Clinical study of serum human-βeta-defensin-1 level for evaluating short-term prognosis in patients with acute-on-chronic liver failure / 中华肝脏病杂志

Objective: To evaluate the diagnostic value of serum human-βeta-defensin-1 level (HBD-1) for short-term (28-day) prognosis in patients with acute-on-chronic liver failure (ACLF). Methods: Fifty cases diagnosed with ACLF were selected. 20 cases with decompensated cirrhosis and 20 cases with compensated cirrhosis who were admitted at the same time were included. Age, gender, serum HBD-1 level, C-reactive protein (CRP), procalcitonin (PCT), neutrophil count/lymphocyte ratio (NLR), blood routine, coagulation function, liver function, kidney function, and other indicators from the three groups of patients were collected. Patients with ACLF were screened for indicators related to the short-term (28-day) prognosis. Patients were divided into an improvement group and a worsening group according to the 28-day disease outcome. The serum HBD-1 level and other above-mentioned indicators were compared between the two patient groups. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of serum HBD-1 levels for short-term prognosis in patients with ACLF. PCT, NLR, and prothrombin activity (PTA) application as a mono indicator and HBD-1 in combination with NLR, PCT, and PTA were compared to evaluate diagnostic efficacy for short-term prognosis in patients with ACLF. The intergroup mean of measurement data was determined using a t-test or analysis of variance. χ (2) test was used for comparison of count data. Spearman's rank correlation analysis was used for correlation analysis. Results: There was no statistically significant difference in age and gender among the three groups: ACLF, decompensated cirrhosis, and compensated cirrhosis (P > 0.05). The expression levels of serum HBD-1 in the ACLF group, decompensated cirrhosis group, and compensated cirrhosis group were (319.1 ± 44.4) ng/ml, (264.5 ± 46.5) ng/ml and (240.1 ± 35.4) ng/ml, respectively, while the ACLF group expression levels were significantly increased, with statistical significance (P < 0.01).The serum HBD-1 level was significantly higher in the ACLF worsening group (346.2 ± 43.6) ng/ml than that in the improvement group (308.5 ± 40.6) ng/ml, and the difference was statistically significant (P < 0.05). Correlation analysis showed that HBD-1, NLR, PCT, prothrombin time (PT), and international standardized ratio (INR) were negatively correlated with the 28-day disease outcome (improvement) of patients (P < 0.05). PTA was positively correlated with 28-day disease outcome (improvement) (P < 0.05). The area under the receiver operating characteristic curve (AUC) for evaluating HBD-1's diagnostic efficacy for short-term prognosis in patients with ACLF was 0.774, with a sensitivity of 0.750, a specificity of 0.786, and a cut-off point of 337.96 ng/ml. PCT, NLR, and PTA had greater diagnostic efficacy. HBD-1 combined with PTA had the highest diagnostic efficacy, with an AUC of 0.802, a sensitivity of 0.778, and a specificity of 0.786. The diagnostic efficacy of HBD-1+PCT, HBD-1+NLR and HBD-1, PCT, and NCR was superior to PTA mono. Conclusion: The serum HBD-1 level gradually increases with the aggravation of liver function injury and is negatively correlated with the short-term prognosis in patients with ACLF. Serum HBD-1 level has high sensitivity and specificity in predicting short-term prognosis in patients with ACLF, and its diagnostic efficacy is superior to that of PCT, NLR, and PTA. The combined application of HBD-1 and PTA has higher diagnostic efficacy; however, when the serum HBD-1 level is greater than 337.96ng/ml, it indicates poor prognosis in patients.

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.

The Relation Between Decay Missing Filled-Teeth (DMF-t), Body Mass Index (BMI) with Salivary Human Beta-3 (HBD-3) Secretion in Children with Caries and Free Caries

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

Serum defensin levels in patients with systemic sclerosis

Abstract Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and lung as well as involvement of kidney, gastrointestinal system and heart. Aetiology and exact mechanism of disease is poorly understood. The association between antimicrobial peptides (AMPs) and other diseases such as idiopathic pulmonary fibrosis, diffuse panbronchiolitis, pulmoner alveolar proteinosis and psoriasis have been reported. A small number of studies have examined the role of AMPs on autoimmune diseases which has not been studied in scleroderma yet. We aimed to investigate AMP serum levels and their association with disease characteristics of SSc. Methods: Forty-two patients (40 female, mean age 42 years) and 38 healthy subjects (32 female, mean age 38 years) were enrolled. For SSc patients, the following data were recorded: disease subset (limited/diffuse), autoantibodies (antinuclear, anti-centromere (ACA), and anti-SCL-70), blood tests, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), modified Rodnan skin score, presence and history of digital ulcers, kidney, gastrointestinal disease and lung involvement assessed by computed tomography and pulmonary function tests. Association between serum AMPs and disease characteristics were analysed. Results: Twenty-nine of the patients had diffuse (69%) and 13 of the patients had limited (31%) systemic sclerosis. Average disease duration was 5.5 years. Pulmonary involvement was detected in 20 patients (47.6%). Serum concentration of alpha defensin was higher than healthy subjects (563 ± 415 vs 377 ± 269 ng/mL, p = 0.02). However, no difference was observed for beta-1 and beta-2 defensins in SSc patients and healthy controls. In sub-group analysis patients with interstitial lung disease had higher levels of alpha defensin than those without lung involvement (684 ± 473 vs 430 ± 299 ng/ml, p = 0.04). There was also correlation between alfa defensin serum concentrations and CRP (r = 0.34). Conclusions: Alpha defensin levels are increased in scleroderma patients and correlated with lung involvement indicating a role in the pathogenesis of disease. Trial registration: This study is not a clinical trial study.(AU)

Expressions of intestinal alpha-defensin-5, beta-defensin-2 in acute liver failure / 中华实用儿科临床杂志

Objective To investigate the dynamic changes in intestinal alpha-defensin-5 (RD-5),beta-defensin-2 (BD-2) mRNA after acute liver failure(ALF),and to explore their role in ALF.Methods A total of 60 C57BL5 mice were divided into 4 groups by means of random number table method:normal control group,ALF group,E.coli via gavage group and ALF + E.coli via gavage group.Intraperitoneal injection of D-galactosamine (500 mg/kg) and lipopolysaccharide(10 μg/kg) to make the model,in addition,ALF mice were fed with E.coli,and the observation time was 6 hours,12 hours,and 24 hours after modeling,and each time point had 6 specimens.Real-time PCR was used to test the RD-5 mRNA and BD-2 mRNA levels in the ileum tissue.Results The levels of RD-5 and BD-2 showed dynamic change in the experiment of ALF.Compared with the levels of RD-5 and BD-2(11.25 ±0.74,23.86 ±0.39) of the normal control group,the levels of RD-5 and BD-2 in ALF group and E.coli via gavage group increased at 6 hours after modeling(14.19 ±0.39,26.79 ± 0.36 and 12.57 ± 0.68,26.45 ± 0.85),and the differences were significant(all P＜0.05);at 12 hours after modeling,the RD-5 and BD-2 reached to the maximum concentration(15.76 ±0.33,29.10 ± 0.61 and 12.90 ± 0.96,27.42 ± 0.71),and the differences were statistically signi-ficant (all P ＜ 0.05).The degree of elevation of BD-2 was higher than RD-5.Later,they gradually declined.Conclusions RD-5 and BD-2 may play an important role in the pathogenesis of intestinal endotoxemia in experimental ALF.

Expression of human β-defensin 1 in peripheral blood of children with community-acquired pneumonia in terms of clinical features / 中国感染与化疗杂志

Objective To investigate the expression of human beta defensin 1 (HBD-1) in peripheral blood of children with community acquired pneumonia (CAP) and its clinical implication.Methods A total of 122 CAP children were included in this study from February 2015 to October 2015.The patients were stratified in terms of age,etiology,and disease severity.Additional 52 patients were included as control in the same period.All patients were classified according to the clinical feature after admission.Serum HBD-1 level was determined by ELISA method.Results Serum HBD-1 level was significantly higher in CAP group than in control group (P＜0.001).HBD-1 level did not vary significantly with pathogen or sex in CAP group.HBD-1 level did not show significant difference between severe pneumonia and mild pneumonia.The HBD-1 level in 6-12 months age group was significantly higher than that in other age groups.HBD-1 level was not correlated to CRP or neutrophils in CAP children.Conclusions HBD-1 plays a role in anti-infective process as part of body innate immunity.It boasts non-specific and broad-spectrum anti-infective immunity against various pathogens.

Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2), transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 <i>in vitro </i>and <i>in vivo</i> and to assess the probability of defensins as a new application for infectious corneal diseases in the future. <p>METHODS: The synthetic rBD-2 DNA fragment was inserted between the <i>Xho</i>I and <i>BamHI</i> restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into <i>E.coli DH5α</i>, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. <p>RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. <p>CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in the transfected cells.

gene polymorphism and Crohn′s disease in Han population Correlation between interferon regulatory factor 5 ,vitamin D receptor ,beta-defensin 1 ,Toll-like receptor 4 / 中华消化杂志

ObjectiveTo investigate the correlation between interferon regulatory factor 5 (IRF5) ,vitamin D receptor (VDR ) ,beta-defensin 1 (DEFB1 ) ,Toll-like receptor 4 (TLR4 ) gene polymorphismand Crohn′s disease (CD) in Chinese Han population .MethodsFrom January 2007 to May 2011 ,thedata and serum samples of 158 CD patients and 246 healthy controls were collected .The genotype of 14tag single-nucleotide polymorphisms (SNP) of IRF5 ,VDR ,DEFB1 and TLR4 were detected .Chi-squaretest was performed for rate comparison between CD group and healthy control group . Multifactordimensionality reduction (MDR) was used to analyze the combined effects of above candidate genes and therelation with susceptibility of CD .ResultsAccording to allele or genotype correlation analysis ,there wasno correlation between IRF5 ,VDR ,DEFB1 ,TLR4 and susceptibility of CD (all P> 0 .05) .The resultsof haplotype correlation analysis indicated that the frequency of GTACC haplotype in IRF5 of CD groupand healthy control group was 0 .046 and 0 .089 ,respectively ,the difference was statistically significant (χ2 = 5 .223 ,P= 0 .022 3) .The results of genotype and clinical type analysis indicated that the genotypesof rs2978880 of DEFB1 in CD patients were C/C ,C/T ,T/T ,the frequency of patients with surgery was0 .235 ,0 .603 and 0 .162 ,respectively ,and the frequency of patients without surgery was 0 .482 ,0 .388and 0 .129 ,respectively .The risk of intestinal surgery in patients with C /C genotype was lower (χ2 =10 .065 ,P= 0 .006 ) .The results of MDR analysis indicated that no interactions were detected betweenabove genes and susceptibility of CD (all P > 0 .05) .ConclusionsThe GTACC haplotype in IRF5 wascorrelated with the susceptibility of CD ,and the C/C genotype of rs2978880 of DEFB1 was correlatedwith CD clinical phenotype in Chinese Han population .

Human beta defensin-4 and keratinocyte growth factor gene expression in cultured keratinocyte and fibroblasts of burned patients

PURPOSE: To evaluate KGF and human beta defensin-4 (HBD-4) levels produced by dermic fibroblasts and keratinocytes cultivated from burned patients' skin samples. METHODS: Keratinocytes and fibroblasts of 10 patients (four major burns, four minor burns and two controls) were primarily cultivated according to standard methods. HBD-4 and KGF genes were analyzed by quantitative PCR. RESULTS: In fibroblasts, KGF gene expression was 220±80 and 33.33±6.67 (M±SD; N=4), respectively for major and minor burn groups. In keratinocytes, KGF gene expression was 11.2±1.9 and 3.45±0.37 (M±SD; N=4), respectively for major and minor burn groups. In fibroblasts, HBD-4 gene expression was 15.0±4.0 and 11.5±0.5 (M±SD; N=4), respectively for major and minor burn. In keratinocyte, HBD-4 gene expression was 0.0±0.0 and 13.4±4.8 (M±SD; N=4), respectively for major and minor burn. CONCLUSIONS: KGF expression was increased in burn patient fibroblasts compared to control group. In keratinocytes culture, KGF suppression is inversely proportional to burn extension; it is active and increased in major burn but decreased in minor burn. HBD-4 expression was increased in fibroblasts and decreased in keratinocytes from all burned patients. .

The Expression of beta-defensin 1 in Various Skin Tumors / 대한피부과학회지

BACKGROUND: Among antimicrobial peptides produced by keratinocytes, beta-defensins and LL-37 are the major human antimicrobial peptides. Because beta-defensins have recently received great attention for its roles associated with host defense and cancer development, we hypothesized that beta-defensin 1 has an influence on the differentiation and malignant changes of various skin tumors originating from keratinocytes and malignant melanomas. OBJECTIVE: This study was conducted to measure the expression of beta-defensin 1 in different skin tumors. METHODS: The expression of beta-defensin 1 was examined by immunohistochemical staining of 57 specimens of skin tumors including 12 cases of seborrheic keratosis, 4 cases of keratoacanthoma, 12 cases of actinic keratosis, 10 cases of basal cell carcinoma, 16 cases of squamous cell carcinoma and 3 cases of malignant melanoma. RESULTS: Immunohistochemical analysis of skin tumor tissue samples revealed significantly higher expression of beta-defensin 1 in benign skin tumors (seborrheic keratoses, keratoacanthomas) and premalignant lesions (actinic keratoses) than in malignant tumors (squamous cell carcinomas, basal cell carcinomas, and malignant melanomas). Among malignant tumors, basal cell carcinomas and malignant melanomas showed lower expression of beta-defensin 1 than squamous cell carcinomas, in which their expression was shown to be decreased with increased dysplasia of the tumor cells. CONCLUSION: These findings showed that beta-defensin 1 has a negative association with malignancies of skin tumors. The role of beta-defensin 1 is suggested in the protection of malignant changes including loss of differentiation and dysplasia in skin tumors.

Ultrastructural immunolocalization of beta-defensin-27 in granulocytes of the dermis and wound epidermis of lizard suggests they contribute to the anti-microbial skin barrier / 대한해부학회지

The high resistance to infections in lizard wounds suggests that these reptiles possess effective antimicrobial peptides in their tissues. The present immunocytochemical study shows the cellular localization of beta-defensin 27 in tail tissues and in the blood, a defensin previously identified in the lizard Anolis carolinensis through biomolecular methods. Beta-defensin-27 immunoreactivity is only observed in some large granules mainly contained in heterophilic granulocytes that are sparse within the dermis of the skin or in the isolated blood. This peptide is absent in other cell types of the skin, in keratinocytes and in subdermal muscle tissue of the tail in normal conditions. Pre-corneous keratinocytes of the regenerating tail epidermis are unlabeled or show a weak labeling for the peptide only in sparse cytoplasmic areas or in the extracellular spaces among corneocytes of the wound and regenerating epidermis. The study suggests that beta-defensin 27 is normally stored in granulocytes present in the blood or in connective tissues while in the epidermis keratinocytes do not show the presence of this peptide unless these cells are stimulated from injury to produce and likely release beta-defensins.

Acne Inversa: Evaluating Antimicrobial Peptides and Proteins

BACKGROUND: Acne inversa is a chronic, suppurative relapsing inflammatory skin disease that primarily affects the axillae, perineum and inframammary regions. Evidence suggests that the innate immune system is involved in the pathogenesis of acne inversa. OBJECTIVE: To investigate the role of the innate immune system in acne inversa. METHODS: Skin biopsies were obtained from inflammatory skin lesions (n=17) and from non-lesional skin (intraindividual control, n=17) of patients with acne inversa. Additional skin lesions were taken from patients with chronic venous leg ulcers (interindividual control, n=5). Quantitative real-time reverse transcription-polymerase chain reaction was used to determine the mRNA levels of antimicrobial peptides and proteins (AMPs), including human beta-defensin (hBD)-1, hBD-2 and hBD-3, LL-37 (cathelicidin) and Ribonuclease 7 (RNase 7). mRNA levels were also determined for inflammatory and anti-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinase-1 (MMP1), interleukin (IL)-1beta, IL-6, IL-8 and IL-10. RESULTS: The mRNA levels of hBD-2, LL-37, IL-1beta, IL-6, IL-8, IL-10 and MMP1 were significantly higher in acne inversa lesions compared to non-lesional skin (p<0.05). A significant positive correlation expression was observed between hBD-2 mRNA expression and LL-37 (rho=0.53, p=0.03), and between hBD-2 and RNAse 7 (rho=0.68, p=0.006). When compared to the chronic venous leg ulcer lesions, acne inversa lesions showed a significantly higher expression of RNase 7 mRNA, while IL-1 beta, IL-6, IL-8, TNF-alpha and MMP1 mRNA expression was significantly higher in the chronic venous leg ulcer lesions (p<0.05). CONCLUSION: The AMP, cytokine milieu and tissue proteases in acne inversa lesions differ significantly from non-lesional skin and chronic venous leg ulcers. The positively correlating up-regulation of AMPs in acne inversa indicates an important role of the innate immune system in the pathogenesis of this disorder.

Expression of mouse beta defensin 2 in Escherichia coli and its broad-spectrum antimicrobial activity

Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95 percent). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86 percent at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.

Expression of mouse beta defensin 2 in Escherichia coli and its broad-spectrum antimicrobial activity

Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86% at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.

Constructed a cell line to express hBD1 stablly and detected the antimicrobial activity of hBD1 to multidrug resistant bacterial strains / 中华微生物学和免疫学杂志

ObjectiveTo established a cell line that expresses hBD1 stably,and detected the antimicrobial activity of the hBD1 to the muhidrug resistant bacterial strains.MethodsRecombinant plasmid was introduced into COS-7 cells by lipofectamine,cells were selected in culture medium containing G418 to acquired the monoclonal cell lines,total RNA were extracted from the cultured cells,expression levels of hBD1 mRNA was identified by RT-PCR,collected the supernatant solution of the cultured cell,expression levels of protein was identified by Western blot.Put the expression products and resistant organisms mixed together,after incubation in different times in 37℃,coating the mixtures in LB flat,then obtained the ratios between colonies number of experimental groups and colonies number of control groups,put those ratios as the survival rate of the drug resistance bacterias.Results The monoclonal cell lines had obtained after screened with G418,the hBD1 gene could be detected both at transcriptional and protein levels,Under the influence of expression product hBD1,survival rate of muhidrug-resistant Acinetobacter baumannii,multidrug-resistant Escherichia coli and multidrug-resistant Klebsiella pneumoniae could reduced to 9%,22% and 50%,but survival rate of multidrug-resistant Stenotrophomonas maltophilia is not have apparente difference with the control group.ConclusionThe stably-transfected cell line of hBD1 was successfully constructed,and the expression products of hBD1 showed the antimicrobial activity toward multidrug resistant bacterial strains.

Prokaryotic expression of mouse interleukin 17A and its ability of stimulating RAW264.7 cell inflammatory factors expression / 中华微生物学和免疫学杂志

Objective To express and purify mouse interleukin 17A(mIL-17A) in E. coli and to analyze its ability of stimulating macrophage inflammatory factors expression. Methods The coding gene of mouse mIL-17A mature protein was amplified from mouse spleen cells by RT-PCR. PCR product was cloned into the prokaryotic expressing vector pET28a, and the resulting recombinant plasmid pET28a/mIL-17a was then transformed into the host E. coli strain BL21(DE3) for expression. The mIL-17A protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by the Ni-NTA affinity chromatography, and was further tested on the stimulation of cytokine and chemokine of RAW264.7 cells by ELISA and real-time quantity PCR in vitro. Results The mIL-17A with bioactivity was over-expressed and purified successfully, and the results of real-time PCR and ELISA showed that recombinant mIL-17A stimulated macrophage mRNA upregulation of IL-6, defensin β2 and Cxcl3 and secretion of defensin β2, Ccl3, Cxcl3,IFN-γ, IL-6 and IL-4. Interestingly, these effects could be blocked by the addition of anti-IL-17A neutralizing antibody partly. After treatment with mIL-17, 74. 87-fold of defensin β2 mRNA expression was increased comparing with that of untreated cells( P ＜0.01 ), while blocking with anti-IL-17A antibody the increase was only 5.4-fold(P ＜ 0.01 ). Conclusion The recombinant mIL-17A has a strong stimulation on secretion of cytokine and chemokine of macrophage, that maybe result to the enhancement of anti-infection ability of macrophage.

Effect of polymorphisms of Crohn disease related NOD2 gene and human beta-defensin 2 on expres-sion of human beta-defensin 2 / 中华消化内镜杂志

Objective To explore the effects of polymorphisms of Crohn's disease related NOD2 gene and human beta-defensin 2 (hBD-2) on transcription of hBD-2 gene and its mechanism. Methods HEK293T cells were transfected with hBD-2 gene and NOD2 eukaryotic expression plasmid, and were then stimulated with LPS, TNF-α, or BAY 11-7082 (antagonist of NF-κB), respectively. Transcriptional activity of hBD-2 was detected afterwards. Results LPS could suppress transcription of hBD-2 (P=0. 020), which was increased by TNF-α in a dose-dependent manner (P =0. 004). In the presence of LPS, there was sig-nificant difference in transcriptional activity of hBD-2 between wild-NOD2 transfected group and mutated NOD2 (P268S) transfected group (P=0. 008), but there was no significant difference between wild hBD-2 transfected group and mutated hBD-2 transfected group (P=0. 053). With the stimulation of TNF-α (5 ng/ml), there was a significant difference between mutated hBD-2 transfected group and wild hBD-2 transfected group (P=0. 006), but no significant difference between wild-NOD2 transfected and mutated NOD2 transfected group was defected (P = 0. 064). Pretreatment with BAY 11-7082 before TNF-α (5 ng/ml) significantly inhibited the transcriptional activity of hBD-2 (P < 0. 001). Conclusion The poly-morphism of NOD2 affects the innate expression of hBD-2, the polymorphism of site in hBD-2 promoter (-233) may lead to significant decline of the inducible expression of hBD-2, and NF-κB might be a key pathway that NOD2 protein mediates the expression of defensin.

TNF-alpha-induced Increase of Human beta-defensin-2 Expression in HaCaT Cell Lines / 대한피부과학회지

BACKGROUND: Several kinds of epithelial cells and focal lymph nodes are known to be involved in the skin's immune reaction. Especially, internal antimicrobial peptide play an important role in protecting microbial agents. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which is produced by epithelial cells after stimulation with microorganisms or inflammatory mediators. hBD-2 participates in the increase of the cell-mediated immune reaction. It also affects the proliferation and differentiation of epithelial cells and fibroblasts, resulting in enhancement of wound healing. However, little is known as to whether the TNF-alpha induces the expression of hBD-2 in HaCaT cells through the NF-kappaB or MAPKs pathways. OBJECTIVE: Research was undertaken to investigate the roles of NF-kappaB and MAPKs transcription factors in the molecular pathway of TNF-alpha-induced hBD-2 expression in HaCaT cell lines. METHODS: The expression of hBD-2 in TNF-alpha-treated HaCaT cells was analyzed by immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). The expression of NF-kappaB was analyzed by Western blot analysis and electrophoretic mobility shift assay (EMSA). RESULTS: Strong positive hBD-2 immunofluorescence staining in TNF-alpha-treated HaCaT cells was observed. According to RT-PCR analysis, the expression of hBD-2 increased TNF-alpha-treated HaCaT cells by dose-dependent and time-dependent manners. In addition, according to Western blot analysis and EMSA, NF-kappaB was also activated in TNF-alpha-treated HaCaT cells. Interestingly, the expression of hBD-2 in TNF-alpha-treated HaCaT cells was attenuated in the presence of NF-kappaB inhibitors, PDTC or MG132. Furthermore, MAPKs inhibitors, especially SB (p38 inhibitor), partially attenuated the TNF-alpha-induced hBD-2 expession, but not PD (ERK inhibitor) and SP (JNK inhibitor). CONCLUSION: These results collectively suggest that hBD-2 is up-regulated in TNF-alpha-treated HaCaT cells through activation of NF-kappaB and p38 MAPKs pathway. Our data regarding the up-regulation of hBD-2 may help us to understand the antimicrobial mechanism in normal skin or in skin diseases.

Immunohistochemical Study of Human beta Defensin-2 Expression in Superficial Mycosis / 대한피부과학회지

BACKGROUND: Normal human skin is resistant to infection with various kinds of microorganisms by producing anti-microbial chemicals. Human beta defensin-2 (hBD-2) is an anti-microbial peptide that has recently been shown to be expressed in various epithelial cells and inflammatory diseases. However, the expression of hBD-2 in fungus-infected skin is not well-known. OBJECTIVE: This study was performed to investigate the expression pattern of hBD-2 in superficial mycosis. METHODS: Using the immunohistochemical method with formalin-fixed, paraffin-embedded sections, we checked the expression levels and localization of hBD-2 in lesional skin samples of tinea capitis (5 patients), tinea corporis (6 patients), candidiasis (3 patients), Malassezia folliculitis (2 patients), and psoriasis (3 patients) as positive control, and normal skin samples from 6 healthy subjects as negative control. RESULTS: The expression of hBD-2 was not observed in normal skin, but moderate to strong expression of hBD-2 was observed in the epidermis, and the papillary dermal infiltrating cells of psoriasis. In tinea capitis, strong hBD-2 expression was found in the upper spinous layer of epidermis and follicular epidermis, and perifollicular inflammatory cells. In tinea corporis and candidiasis, mild to strong expression of hBD-2 was found in the horny or spinous layer of epidermis and infiltrating inflammatory cells. Strong hBD-2 expression was found in the follicular epidermis and perifollicular inflammatory cells of Malassezia folliculitis. CONCLUSION: These results suggest that hBD-2 plays an important role in cutaneous innate immune defense against fungal infection.

Expression of Human beta-defensin 2 mRNA by Lipopolysaccharide in Human Corneal Epithelial Cells

Recently the transcriptional up-regulation of human beta-defensin 2 (HBD-2) by lipopolysaccharide (LPS) was found to be associated with NF-kappaB binding site. Although the general mechanisms of NF-kappaB activation by LPS stimulation are well understood, less is known about the signal transduction pathway leading to LPS-induced NF-kappaB activation in human corneal epithelial (HCE) cells. The aim of this study was to investigate the intracellular signals involved in LPS-induced HBD-2 mRNA expression in HCE cells. Pretreatments of inhibitors for NF-kappaB, protein tyrosine kinase, p38 mitogen activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) attenuated the LPS-induced NF-kappaB DNA binding activity and HBD-2 mRNA expression. Furthermore, pretreatments with inhibitors for protein kinase C (PKC), phosphatidylcholine-phospholipase C, phosphatidylinositol-phospholipase C, or phosphatidate phosphohydrolase prevented LPS-induced HBD-2 mRNA expression and HBD-2 prmoter-driven luciferase activity. However, the increased expression of HBD-2 mRNA and the increased DNA binding activity of NF-kappaB induced by LPS were not changed by the blockage of extracellular signal-regulated kinase (ERK) and of addition of antioxidants. Forskolin, a protein kinase A (PKA) agonist did not induce HBD-2 mRNA expression. These data demonstrate that LPS-induced HBD-2 mRNA expression via NF-kappaB is, at least in part, dependent on PKC, p38 MAPK, JNK, and protein tyrosine kinase status, but appears to be independent on PKA, ERK and ROS in HCE cells. Taken together, there may be more than one signaling pathways that lead to LPS-induced up-regulation of HBD-2 mRNA expression in HCE cells.