Human Beta Defensin-2 mRNA and Proteasome Subunit ß Type 8 mRNA Analysis, Useful in Differentiating Skin Biopsies from Atopic Dermatitis and Psoriasis Vulgaris Patients.

(1): Atopic dermatitis and psoriasis vulgaris are chronic, inflammatory diseases. Clinical presentation usually leads to a proper diagnosis, but sometimes neither clinical examination nor histopathological evaluation can be conclusive. Therefore, we aimed to build up a novel diagnostic tool and check it for accuracy. The main objective of our work was to differentiate between healthy skin (C), atopic dermatitis (AD) and psoriasis vulgaris (PV) biopsies on the base of involucrin (IVL) and human ß-defensin-2 (hBD-2) concentrations and their mRNA, as well as mRNA for TPP2 and PSMB8. (2): ELISA for IVL and hBD-2 proteins and Real-time PCR for the relative expression of mRNA for: IVL (IVL mRNA), hBD-2 (hBD-2 mRNA), PSMB8 (PSMB8 mRNA) and TPP2 (TPP2 mRNA), isolated from skin biopsies taken from AD and PV patients and healthy volunteers were performed. (3): hBD-2 mRNA and PSMB8 mRNA correlated with some parameters of clinical assessment of inflammatory disease severity. hBD-2 mRNA expression, exclusively, was sufficient to distinguish inflammatory skin biopsies from the healthy ones. (4): hBD-2 mRNA and PSMB8 mRNA analysis were the most valuable parameters in differentiating AD and PV biopsies.

Bacillus subtilis Induces Human Beta Defensin-2 Through its Lipoproteins in Human Intestinal Epithelial Cells.

Human intestinal epithelial cells (IECs) play an important role in maintaining gut homeostasis by producing antimicrobial peptides (AMPs). Bacillus subtilis, a commensal bacterium, is considered a probiotic. Although its protective effects on intestinal health are widely reported, the key component of B. subtilis responsible for its beneficial effects remains elusive. In this study, we tried to identify the key molecules responsible for B. subtilis-induced AMPs and their molecular mechanisms in a human IEC line, Caco-2. B. subtilis increased human beta defensin (HBD)-2 mRNA expression in a dose- and time-dependent manner. Among the B. subtilis microbe-associated molecular patterns, lipoprotein (LPP) substantially increased the mRNA expression and protein production of HBD-2, whereas lipoteichoic acid and peptidoglycan did not show such effects. Those results were confirmed in primary human IECs. In addition, both LPP recognition and HBD-2 secretion mainly took place on the apical side of fully differentiated and polarized Caco-2 cells through Toll-like receptor 2-mediated JNK/p38 MAP kinase/AP-1 and NF-κB pathways. HBD-2 efficiently inhibited the growth of the intestinal pathogens Staphylococcus aureus and Bacillus cereus. Furthermore, LPPs pre-incubated with lipase or proteinase K decreased LPP-induced HBD-2 expression, suggesting that the lipid and protein moieties of LPP are crucial for HBD-2 expression. Q Exactive Plus mass spectrometry identified 35 B. subtilis LPP candidates within the LPP preparation, and most of them were ABC transporters. Taken together, these results suggest that B. subtilis promotes HBD-2 secretion in human IECs mainly with its LPPs, which might enhance the protection from intestinal pathogens.

Periodontal health status in patients with lung cancer: Case-control study.

Objective: The objective of this study was to assess the periodontal health status of individuals with lung cancer in the North Indian population. In addition, the study aimed to determine the levels of human beta-defensin2 (Hbd-2) in the gingival crevicular fluid (GCF) and serum samples collected from the participants. Methods: The study consisted of a total of 90 participants, who were categorized into three groups: Group 1 included 30 healthy individuals, Group 2 comprised 30 patients with chronic periodontitis, and Group 3 involved 30 patients diagnosed with both lung cancer and chronic periodontitis. Various periodontal parameters, including plaque index, gingival index, probing pocket depth, and clinical attachment level (CAL), were assessed in addition to the analysis of human beta defensin2 levels in both the GCF and serum samples of all participants. Results: The study results revealed that all clinical parameters assessed were higher in Group 3 compared to both Group 2 and Group 1. Specifically, the levels of hBD-2 in the GCF were measured as 52.29 ± 46.41 pg/mL in Group 1, 27.15 ± 28.76 pg/mL in Group 2, and 86.01 ± 68.82 pg/mL in Group 3. When comparing the hBD-2 levels in serum, the values were found to be 813.72 ± 269.43 pg/mL in Group 1, 591.50 ± 263.91 pg/mL in Group 2, and 1093.04 ± 674.55 pg/mL in Group 3. These intergroup comparisons indicate variations in hBD-2 levels among the different groups. Conclusions: The study findings demonstrated significantly higher clinical and biochemical markers in patients with both lung cancer and chronic periodontitis, in comparison to individuals with chronic periodontitis alone and healthy participants. These results suggest that Hbd-2 could potentially serve as a valuable diagnostic biomarker for identifying and distinguishing individuals with both lung cancer and chronic periodontitis.

Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin.

(1) Background: Atopic dermatitis is one of the most common inflammatory skin diseases characterized by T helper (Th) 2 and Th22 cells producing interleukin (IL)-4/IL-13 and IL-22, respectively. The specific contribution of each cytokine to the impairment of the physical and the immune barrier via Toll-like receptors (TLRs) is poorly addressed concerning the epidermal compartment of the skin. (2) Methods: The effect of IL-4, IL-13, IL-22, and the master cytokine IL-23 is evaluated in a 3D model of normal human skin biopsies (n = 7) at the air-liquid interface for 24 and 48 h. We investigated by immunofluorescence the expressions of (i) claudin-1, zonula occludens (ZO)-1 filaggrin, involucrin for the physical barrier and (ii) TLR2, 4, 7, 9, human beta-defensin 2 (hBD-2) for the immune barrier. (3) Results: Th2 cytokines induce spongiosis and fail in impairing tight junction composition, while IL-22 reduces and IL-23 induces claudin-1 expression. IL-4 and IL-13 affect the TLR-mediated barrier largely than IL-22 and IL-23. IL-4 early inhibits hBD-2 expression, while IL-22 and IL-23 induce its distribution. (4) Conclusions: This experimental approach looks to the pathogenesis of AD through molecular epidermal proteins rather than cytokines only and paves the way for tailored patient therapy.

Human beta defensin-2 loaded PLGA nanoparticles impregnated in collagen-chitosan composite scaffold for the management of diabetic wounds.

Diabetic wound (DW) is the most devastating complication resulting in significant mortality and morbidity in diabetic patients. The standard treatment of DW care fails to address the prerequisites of treating DW owing to its multifactorial pathophysiology. Henceforth, developing a single treatment strategy to handle all the loopholes may effectively manage DW. The objective of the current study was to formulate Human beta defensin-2 (HBD-2) loaded Poly (lactic-co-glycolic acid) (PLGA) nanoparticle impregnated in collagen/chitosan (COL-CS) composite scaffolds for the accelerated healing of DW. Upon investigation, the developed biodegradable crosslinked scaffold possesses low matrix degradation, optimum porosity, and sustained drug release than the non-crosslinked scaffold. In vitro studies revealed that the HBD-2 COL-CS scaffold was biocompatible and accelerated cell migration and angiogenesis. The HBD-2 COL-CS scaffold showed significant antimicrobial activity in S. aureus, E. coli, and P. aeruginosa. The in vivo studies revealed that the HBD-2 COL-CS treated group accelerated healing compared to those in COL-CS and control groups. The ELISA results indicated a significant decrease in MMP-9, TNF-α, MPO, NAG, and NO with an increase in IL-10 in HBD-2 COL-CS treated group. The accelerated healing in HBD-2 COL-CS treated group might be due to the synergistic effects of PLGA (collagen synthesis and deposition and positive angiogenic effect), HBD-2 (anti-inflammatory, antibacterial, positive angiogenic effect, cell proliferation, and migration), COL (established wound healer and stabilizer) and CS (antibacterial, controlled drug release).

Reuterin Isolated from Lactobacillus reuteri Indonesian Strain Affected Interleukin-8 and Human Beta Defensin-2 on Pathogens Induced-HaCat Cells.

Probiotic Lactobacillus reuteri has positive effects on health through inhibiting pathogenic bacteria and the ability to reduce inflammation. This study investigates the ability of reuterin isolated from L. reuteri Indonesian strain for increasing mRNA expression of interleukin (IL)-8 and human beta-defensin (hBD)-2 gene by epithelial cells, after exposure to oral bacteria. L. reuteri isolated from Indonesian's saliva, and species was confirmed by PCR, using 16S rRNA specific gene. To produce reuterin, the isolate was mixed in glycerol-containing MRS broth. Reuterin molecule's weight was counted by SDS-PAGE. Streptococcus mutans ATCC-25175 and Porphyromonas gingivalis ATCC-33277 were put in water (80°C) for 30 min, and each killed bacterial (107 CFU/mL) was inoculated into HaCat cell line (105 cell/mL). Reuterin was added in different concentrations (100%, 50%, 25%, 12,5%) and different incubation time at 37°C, 5% CO2. RNA was extracted, and a reverse transcription procedure was performed to obtain cDNA. Subsequently, a quantitative PCR method was performed to analyse the transcription level of IL-8 and HBD-2 mRNA expressed by inflamed HaCat cells. All results were statistically analysed by ANOVA test. PCR assays showed that clinical isolates were L. reuteri. Quantitative PCR results showed reuterin decreased the expression of IL-8 and increased the expression of hBD-2 in all concentrations and time periods set in this study (p < 0.05). Reuterin isolated from L. reuteri Indonesian strain increased expression of human beta defensin-2 as antimicrobial peptide and may be useful in combating inflammation.

Increased fecal human beta-defensin-2 expression in preterm infants is associated with allergic disease development in early childhood.

Background: This study aimed to investigate whether fecal human beta-defensins (HBD)-2 and eosinophil cationic protein (ECP) expression in preterm infants are associated with allergic disease development by age 2 years. Methods: Preterm infants' stool samples were collected at the age of 6 and 12 months postnatally. Information regarding medication exposure histories (antibiotics, antipyretics, probiotics) and physician-diagnosed allergic diseases was obtained using age-specific questionnaires and medical records. We compared the 6-month and 12-month fecal HBD-2 and ECP concentrations between the medication exposure and non-exposure group, respectively, and between children who developed allergic diseases and those who did not by 2 years of age. Univariate and multivariable logistic regression analyses were performed to investigate independent variables related to physician-diagnosed allergic diseases by 2 years of age. Results: Seventy-four preterm infants (gestational age, 31-36 weeks) were included. Fecal HBD-2 levels were significantly increased at 12 months of age among children who developed allergic diseases compared to those who did not (37.18 ± 11.80 ng/g vs. 8.56 ± 4.33 ng/g, P = 0.011). This association was more apparent among allergic children given antibiotics (50.23 ± 16.15 ng/g vs. 9.75 ± 7.16 ng/g, P = 0.008) or antipyretics (46.12 ± 14.22 ng/g vs. 10.82 ± 6.81 ng/g, P = 0.018) during the first year, whereas among allergic children who were previously not exposed to antibiotics or antipyretics, the differences were not significant. Results of the multivariable logistic regression analysis indicated that HBD-2 concentration in 12-month stools was an independent indicator associated with physician-diagnosed allergic diseases by 2 years of age (adjusted odds ratio: 1.03 [95% confidence interval: 1.00-1.05], P = 0.036). Our data revealed a lack of association between fecal ECP and allergic diseases. Conclusions: We found that preterm infants who expressed high fecal HBD-2 at 12 months of age were associated with physician-diagnosed allergic diseases by the age of 2 years. Further studies are needed to determine the role of fecal HBD-2 in the development of allergic diseases.

Clinical Efficacy of Maxingshigantang Enema in Treating Infants with Pneumonia and Asthma and Intervention Mechanism of Lung-intestine Microenvironment / 中国实验方剂学杂志

ObjectiveTo observe the clinical efficacy of Maxingshigantang enema in the treatment of infant viral pneumonia by comparing related indicators， and comprehensively evaluate the effect of traditional Chinese medicine （TCM） enema on the intestinal microenvironment. MethodSixty infants with viral pneumonia were selected and randomly divided into 3 groups. The dosage of enema drugs in high- （0.117 g·mL-1） and low-concentration （0.07 g·mL-1） TCM enema groups was same （3.5 g per time）， and the control group received normal saline enema， once a day for 7 days. Finally， the curative effect， total symptom score， salivary secretory immunoglobulin A （sIgA）， human beta defensin 2 （hBD2） and fecal calprotectin （CALP） of each group were statistically analyzed by SPSS 21.0， and the clinical efficacy of TCM enema in treating children with pneumonia and asthma was comprehensively evaluated. ResultThe curative effect of high-concentration TCM enema group （total effective rate 100%， χ2=7.059） was equivalent to that of low-concentration TCM enema group （total effective rate 95%， χ2=4.329）， higher than that of control group （total effective rate 70%） （P<0.017）. After treatment， compared with control group and low-concentration TCM enema group， high-concentration TCM enema group had higher total symptom score of children （P<0.05， P<0.01）. The proportion of coccobacillus was reduced in three groups， with high- and low-concentration TCM enema groups lower than control group （P<0.05）. The salivary sIgA concentration was increased in three groups （P<0.05）， with high-concentration TCM enema group higher than the other groups （P<0.01）. The hBD2 concentration was decreased in three groups， with high- and low-concentration TCM enema groups lower than control group （P<0.05）. The three groups reduced the fecal CALP concentration， and high-concentration TCM enema group had the highest reduction， followed by low-concentration TCM enema group （P<0.01）. ConclusionTCM enema outweighs western medicine in improving clinical symptoms， intestinal flora， and mucosal immune function， and reducing inflammation in children， and the high-concentration TCM enema group has better curative effect. Therefore， with easiness to operate， high compliance， and significant therapeutic effect， TCM enema is worthy of clinical promotion.

A combination of fecal calprotectin and human beta-defensin 2 facilitates diagnosis and monitoring of inflammatory bowel disease.

Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) show a large overlap in clinical presentation, which presents diagnostic challenges. As a consequence, invasive and burdensome endoscopies are often used to distinguish between IBD and IBS. Here, we aimed to develop a noninvasive fecal test that can distinguish between IBD and IBS and reduce the number of endoscopies.We used shotgun metagenomic sequencing to analyze the composition and function of gut microbiota of 169 IBS patients, 447 IBD patients and 1044 population controls and measured fecal Calprotectin (FCal), human beta defensin 2 (HBD2), and chromogranin A (CgA) in these samples. These measurements were used to construct training sets (75% of data) for logistic regression and machine learning models to differentiate IBS from IBD and inactive from active IBD. The results were replicated on test sets (remaining 25% of the data) and microbiome data obtained using 16S sequencing.Fecal HBD2 showed high sensitivity and specificity for differentiating between IBD and IBS (sensitivity = 0.89, specificity = 0.76), while the inclusion of microbiome data with biomarkers (HBD2 and FCal) showed a potential for improvement in predictive power (optimal sensitivity = 0.87, specificity = 0.93). Shotgun sequencing-based models produced comparable results using 16S-sequencing data. HBD2 and FCal were found to have predictive power for IBD disease activity (AUC ≈ 0.7).HBD2 is a novel biomarker for IBD in patients with gastro-intestinal complaints, especially when used in combination with FCal and potentially in combination with gut microbiome data.

Molecular dynamics simulations and functional studies reveal that hBD-2 binds SARS-CoV-2 spike RBD and blocks viral entry into ACE2 expressing cells.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and long-term medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical and biochemical assays confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (KD ~ 300 nM), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSV-G mediated infection, of ACE2 expressing human cells with an IC50 of 2.4± 0.1 µM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as novel agents to prevent SARS-CoV-2 infection.

Comparison of gingival crevicular fluid and serum human beta-defensin-2 levels between periodontal health and disease.

BACKGROUND: The aim of this study was to evaluate the levels of serum and gingival crevicular fluid (GCF) human beta-defensin-2 (hBD-2), an antimicrobial peptide that takes roles in inflammatory diseases, in patients with chronic periodontitis (CP). SUBJECTS AND METHODS: A total of one hundred and one individuals, 59 controls and 42 patients with CP, participated in this study. Clinical index measurements were recorded during the periodontal examination, and radiographic evaluation was also performed. The serum and gingival crevicular fluid (GCF) samples were taken from all of the participants, and the hBD-2 levels were determined biochemically by enzyme-linked immunosorbent assay (ELISA). RESULTS: In our study, hBD-2 GCF levels in CP (stages II-IV periodontitis based on the new 2018 classification of periodontal diseases) group (2.77 ng/30 s) were higher than in the periodontally healthy (2.51 ng/30 s; p = .047) individuals. In contrast, serum hBD-2 levels in CP (2.92 ng/ml) were lower compared with those in healthy controls (7.75 ng/ml, p < .001). CONCLUSION: Interestingly, our results showed that while higher hBD-2 GCF levels are associated with CP, lower serum hBD-2 levels were detected in CP.

The Improvement of Immune Effect of Recombinant Human Beta-Defensin 2 on Hepatitis B Vaccine in Mice.

Immunization with hepatitis B vaccine is an effective measure for prevention and control of hepatitis B Virus (HBV) infection. Although lots of efforts to improve the effect of hepatitis B vaccine have been made, the function of human beta defensin 2 (hBD2) on hepatitis B vaccine keeps unclear. In this article, we report that hBD2 not only promoted the activation and maturation of immature dendritic cells (iDCs) by increasing MHC II and CD86 expression, but it also significantly upregulated the mRNA level of IL-6 and IL-12B in mouse bone marrow-derived dendritic cells. The serum concentrations of IFN-γ in mice stimulated with 300 ng hBD2 increased from 25.21 to 42.04 pg/mL, with a time extension from 4 to 12 h post-injection. During the process of three times immunization (1, 14, 28 days) with 3 µg hepatitis B vaccine combined with or without 300 ng hBD2 with a 2 week interval in BALB/c mice, the antibody against HBsAg (HBsAb) concentration in serum at every time point of observation in the combined group was statistically higher than the hepatitis B vaccine group. The serum concentration of IgG2a subclass HBsAb on the 14th day post last injection in the combined group was significantly higher than the hepatitis B vaccine group. Further, the splenic cells from the mice treated with both hBD2 and hepatitis B vaccine possessed a greater ability to produce a surface antigen of hepatitis B virus (HBsAg) specific IFN-γ than those treated with hepatitis B vaccine alone. The percentages of CD3+/CD4+ T cells and CD3+/CD8+ T lymphocytes in spleens from the mice treated with 300 ng hBD2 were statistically higher than the phosphate buffered saline group. These data suggest that hBD2 improves iDC maturation and the immune efficiency of hepatitis B vaccine in BALB/c mice.

Copy number variation, gene expression and histological localization of human beta-defensin 2 in patients with adeno-tonsillar hypertrophy.

Both bacterial infections and innate oral immunity response participate in development of adeno-tonsillar hypertrophy (ATH). ATH can lead to obstructive sleep apnea. We investigated the beta-defensin 2 (hBD-2) encoding gene, DEFB4, by analyzing the copy number variations (CNVs) of the defensin gene cluster in patients with ATH and by correlating CNV with DEFB4 gene expression. We enrolled 79 patients with ATH, 21 of whom presented with only adenoid hypertrophy, while 58 exhibited hypertrophy of both adenoid and tonsil. CNVs of the defensin gene cluster, DEFB4 mRNA, and hBD-2 protein expression were assessed. Also, beta-defensin 2 was localized histologically using immunohistochemistry. The distribution of defensin gene cluster CNV was similar among the 79 subjects. DEFB4 expression analysis exhibited considerable inter-individual variability, but with neither specific differences among subjects nor correlation with the CNV number. Immunohistochemistry enabled localization of hBD-2 in the tonsil and adenoid epithelium. No differences in localization between the two ATH presentations were found. Inducible antimicrobial defensin peptides exhibited great inter-individual variability in terms of both CNV and gene expression, but no correlation with presentation of ATH was found.

Efficacy of antimicrobial photodynamic and photobiomodulation therapy against Treponema denticola, fusobacterium nucleatum and human beta defensin-2 levels in patients with gingivitis undergoing fixed orthodontic treatment: A clinic-laboratory study.

BACKGROUND: Evidence suggests that molecular pathways are involved in human ß-defensin (hBD)-2 mRNA regulation in human gingival epithelial cells stimulated with periodontal bacteria. This clinical and laboratory study evaluated the efficacy of two laser therapies including antimicrobial photodynamic therapy (aPDT) and photobiomodulation (PBM) therapy as an adjunct to ultrasonic scaling (US) on the gingival crevicular fluid (GCF) levels of hBD-2 and subgingival Treponema denticola (T. denticola) and Fusobacterium nucleatum (F. nucleatum) spp., in patients undergoing fixed orthodontic therapy and gingivitis. MATERIALS AND METHODS: Forty-five patients undergoing fixed orthodontic treatment were randomly divided into three groups based on the type of treatment rendered: Group-I: aPDT as an adjunct to US, Group-II: PBM as an adjunct to US and, Group-III: US alone. Full-mouth plaque scores (FMPS), bleeding on probing (FMBOP) and probing depth (PD) were assessed. GCF was collected for estimation of hBD-1 using enzyme-linked immunosorbent assay. Plaque samples were used to quantify T. denticola and F. nucleatum spp by quantitative polymerase chain reaction. All clinical and laboratory investigations were carried out at baseline (T0), day 30 (T30) and day 60 (T60). RESULTS: FMPS and FMBOP showed statistically significant reduction in all groups at T30 and T60 from T0. No inter-group differences were observed between any groups at follow-up. Mean PD remained stable for Group-II and Group-III, while Group-I showed progressive reduction at T60. The GCF levels of hBD-2 progressively decreased in Group-I (aPDT) while the levels increased slightly at T60 in Group-III. The levels in Group-II (PBM) remained stable from T30 to T60. Statistically significant reduction was seen for Group-I when compared with Group-II and Group-III at T60 (p = 0.045). A significant reduction was observed for T. denticola in only Group-I patients at T30 (p = 0.031) and T60 (p = 0.047). A significant reduction was seen in both Group-I and Group-II patients at T30 and T60. The number of sites with BOP was correlated with both bacterial species (Table 4). Only T. denticola showed positive correlation to mean BOP after correcting for multiple testing. CONCLUSION: aPDT and PBM showed similar improvement in gingival inflammatory and microbiological parameters compared to US. aPDT assisted in modest reduction of hBD-2 in patients undergoing fixed orthodontic treatment.

Specific gut microbiome members are associated with distinct immune markers in pediatric allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Increasing evidence reveals the importance of the microbiome in health and disease and inseparable host-microbial dependencies. Host-microbe interactions are highly relevant in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT), i.e., a replacement of the cellular components of the patients' immune system with that of a foreign donor. HSCT is employed as curative immunotherapy for a number of non-malignant and malignant hematologic conditions, including cancers such as acute lymphoblastic leukemia. The procedure can be accompanied by severe side effects such as infections, acute graft-versus-host disease (aGvHD), and death. Here, we performed a longitudinal analysis of immunological markers, immune reconstitution and gut microbiota composition in relation to clinical outcomes in children undergoing HSCT. Such an analysis could reveal biomarkers, e.g., at the time point prior to HSCT, that in the future could be used to predict which patients are of high risk in relation to side effects and clinical outcomes and guide treatment strategies accordingly. RESULTS: In two multivariate analyses (sparse partial least squares regression and canonical correspondence analysis), we identified three consistent clusters: (1) high concentrations of the antimicrobial peptide human beta-defensin 2 (hBD2) prior to the transplantation in patients with high abundances of Lactobacillaceae, who later developed moderate or severe aGvHD and exhibited high mortality. (2) Rapid reconstitution of NK and B cells in patients with high abundances of obligate anaerobes such as Ruminococcaceae, who developed no or mild aGvHD and exhibited low mortality. (3) High inflammation, indicated by high levels of C-reactive protein, in patients with high abundances of facultative anaerobic bacteria such as Enterobacteriaceae. Furthermore, we observed that antibiotic treatment influenced the bacterial community state. CONCLUSIONS: We identify multivariate associations between specific microbial taxa, host immune markers, immune cell reconstitution, and clinical outcomes in relation to HSCT. Our findings encourage further investigations into establishing longitudinal surveillance of the intestinal microbiome and relevant immune markers, such as hBD2, in HSCT patients. Profiling of the microbiome may prove useful as a prognostic tool that could help identify patients at risk of poor immune reconstitution and adverse outcomes, such as aGvHD and death, upon HSCT, providing actionable information in guiding precision medicine.

Human ß-Defensin 2 Expression in Oral Epithelium: Potential Therapeutic Targets in Oral Lichen Planus.

Human ß-defensin 2 (hBD-2) is a potent antimicrobial peptide that participates in defense against invading bacteria. We recently showed that bacterial components and histamine, through histamine H4 receptor (H4R), are involved in the pathogenesis of the potentially malignant lesion, oral lichen planus (OLP). However, the underlying mechanisms remain unknown. We, therefore, investigated the role of hBD2-histamine crosstalk signaling in promoting OLP pathology. Biopsies from OLP and oral tongue squamous cell carcinoma (OTSCC) patients, and healthy controls were used. Two OTSCC cell lines and normal human oral keratinocytes (HOKs) were used. HBD-2 and other targets were mapped by immunostaining and analyzed by ImageJ2 software. The highly sensitive droplet-digital PCR technology and qRT-PCR were utilized to study the clinically derived and in vitro samples, respectively. H4R was challenged with the specific agonist HST-10 and inverse agonist ST-1007. HBD-2 was highly induced in OLP lesions. In contrast, hBD2 expression was attenuated in OTSCC tissues, while very low levels of hBD-2 messenger RNA (mRNA) were observed in OTSCC cells. Together with tumor necrosis factor-α (TNF-α), histamine upregulated hBD-2 mRNA expression in HOKs. Activation of H4R seems to modulate the expression of epithelial hBD-2. These findings suggest the involvement of hBD-2 in the pathogenesis of OLP and may, thus, be harnessed for therapeutic interventions in OLP.

The guardians of the periodontium-sequential and differential expression of antimicrobial peptides during gingival inflammation. Results from in vivo and in vitro studies.

AIM: To evaluate the sequential and differential expression of a variety of antimicrobial peptides (AMPs) during the development of an experimentally induced gingivitis in humans. MATERIAL AND METHODS: In twenty healthy volunteers, gingival inflammation was induced by abstention from oral hygiene at 6 teeth. Bleeding on probing (BOP) and plaque index (PI) were assessed, and gingival biopsies and gingival crevicular fluid (GCF) were collected at 8 different time points (t0-t35). Gingival epithelial cells (GECs) were stimulated with various receptor agonists. In biopsies and GECs, mRNA expression of human beta-defensins (hBD-2, hBD-3), CC-chemokine ligand 20 (CCL20), S100A7/psoriasin (S100A7), and calgranulin A/B (S100A8, S100A9) was evaluated using real-time PCR, and protein profiles were measured by ELISA. Statistical analysis was performed using non-parametric tests. RESULTS: The clinical parameters BOP, PI and GCF increased over time (p < 0.0001). Tissue AMP mRNA expression was elevated, but at different and AMP-specific time points (p < 0.05). Protein analysis revealed a similar expression pattern for hBD-2 and CCL20 in GCF (p < 0.05). In GECs, multiple receptor stimulation was required to induce AMP gene expression (p < 0.0001). CONCLUSIONS: For the first time, this study showed the sequential and differential expression of AMPs during a developing inflammation in vivo providing further evidence for their role as guardians of a healthy periodontium.

Water-based extracts of Zizania latifolia inhibit Staphylococcus aureus infection through the induction of human beta-defensin 2 expression in HaCaT cells.

Zizania latifolia is a perennial herb belonging to the family Gramineae that has been used as a health food in Asian countries. In this study, we investigated the antimicrobial effect of Z. latifolia, which increased human beta-defensin 2 (hBD2) expression in HaCaT cells. hBD2 expression was further increased in cells treated with Z. latifolia extracts and subsequently infected with Staphylococcus aureus. Inversely, S. aureus infection decreased after treatment. The induction of hBD2 in HaCaT cells was mediated by the Toll-like receptor 2 (TLR2) signaling pathway, including the activation of extracellular signal-regulated kinase (ERK) and activator protein 1 (AP-1). Further study using siRNA revealed that hBD2 played an important role in the inhibition of S. aureus infection in HaCaT cells. Our data suggest that Z. latifolia extracts can be used as an antimicrobial ingredient for skin treatment formulas.

Evaluation of the effect of pulsed dye laser on chronic psoriatic plaque.

BACKGROUND: Psoriasis is one of the most prevalent immune mediated skin diseases. Selective vascular destruction by pulsed dye laser is considered one of effective laser treatments. OBJECTIVE: Evaluation of the therapeutic effect of pulsed dye laser in treatment of chronic psoriatic plaque lesions and changes in human beta defensin-2(HBD-2) expression in the psoriatic patients before and after treatment, with correlation to the clinical improvement. METHODS: Twenty patients with psoriatic plaque on right side treated by laser and another plaque on left side similar in size and its severity score served as control receiving no treatment. Each patient received 3-4 sessions of pulsed dye laser 595 nm. Follow up was done for three months. RESULTS: There was statistically significant difference between the two psoriatic plaques (untreated and treated; A and B; p = .004). There was significant decrease in HBD-2 expression after treatment. No recurrence was observed during follow up period. CONCLUSION: The pulsed dye laser is safe, effective, and tolerable treatment for resistant stable localized plaque psoriasis with minimal side effects and prolonged recurrence period.

Serum Human Beta-Defensin-2 Is a Possible Biomarker for Monitoring Response to JAK Inhibitor in Psoriasis Patients.

AIMS: To analyse the correlation between serum human beta-defensin-2 (hBD-2) levels and response to JAK inhibitor in psoriasis. METHODS: We evaluated the psoriasis area and severity index (PASI) and serum hBD-2 levels of 18 psoriasis patients randomized to receive placebo or tofacitinib 5 or 10 mg b.i.d. at baseline, week 8, and week 16. Serum hBD-2 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The PASI achieved a dramatic reduction after tofacitinib 5 or 10 mg b.i.d. treatment for 16 weeks (p < 0.05). Serum hBD-2 levels significantly decreased in patients treated with tofacitinib 10 mg b.i.d. compared with baseline and the placebo-treated patients (p < 0.05). A significant correlation was found between hBD-2 levels and PASI (r = 0.52, p < 0.01). A serum hBD-2 level of 1,255.45 pg/mL was a cut-off between mild and moderate-to-severe psoriasis in ROC analysis. CONCLUSIONS: Serum hBD-2 level might be a possible biomarker for monitoring psoriasis treatment response and differentiating mild from moderate-to-severe psoriasis.