs2738047 polymorphism showed the same results with ~3-fold increased correlation with CS (OR = 2.788; P = 0.03448), compared with the cigarette smokers category. No significant association was shown in genotypic distributions and allelic frequencies of rs1047031. Further investigations, including large study samples, are required to investigate the effects of shisha on human beta-defensin expression and protein levels.

Gold Nanoparticles Combined Human β-Defensin 3 Gene-Modified Human Periodontal Ligament Cells Alleviate Periodontal Destruction via the p38 MAPK Pathway.

Periodontitis is a chronic inflammatory disease with plaques as the initiating factor, which will induce the destruction of periodontal tissues. Numerous studies focused on how to obtain periodontal tissue regeneration in inflammatory environments. Previous studies have reported adenovirus-mediated human β-defensin 3 (hBD3) gene transfer could potentially enhance the osteogenic differentiation of human periodontal ligament cells (hPDLCs) and bone repair in periodontitis. Gold nanoparticles (AuNPs), the ideal inorganic nanomaterials in biomedicine applications, were proved to have synergetic effects with gene transfection. To further observe the potential promoting effects, AuNPs were added to the transfected cells. The results showed the positive effects of osteogenic differentiation while applying AuNPs into hPDLCs transfected by adenovirus encoding hBD3 gene. In vivo, after rat periodontal ligament cell (rPDLC) transplantation into SD rats with periodontitis, AuNPs combined hBD3 gene modification could also promote periodontal regeneration. The p38 mitogen-activated protein kinase (MAPK) pathway was demonstrated to potentially regulate both the in vitro and in vivo processes. In conclusion, AuNPs can promote the osteogenic differentiation of hBD3 gene-modified hPDLCs and periodontal regeneration via the p38 MAPK pathway.

Effect of the Cellular Extract of Co-cultured Lactobacillus Casei on BAX and Human β-Defensin 2 Genes Expression in HT29 Cells

Aims: Defensins are cysteine-rich antimicrobial cationic peptides and BAX is a proapoptotic gene that can cause cell death. This study aimed to investigate the effect of cellular extract of co-cultured Lactobacillus casei on the expression of BAX and human β-defensin 2 (hBD-2) genes in HT29 cells. Methods & Materials: This experimental study was conducted in the Research Center for Pharmaceutical Nanotechnology of Tabriz University of Medical Sciences in 2017. The HT29 cell line was obtained from the Pasteur Institute of Iran, and cells were assessed using Microculture Tetrazolium Test (MTT) after culturing. DNA was extracted from the treated cells, and then the DNA ladder assay was carried out. After preparing cDNA, the expression levels of BAX and hBD-2 genes in the HT29 cell line were measured using a real-time Polymerase Chain Reaction (PCR) method. Findings: The results of the MTT assay indicated that Lactobacillus casei inhibited the proliferation of HT29 cells and induced apoptosis in these cells. Results of DAPI staining and DNA ladder assay obtained from treating HT29 cells by Lactobacillus casei showed qualitative changes in cell apoptosis. Moreover, realtime PCR results indicated that Lactobacillus casei bacteria significantly increased the expression of the hBD-2 gene in HT29 colon cancer cells within 12-24 hours (P= 0.023), while BAX gene expression showed no significant change in the first 24 hours (P= 0.37). Conclusion: The extract of Lactobacillus casei can be used to stimulate cancer cells to produce β-defensins, inhibit pathogens, prevent the stimulation of cellular signaling, and fight antibiotic-resistant bacteria.

Vivostat Platelet-Rich Fibrin® for Complicated or Chronic Wounds-A Pilot Study.

Vivostat Platelet-Rich Fibrin® (PRF) is an autologous platelet concentrate used for the local treatment of chronic or complicated wounds. Still, its application for this indication is not evidence-based. Therefore, we performed this monocentric retrospective pilot study investigating the clinical outcome of a local treatment of chronic or complicated wounds in 35 patients (23 male, 12 female, mean age 68.7 years) treated with Vivostat PRF®. This study population is the largest among published studies analyzing the clinical efficacy of Vivostat PRF® on chronic wounds so far. Using the perpendicular method we divided the wounds into three sizes (<10, 10–30, and >30 cm2). The clinical efficacy of the Vivostat PRF treatment was the primary endpoint and was divided into three groups of increasing degrees of wound improvement: (1) no improvement of the wound (wound area was not reduced > 10% under Vivostat PRF® treatment), (2) improvement of the wound (reduced area > 10% under Vivostat PRF® treatment) and (3) complete epithelialization (wounds that were completely re-epithelialized after Vivostat PRF® treatment). We included patients’ diagnosis and concomitant diseases (peripheral arterial occlusive disease (PAOD)), chronic venous insufficiency (CVI)), diabetic foot syndrome (DFS)) in our data analysis in order to investigate their potential impact on the wound healing capacity of Vivostat PRF®. Our results show that in the entire study population, 13 out of 35 (37.1%) patients experienced wound improvement and 14 out of 35 (40%) patients showed complete epithelialization of their wound under Vivostat PRF® treatment. In summary, 77.1% of the treated patients benefited from the Vivostat PRF® therapy. These positive wound healing effects were all observed within the first three to six Vivostat PRF® applications. Subgroup analyses showed that Vivostat PRF® appeared to be more efficient in patients without CVI in comparison to patients with CVI (p = 0.02). Moreover, Vivostat PRF® treatment seems to be particularly efficient in PAOD-related wounds with a reduced crural arterial blood supply (p = 0.01). Additionally, we performed an experimental human in vivo study on ten male students where we artificially generated bilateral gluteal wounds and analyzed the influence of the Vivostat PRF® treatment on the expression of two genes (human beta Defensin-2, ((hBD-2) and human beta-Defensin-3 (hBD-3)) in keratinocytes of resected wound specimens that are induced during wound healing. Interestingly, this analysis revealed that only seven of out ten individuals showed a relevant hBD-2 and hBD-3 gene induction after Vivostat PRF® treatment. This led to the novel “key-lock-hypothesis”. With the goal of an individualized precision medicine approach with optimized wound treatment strategies in the future, this is an important observation that demands further experimental and clinical studies.

Influence of resveratrol application with pulp-capping materials on the genetic expression levels of stem cells.

To evaluate in a laboratory setting the response of human mesenchymal stem cells (MSCs) to pulp-capping materials with and without resveratrol (RSV). Five materials, Calcimol LC, Life, TheraCal LC, ProRoot MTA and Biodentine, were prepared according to the manufacturers' instructions. Human MSCs were then exposed to these materials, with and without RSV, for 24h (n=8). Cell viability was evaluated using the MTT assay, and total cell death was quantified by annexin V-FITC staining with flow cytometry. The expression levels of the IL-8, IL-10, HBD-2 and BCL-2 genes were investigated using real-time polymerase chain reaction (RT-PCR). Data obtained from MTT test were analysed using one-way anova, and Tukey's multiple-comparison test. The paired Student t test was employed to compare the effects of materials on gene expression (significance level of 5%). The group cell viabilities were Calcimol LC 53%, Life 43%, TheraCal LC 78%, ProRoot MTA 75% and Biodentine 78%. Calcimol LC and Life exhibited significant differences compared with the control groups (P<0.05). The percentages of necrotic/late apoptotic cells associated with Calcimol LC and TheraCal LC were greater than in the other materials. However, when RSV was added to wells containing materials, cell viability increased to Calcimol LC 63%, Life 52%, TheraCal LC 82%, ProRoot MTA 91% and Biodentine 96%, and the percentages of early apoptotic and late apoptotic/necrotic cells decreased. Calcimol LC+RSV and Life+RSV differed significantly from the control group (P<0.05). The expression of IL-8 gene was high for all materials, ProRoot MTA caused significant overexpression, and the addition of RSV reduced the expression of IL-8 in the Calcimol LC, TheraCal LC and ProRoot MTA groups and led to increased expression of IL-10 in the Calcimol LC, Life and Biodentine groups. HBD-2 and BCL-2 exhibited increased expression in ProRoot MTA with RSV (P<0.05). The addition of RSV exerted a protective effect on MSCs and regulated the inflammatory process by altering the expression levels of pro- and anti-inflammatory genes.

Cervical Gene Delivery of the Antimicrobial Peptide, Human β-Defensin (HBD)-3, in a Mouse Model of Ascending Infection-Related Preterm Birth.

Approximately 40% of preterm births are preceded by microbial invasion of the intrauterine space; ascent from the vagina being the most common pathway. Within the cervical canal, antimicrobial peptides and proteins (AMPs) are important components of the cervical barrier which help to prevent ascending vaginal infection. We investigated whether expression of the AMP, human β-defensin-3 (HBD3), in the cervical mucosa of pregnant mice could prevent bacterial ascent from the vagina into the uterine cavity. An adeno-associated virus vector containing both the HBD3 gene and GFP transgene (AAV8 HBD3.GFP) or control AAV8 GFP, was administered intravaginally into E13.5 pregnant mice. Ascending infection was induced at E16.5 using bioluminescent Escherichia coli (E. coli K1 A192PP-lux2). Bioluminescence imaging showed bacterial ascent into the uterine cavity, inflammatory events that led to premature delivery and a reduction in pups born alive, compared with uninfected controls. Interestingly, a significant reduction in uterine bioluminescence in the AAV8 HBD3.GFP-treated mice was observed 24 h post-E. coli infection, compared to AAV8 GFP treated mice, signifying reduced bacterial ascent in AAV8 HBD3.GFP-treated mice. Furthermore, there was a significant increase in the number of living pups in AAV HBD3.GFP-treated mice. We propose that HBD3 may be a potential candidate for augmenting cervical innate immunity to prevent ascending infection-related preterm birth and its associated neonatal consequences.

HUMAN BETA-DEFENSIN-3 GENE EXPRESSION IN THE NASAL AND SINONASAL MUCOSA

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

Beta-defensins and analogs in Helicobacter pylori infections: mRNA expression levels, DNA methylation, and antibacterial activity.

Antimicrobial peptides can protect the gastric mucosa from bacteria, but Helicobacter pylori (H. pylori) can equally colonize the gastric apparatus. To understand beta-defensin function in H. pylori-associated chronic gastritis, we investigated susceptibility, human beta-defensin mRNA expression, and DNA methylation changes to promoters in the gastric mucosa with or without H. pylori infection. We studied the expression of HBD2 (gene name DEFB4A), HBD3 (DEFB103A), and HBD4 (DEFB104) using real-time PCR in 15 control and 10 H. pylori infection patient gastric specimens. This study demonstrates that H. pylori infection is related to gastric enhancement of inducible HBD2, but inducible HBD3 and HBD4 expression levels remained unchanged. HBD2 gene methylation levels were overall higher in H. pylori-negative samples than in H. pylori-positive samples. We also assessed antimicrobial susceptibility using growth on blood agar. The H. pylori strain Tox+ was susceptible to all defensins tested and their analogs (3N, 3NI). These results show that HBD2 is involved in gastritis development driven by H. pylori, which facilitates the creation of an epigenetic field during H. pylori-associated gastric tumorigenesis.

Human β-Defensin-2 Improves Hyperoxia-Induced Lung Structural and Functional Injury in Neonatal Rats.

BackgroundBronchopulmonary dysplasia (BPD) is a major complication of extreme prematurity, characterized by alveolar simplification and pulmonary malfunction. Hyperoxia-induced lung injury in neonatal rats has been used as a model of BPD, as indicated by lung architectural change and alveolar simplification that resembles clinical feature of BPD. β-defensin-2 (BD2) plays an important role in lung diseases by inhibiting inflammation response. However, little is known about its role in BPD. The aim of this study was to determine the effect of human BD2 (hBD2) gene on hyperoxia-induced animal model of BPD.Material/MethodsThe neonatal rats were exposed to 90% oxygen (O2) continuously for 14 days to mimic the BPD-like lung injury. These rats were then randomly assigned to the following four groups: in room air (air), in 90% O2, in 90% O2 with null adenovirus vector infection (O2+Ad), and in 90% O2 with gene therapy through adenovirus transfected hBD2 (O2+Ad-hBD2). Morphology of lungs, pulmonary function and expression of inflammatory cytokines on P7, P10, P14, and P21 were documented and compared across the 4 groups.ResultsThe overexpression of hBD2 mediated by the adenovirus vector was successfully constructed. hBD2 gene therapy increased hBD2 mRNA expression, increased radial alveolar count (RAC), lung volume and compliance, decreased mean linear intercept (MLI), tissue damping, and elastance. Furthermore, pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were inhibited and anti-inflammatory cytokines IL-10 was increased in the lungs of rats in O2+Ad-hBD2 group.ConclusionsIn hyperoxia-induced rat models of BPD, hBD2 promotes alveolarization and improves pulmonary function. The mechanism may contribute in alleviating inflammation response and inhibiting pro-inflammatory factors including IL-1β, IL-6, and TNF-α.

Candida albicans-Cell Interactions Activate Innate Immune Defense in Human Palate Epithelial Primary Cells via Nitric Oxide (NO) and β-Defensin 2 (hBD-2).

The presence of Candida albicans in the biofilm underlying the dental prosthesis is related to denture stomatitis (DS), an inflammatory reaction of the oral mucosa. The oral epithelium, a component of the innate immune response, has the ability to react to fungal invasion. In this study, we evaluated the in vitro effect of viable C. albicans on the apoptosis, nitric oxide (NO) production, and β-defensin 2 (hBD-2) expression and production of human palate epithelial cells (HPECs). We further determined whether or not these effects were correlated with fungal invasion of epithelial cells. Interaction between HPEC primary culture and C. albicans was obtained through either direct or indirect cell–cell contact with a supernatant from a hyphal fungus. We found that the hyphae supernatants were sufficient to induce slight HPEC apoptosis, which occurred prior to the activation of the specific mechanisms of epithelial defense. The epithelial defense responses were found to occur via NO and antimicrobial peptide hBD-2 production only during direct contact between C. albicans and HPECs and coincided with the fungus’s intraepithelial invasion. However, although the hBD-2 levels remained constant in the HPEC supernatants over time, the NO release and hBD-2 gene expression were reduced at a later time (10 h), indicating that the epithelial defense capacity against the fungal invasion was not maintained in later phases. This aspect of the immune response was associated with increased epithelial invasion and apoptosis maintenance.

Relationship between β-defensin-1 gene polymorphism and susceptibility and prognosis of acute respiratory distress syndrome.

Objective:The 1st exon 5′ noncoding region rs1799946 (-52A/G), rs1800972 (-44C/G), rs11362 (-20A/G) 3 single-nucleotide polymorphisms (SNPs) on human β-defensin-1 (HBD-1) gene affect its transcription and posttranscriptional mRNA stability then affect the activity of HBD-1. This study was to investigate the effects of HBD-1 gene rs1799946, rs1800972, and rs11362 locus SNPs on genetic susceptibility and prognosis of acute respiratory distress syndrome (ARDS).Methods:A total of 300 patients with ARDS (ARDS group) and 240 patients who were admitted to the intensive care unit and had a high risk of ARDS but did not progress to ARDS (control group) were included in this study. The genotypes of HBD-1 gene rs1799946, rs1800972, and rs11362 locus and serum HBD-1 were detected. Patients were followed for 60 days with development of ARDS as a primary outcome, ARDS-related mortality and organ dysfunction were secondary outcomes.Results:HBD-1 gene rs1799946 and rs11362 gene mutations were not risk factors for ARDS (P > .05). Mutation allele G of rs1800972 locus in HBD-1 gene was a risk factor for ARDS. There was no significant difference in serum HBD-1 levels between patients with different genotypes of rs1799946 and rs11362 locus in the HBD-1 gene (P > .05). HBD-1 gene rs1800972 locus wild type, heterozygous, and mutant homozygous serum levels of HBD-1 gradually decreased, the difference was statistically significant (P < .001). The 60-day survival rate of subjects with wild type, heterozygous, and mutant homozygote at the rs1800972 locus of HBD-1 gene decreased sequentially (81.7%, 48.9%, and 39.7%), and the difference was statistically significant (P < .05).Conclusion:The SNP of rs1800972 (-44C/G) in HBD-1 gene is associated with the risk of ARDS. The rs1800972 locus G allele carriers are more likely to develop ARDS and have a poor prognosis.

Promoter DNA methylation contributes to human β-defensin-1 deficiency in atopic dermatitis

ABSTRACTAtopic dermatitis (AD) is a chronic inflammatory skin disease caused by epidermal barrier dysfunction and dysregulation of innate and adaptive immunity. Epigenetic regulation of human β-defensin-1 (HBD-1) might be associated with a variety of defects in the innate immune system during AD pathogenesis. We investigated the possible mechanism of decreased HBD-1 gene expression in AD and demonstrated the restoration of HBD-1 transcription in undifferentiated normal human epidermal keratinocyte cells after treatment with a DNA methyltransferase inhibitor. We also conducted an in vitro methylated reporter assay using a reporter containing 14 CpG sites. Methylation of the 14 CpG sites within the HBD-1 5′ region resulted in an approximately 86% reduction in promoter activity and affected HBD-1 transcriptional regulation. We then compared methylation frequencies at CpG 3 and CpG 4 between non-lesional and lesional epidermis samples of patients with severe AD and between these paired tissues and healthy control epidermis from normal volunteers without AD history. Bisulfite pyrosequencing data showed significantly higher methylation frequencies at the CpG 3 and 4 sites in AD lesional samples than in non-lesional AD skin and normal skin samples (P < 0.05). These results suggest that the DNA methylation signature of HBD-1 is a novel diagnostic/prognostic marker and a promising therapeutic target for the compromised stratum corneum barrier attributed to HBD-1 deficiency.

STUDY OF EXPRESSION OF HBD-1 AND HBD-2 GENES IN EPITHELIAL CELLS OF MUCOUS UPPER AIRWAY IN NEWBORNS WITH PNEUMONIA DEPENDING ON THE CAUSATIVE AGENT

Β-defensins play an important role in protecting the fetus from infection, so the expression of these antimicrobial peptides in the respiratory tract in newborns is really important. In this regard, we set a task of studying the expression of the HBD-1 and HBD-2 genes in the epithelial cells of the mucous of the upper airway in newborns with pneumonia and in healthy newborns, depending on the causative agent. Also, the polymorphic marker G(-20) A in the DEFB1 gene was associated with infectious pathology of newborns (in particular pneumonia). Methods: The microflora and the factors of congenital immunity on the mucous membranes of the upper airway have been studied in two groups: newborns with ventilator-associated and congenital pneumonia. The biological material was scrapings of epithelial cells of the mucous membrane of the upper airway of newborns and puerperas and blood. Results: It was found that the expression of the HBD-2 gene increases 2.3-fold in children who have an infectious agent, but there are no clinical manifestations of pneumonia. A significant decrease in HBD-2 (3.2 times) in patients with pneumonia caused by K. pneumonia was shown. The frequencies of alleles of the DEFB1 gene in the fetal infection group and in the comparison group: allele G - 0.66, 0.79, allele A - 0.34, 0.21, respectively. The frequencies of the genotypes of the test marker in mothers in the ventilator-associated, congenital pneumonia and the comparison group were as follows: GG - 0.78, 0.58, 0.58; AA is 0, 0.25, 0; AG - 0.22, 0.17, 0.42, respectively. In newborns allele G dominated among alleles (frequency was higher than 0.73 in all groups) and genotype GG (frequency exceeded 0.52). Conclusion: In the course of the study, it was confirmed that β-defensins protect the mucous from infectious agents. The results indicate that the genetic marker G (-20) A of the DEFB1 gene is associated with the risk of developing the child's UTI.

432 The epidermal growth factor receptor is critical for the Staphylococcus aureus-mediated induction of human beta-defensin-3 in keratinocytes

Antimicrobial peptides (AMP) contribute to cutaneous defense due to their potent antimicrobial activity. A feature of many AMP expressed by keratinocytes is their inducibility by cytokines and microbial factors. There is increasing evidence that several skin infections may be associated with a dysregulation of AMP. Patients receiving anti-EGFR (epidermal growth factor receptor) therapy often suffer from cutaneous infection caused by Staphylococcus (S.) aureus. This is in line with recent studies indicating a pivotal role of the EGFR to mediate the expression of various AMP in keratinocytes. Human beta-defensin-3 (hBD-3) is an important AMP expressed by keratinocytes to control the growth of S. aureus. Since S. aureus is able to induce the expression of hBD-3 in keratinocytes we analyzed if the EGFR is involved in this induction. To this end we stimulated human primary keratinocytes with S. aureus in the presence of the anti-EGFR antibody cetuximab. Inactivation of the EGFR by cetuximab already resulted in a decreased constitutive hBD-3 gene expression as measured by real-time PCR. Simulation of the keratinocytes with living S. aureus or their culture supernatants resulted in an increased hBD-3 gene expression which was abrogated in the presence of cetuximab. Accordingly, the EGFR inhibitor AG1478 also inhibited the S. aureus-mediated hBD-3 induction. Induction of hBD-3 in keratinocytes treated with the EGFR ligand TGF-alpha confirmed the EGFR-dependent induction of hBD-3. Treatment of a 3D skin equivalent with living S. aureus also resulted in an increased expression of hBD-3. This S. aureus-mediated hBD-3 induction was decreased in skin equivalents treated with cetuximab. Together, these data further highlight the importance of the EGFR-pathway in cutaneous defense against S. aureus. Our data suggest that a failure of S. aureus-mediated hBD-3 induction in patients receiving anti-EGFR therapy may contribute to the increased S. aureus infection rate seen in these patients.

Arabinogalactan Proteins From Baobab and Acacia Seeds Influence Innate Immunity of Human Keratinocytes In Vitro.

Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However, little is currently known regarding their potential activity toward skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability. Moreover, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. J. Cell. Physiol. 232: 2558-2568, 2017. © 2016 Wiley Periodicals, Inc.