Impact of smoking on protein levels of beta-defensins in periodontal disease

Abstract Antimicrobial peptides (AMPs) are important components of the host response against invading pathogens. In addition to their direct antimicrobial activity, they can also participate in the immune system modulation. However, the role of AMPs in the etiopathogenesis of periodontal disease and the risk factors that may influence their expression in the oral cavity are not fully understood. The aim of this study was to determine the impact of smoking on beta-defensin (hBD) 1 and 2 levels analyzing samples from periodontitis patients. Fifty patients with periodontitis, 25 smokers and 25 non-smokers, and 20 periodontally healthy patients were recruited. After periodontal clinical evaluation, gingival crevicular fluid (GCF) samples were collected from healthy sites of patients without periodontal disease and from healthy and diseased sites of patients with periodontitis. Peptides quantification was performed by sandwich ELISA technique. Smokers showed reduced GCF hBD 1 levels and increased hBD 2 levels compared to non-smokers in diseased sites (p <0.05). Higher levels of hBD 1 were observed in healthy sites of patients without periodontal disease than in healthy sites of patients with periodontitis (p<0.0001). Diseased sites of non-smokers presented higher levels of hBD 2 than healthy sites (p <0.05). These results reveal that protein levels of hBDs 1 and 2 can be impaired by cigarette smoking in the presence of periodontal disease.

Resumo Peptídeos antimicrobianos (PAMs) são componentes importantes da resposta do hospedeiro contra patógenos invasores. Além de sua atividade antimicrobiana direta, eles também podem participar da modulação do sistema imunológico. No entanto, o papel dos PAMs na etiopatogenia da doença periodontal e os fatores de risco que podem influenciar a sua expressão na cavidade oral não são totalmente compreendidos. O objetivo deste estudo foi determinar o impacto do tabagismo nos níveis de beta-defensina (hBD) 1 e 2 analisando amostras de pacientes com periodontite. Cinquenta pacientes com periodontite, 25 fumantes e 25 não fumantes e 20 pacientes periodontalmente saudáveis foram recrutados. Após avaliação clínica periodontal, amostras de fluido crevicular gengival (FCG) foram coletadas de sítios saudáveis de pacientes sem doença periodontal e de sítios saudáveis e doentes de pacientes com periodontite. A quantificação dos peptídeos foi realizada pela técnica de ELISA sanduíche. Fumantes apresentaram níveis reduzidos de hBD 1 no FCG e níveis aumentados de hBD 2 em comparação com não fumantes em locais doentes (p <0,05). Níveis mais elevados de hBD 1 foram observados em sítios saudáveis de pacientes sem doença periodontal do que em sítios saudáveis de pacientes com periodontite (p<0,0001). Os sítios doentes de não fumantes apresentaram níveis mais elevados de hBD 2 do que os sítios saudáveis (p<0,05). Esses resultados revelam que os níveis das hBDs 1 e 2 podem ser prejudicados pelo tabagismo na presença de doença periodontal.

The level of antimicrobial peptides in gingival crevicular fluid and its correlation with periodontal clinical indexes in elderly patients with type 2 diabetic periodontitis / 口腔疾病防治

Objective@#To investigate the changes and significance of human beta-defensin-2 (HBD-2) and LL-37 in the gingival crevicular fluid of patients with periodontitis and type 2 diabetes mellitus (T2DM).@*Methods@#This study was conducted among 45- to 85-year-old patients in the Department of Stomatology and Internal Medicine of Shenzhen Center for Chronic Disease Control, including a healthy control group of 22 people, a systemically healthy control group of 19 people with periodontitis, a T2DM periodontal health group of 15 people, and a T2DM group of 21 people with periodontitis. The Florida periodontal probe was used for periodontal examination, and the clinical indexes, including probing depth (PD), clinical attachment level (CAL) and probing on bleeding (BOP), were recorded. The concentrations of HBD-2 and Ll-37 in gingival crevicular fluid were determined by ELISA. The differences in HBD-2, LL-37 and periodontal clinical indexes between the groups were compared, and correlation analysis was conducted.@*Results@#The PD values in T2DM with the periodontitis group were higher than those of the systemically healthy controls with periodontitis group (P < 0.05); the levels of HBD-2 and LL-37 in gingival crevicular fluid in systemically healthy controls with periodontitis group were significantly higher than those in the healthy control group (P < 0.05), the level of HBD-2 in gingival crevicular fluid in systemically healthy controls with periodontitis group was significantly higher than that in T2DM with periodontitis group (P < 0.05); and the antimicrobial peptides HBD-2 and LL-37 in gingival crevicular fluid were significantly positively correlated with the PD and CAL in systemically healthy controls with periodontitis group (P < 0.05), and there was no significant correlation between the antimicrobial peptides HBD-2, LL-37 in gingival crevicular fluid and PD, CAL in T2DM with periodontitis group (P > 0.05).@*Conclusion@#The levels of antimicrobial peptides HBD-2 and LL-37 in gingival crevicular fluid of middle-aged and elderly patients with T2DM periodontitis were lower, and there was no significant correlation with PD and CAL in periodontal clinical indicators.

Quantification of Beta-Defensins (DEFB) Gene Copy Number Variations in Relation to Inflammation in Type 2 Diabetes Mellitus and Diabetic Nephropathy Patients

@#Introduction: Association studies between single nucleotide polymorphisms (SNPs) and type 2 diabetes mellitus (T2DM) have been abundant. However, there are limited reports on copy number variations (CNVs) of beta-defensins (DEFB) gene in relation to T2DM. In this study, DEFB copy numbers were quantified in T2DM with nephropathy, T2DM without nephropathy and non-diabetic control groups to investigate its influence in chronic inflammation in Malaysian individuals. Methods: DEFB copy number in Malaysian individuals were quantified by using paralogue ratio tests (PRT) which allow direct quantification of gene copy number by using PRT107A and HSPD21 PRT primers. The copy number generated was then validated from insertion/deletion ratio measurement 5DEL (rs5889219) and two microsatellite analyses (EPEV-1 and EPEV-3). Results: DEFB copy number was found extending from 2 to 8 copies in the non-diabetic group (n=146), while in T2DM group (n=392), copy numbers were more extensive, ranging between 1 and 12 copies; with 1, 10 and 12 copies detected in T2DM with nephropathy group (n=202). Statistically, there is no significant difference in DEFB copy number between T2DM and the non-diabetic group (p=0.209) as well as between diabetic nephropathy and without nephropathy of the T2DM group (p=0.522). However, significant white blood cell (WBC) count was found between T2DM groups with and without diabetic nephropathy (p=0.000). Conclusion: Extreme DEFB copy numbers in T2DM with nephropathy group suggest future studies with bigger sample size are necessary to elucidate the true impact of CNVs of DEFB gene in promoting early onset of nephropathy in T2DM.

Associação entre beta-defensinas e periodontite / Association between beta-defensins and periodontitis: literature review

Objetivo: as beta-defensinas humanas (hBDs) podem ter um papel-chave na susceptibilidade às doenças na cavidade bucal. Além do efeito antimicrobiano direto, as hBDs aumentam a imunidade adaptativa. O objetivo deste estudo foi realizar uma revisão de literatura científica sobre a relação entre beta-defensinas (hBD) e periodontite. Material e métodos: foi realizada uma pesquisa bibliográfica na base de dados PubMed sobre a expressão de hBDs em indivíduos com periodontite. Os termos beta defensins e periodontitis foram utilizados nessa busca. Resultados: foram selecionados, por um revisor, sete artigos para serem incluídos nessa revisão de literatura: dois estudos de intervenção e cinco estudos transversais. Conclusão: o número de estudos sobre a expressão de beta-defensinas em indivíduos com periodontite é reduzido. O conhecimento sobre o papel das beta-defensinas na periodontite pode trazer um maior entendimento de sua etiopatogenia, além de possibilitar novos indicadores de risco e terapias. Estudos adicionais são necessários para a elucidação da relação entre esses peptídeos antimicrobianos e a periodontite.

Objective: human beta-defensins (hBDs) may play a key role in the susceptibility to diseases in the oral cavity. In addition to the direct antimicrobial effect, hBDs enhance adaptive immunity. The objective of this study was to investigate the literature on the relationship between hBD and periodontitis. Material and methods: a literature review was conducted in the PubMed database on the expression of hBDs in subjects with periodontitis. The terms "beta-defensins" and "periodontitis" were used in this search. Results: seven articles were selected being: two intervention studies and fi ve cross-sectional studies. Conclusion: the number of studies on the expression of beta-defensins in individuals with periodontitis is reduced. Knowledge about the role of beta-defensins in periodontitis may lead to a better understanding of their etiopathogenesis, in addition to providing new risk indicators and therapies. Additional studies are needed to elucidate the relationship between these antimicrobial peptides and periodontitis.

An investigation of human beta-defensins and cathelicidin expression in patients with pterygium / Uma investigação da expressão de beta-defensinas humanas e de catelidicina em pacientes com pterígio

ABSTRACT Purpose: To investigate human beta-defensins (HBDs) and cathelicidin LL-37 (LL-37) expressions in patients with pterygium. Methods: In this retrospective consecutive case series, 26 pterygium specimens and 15 normal conjunctival specimens of 15 control subjects were in vestigated. Expressions of HBD-1, HBD-2, HBD-3, and LL-37 were assessed using immuno histochemical staining. A brown color in the cytoplasm and/or nuclei of epithelial cells indicated positive staining for HBDs and LL-37. For each antibody, the intensity of the reaction (negative [-], weak [1+], moderate [2+], or strong [3+]) was determined to describe the immunoreactions. Results: The median age was 52 years in both groups. There were no significant differences in age and sex between the groups (p=0.583, p=0.355, respectively). Of the 26 pterygium specimens, 15 (57.7%) (14 weak, 1 moderate staining) showed HBD-2 expression, which was not observed in any of the control specimens. One (3.8%) pterygium and one (6.7%) control specimen demonstrated weak staining for HBD-3. HBD-2 expression was significantly higher in the pterygium specimens than in the controls (p=0.002). None of the tissue specimens had positive staining for HBD-1 or LL-37 in either group (both; p=1.00). Conclusions: HBD-2 expression was higher in pterygium specimens than in the controls. HBD-2 expression that might be stimulated by inflammatory cytokines may be related to inflammation and fibrovascular proliferation and may play a role in pterygium pathogenesis.

RESUMO Objetivo: Investigar as expressões beta defensinas humanas (HBD) e catelicidina em pacientes com pterígio. Métodos: Nesta série de casos retrospectivos consecutivos, 26 espécimes de pterígio e 15 espécimes conjuntivais normais de 15 indivíduos controle foram investigados. As expressões de HBD-1, HBD-2, HBD-3 e catelicidina (LL-37) foram avaliadas por coloração imuno-histoquímica. Uma cor castanha no citoplasma ou nos núcleos de células epiteliais foi definida como coloração positiva para HBDs e LL-37. Para cada anticorpo foi determinada a intensidade da reação (negativo [-], fraco [1+], moderado [2+] ou forte [3+]) para descrever as imunoreações. Resultados: A idade média foi de 52 anos em ambos os grupos. Não houve diferença significativa entre os grupos em termos de idade e sexo (p=0,583, p=0,355, respectivamente). Das 26 amostras de pterígio, 15 (57,7%) (14 fracas e 1 moderada) demonstraram a expressão de HBD-2 enquanto não foi encontrada em nenhum dos espécimes de controlo. Um dos pterígios (3,8%) e um dos espécimes de controlo (6,7%) demonstraram fraca coloração para HBD-3. A expressão de HBD-2 foi significati vamente maior nos espécimes de pterígio do que nos controles (p=0,002). Nenhum dos espécimes de tecido apresentou coloração positiva para HBD-1 ou LL-37 em ambos os grupos (ambos p=1,00). Conclusão: Encontramos aumento da expressão de HBD-2 em espécimes de pte rígio em relação aos controles. A expressão de HBD-2 que pode ser estimulada por citocinas inflamatórias pode estar relacionada com inflamação e proliferação fibrovascular e pode desempenhar um papel na patogênese do pterígio.

TLR4/NF-κB signal pathway mediate HBD-2 in patients with preterm premature rupture of membrane associated with histologic chorioamnionitis / 中国综合临床

Objective To observe the effect of human β-defensins-2(HBD-2) for chorioamnionitis(HCA) pregnant women before term premature rupture of fetal membrane(PROM) process,and explore toll-like receptor 4 / nuclear factor-κ B (TLR4 / NF-κB) predominate role in the process of signal transduction pathway in the mechanism.Methods Fifty five women with PROM were enrolled in the study.According to the Results of pathological diagnosis of membranes,pregnant women with PROM divided into histological chorioamnionitis,HCA and non-HCA.The same sample without PROM pregnancies matching the same gestational ages were recruited as control group.We examined the messenger RNA(mRNA) of TLR4,NF-κB p65 and HBD-2 in placenta and fetal membrane real-time reverse transcription polymerase chain reactions by dsDNA-binding dyes of SYBR Green.Results (1)In the placenta,the level of TLR-4(17.15±4.52),NF-κB p65(47.11±14.23),HBD-2mRNA(27.35±2.67) in PROM group were significantly higher than the level of TLR-4(7.21±3.25),NF-κB p65(30.51±13.05),HBD-2mRNA(13.55±0.8) in control group(t=-1.966,-1.474,-1.754,P0.05).Conclusion Linear positive correlation of TLR4,NF-κB and HBD2 indicated that TLR4/NF-κB/HBD2 signal transduction pathway may be involved in the development of preterm premature rupture of membrane associated with histologic chorioamnionitis.

Construction and identification of recombinant lentivirus carrying human beta defensins-3 and connective tissue growth factor gene / 军事医学

Objective To construct a recombinant lentivirus containing human beta defensins -3 ( hBD3 ) , connective tissue growth factor gene (CTGF) and enhanced green fluorescent protein (EGFP), and to detect its translation in rabbit bone marrow mesenchymal stem cells (BMSC).Methods The lentivirus containing hBD3, CTGF and EGFP genes was constructed in vitro.The titer of lentivirus was tested with end-paint dilution assay .Rabbit BMSCs were transfected with recombinant virus.The best value of multiplicity of infection (MOI) was tested.The expression condition, transfection efficacy and genetic stability of the target genes were evaluated by using fluorescence microscopy and flow cytometry . Western blotting was used to detect the expression of the target protein .Results Recombinant lentivirus vectors: Lenti-CTGF-hBD3-EGFP, Lenti-hBD3-EGFP, and Lenti-EGFP, were successfully obtained . The titer of the recombinant lentiviruses was 3.21 ×108, 5.80 ×108, and 1.16 ×109, respectively.The best MOI value to transfect BMSCs was 150. The transfection efficacy of these lentivirus vectors was high , reaching 79.72%as assessed by flow cytometry , and it could be stably inherited .Western blotting displayed that target protein expression was successful .Conclusion The construction of recombinant lentiviruses carrying hBD3 and CTGF genes is successful and can be effectively transfected into BMSCs .

Study of anti-inflammatory function of human bea defensin 2 on rats' acute rhinosinusitis / 中国耳鼻咽喉头颈外科

OBJECTIVE To study the antiinflammatory function of human beta defensin 2(hβD-2) on acute rhinosinusitis in rats,in order to provide a new therapy for acute rhinosinusitis.METHODS Acute rhinosinusitis model were established on SD rats before and after the transfection of plasmid,the rats in experimental group were dropped with recombinant hβD-2 plasmid mixture in nose,while with empty plasmid mixture in control group.Immunohistochemistry method was used to prove the transfection results,nasal mucosa were hematoxylineosin stained to compare the pathological difference of nasal mucosa,nasal lavage fliud was collected and cultured to compare the colony number of the bacteria.RESULTS The expression of hβD-2 was confirmed by immunohistochemistry method,which mainly distributed in mucosal epithelium and gland,pathological results showed that the inflammation of nasal mucosa in experimental group was significantly relieved than that in control group.The number of Staphylococcus auresus colony number was significantly decreased in experimental group,while there was no significantly change in the control group.CONCLUSION Recombinant hβD-2 plasmid can be successfully transfected into the nasal mucosa of rats and expressed effectively.The anti-inflammatory ability of nasal mucosa was increased after the transfection,which is expected to provide a new treatment approach for acute rhinosinusitis.

Effect of subcutaneous immunotherapy on serum levels of human beta defensin-2 in children with allergic rhinitis / 中国耳鼻咽喉头颈外科

OBJECTIVE To investigate the effect of subcutaneousimmunotherapy(SCIT) on levels of the serum human beta defensin-2 in children with allergic rhinitis. METHODS 30 cases of children with allergic rhinitis who were treated by SIT were selected as the treatment group, 20 cases of healthy children as the control group. Serum HBD-2 concentration of the control group was tested. Serum HBD-2 concentration of the treatment group was tested at three different time points: before SCIT, half a year after SCIT and one year after SCIT. And total nasal symptom scores(TNSS) and medication scores were recorded at each time point. RESULTS The serum HBD-2 concentration of the control group, that of the treatment group before SIT, half a year after SIT and one year after SIT were 4.62[4.08; 4.87], 3.74[3.37; 4.61], 4.62[4.13; 5.54], 4.79[4.45;6.19]ng/ml. The HBD-2 concentration gradually increased after SCIT. The TNSS of the treatment group before SCIT, half a year after SCIT and one year after SCIT were 7.43±2.15, 4.17±2.16, 4.20±1.92, The medication scores of the treatment group before SCIT, half a year after SCIT and one year after SCIT were 1.25[0.75; 1.38], 0.25[0; 0.75, 0.25[0; 0.75].There was no correlation (all P>0.05) between the serum HBD-2 concentration and TNSS or medication scores of the treatment group. CONCLUSION The serum levels of HBD-2 in patients with allergic rhinitis were lower than those in normal persons. The specific immunotherapy raised the serum HBD-2 levels of allergic rhinitis patients.

Repair of staphylococcus aureus-infected wound with gene-modified C3H10T1/2 cells expressing BPI-BD3 fusion antibiotic peptide / 解放军医学杂志

Objective To study the antibacterial and tissue reparative effect of BPI-BD3 gene-modified mesenchymal stem cells in a mouse model of wound infection. MethodsC3H10T1/2 cells were transfected with recombinant adenovirus vector pAdxsi-BPI-BD3, the expression of BPI-BD3 fusion protein was verified by RT-PCR and Western blotting. Excision wound with a diameter of 1cm was inoculated with Staphylococcus aureus was made on the back of 30 mice. The mice were randomly divided into 3 groups (10 each). Mice in group T were injected with BPI-BD3 gene-modified C3H10T1/2 cells through caudal vein, those in group C were injected with unmodified C3H10T1/2 cells, and in group N were injected with PBS as control. The wound repair result was evaluated by estimation of the percentage of remaining wound area and the amount of wound bacteria under the scar, followed by observation of pathological changes. Inflammatory reactions of the wounds were assessed accordingly. Results The amount of bacteria under the scar was less in group T than in the other two groups (P<0.05). It was also found that the wound healing process was faster in group T than in group C and group N. Pathological observation showed that the inflammatory reaction in group T was also significantly milder than in the other two groups. Conclusion BPI-BD3 gene-modified mesenchymal stem cells may enhance wound repair by controlling infection and promoting tissue regeneration, thus it may be promising in clinical application.

Expression of TIM-3, Human beta-defensin-2, and FOXP3 and Correlation with Disease Activity in Pediatric Crohn's Disease with Infliximab Therapy

BACKGROUND/AIMS: This study investigated the expression of T cell immunoglobulin- and mucin-domain-containing molecule 3 (TIM-3), human beta-defensin (HBD)-2, forkhead box protein 3 (FOXP3), and the frequency of CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) in children with Crohn's disease (CD) during infliximab therapy. METHODS: We enrolled 20 CD patients who received infliximab treatment for 1 year. Peripheral blood and colonic mucosal specimens were collected from all CD patients and from healthy control individuals. RESULTS: A significant difference in TIM-3 mRNA expression was evident in peripheral blood mononuclear cells and colonic mucosa between CD patients before infliximab therapy and the healthy controls (p<0.001 and p=0.005, respectively). A significant difference in HBD-2 mRNA expression was found in colonic mucosa between CD patients before infliximab therapy and the healthy controls (p=0.013). In the active phase of CD, at baseline, the median percentage of T cells that were CD25+ FOXP3+ was 1.5% (range, 0.32% to 3.49%), which increased after inflixmab treatment for 1 year to 2.2% (range, 0.54% to 5.02%) (p=0.008). CONCLUSIONS: Our study suggests that both the adaptive and innate immune systems are closely linked to each other in CD pathogenesis. And the results of our study indicate that it could be a useful therapeutic tool, where restoration of TIM-3, HBD-2 and the function of Tregs may repair the dysfunctional immunoregulation in CD.

The Expression and Clinical Significance of HBD-2 in Gastric Mucosa of H. pylori Associated Gastric MALT Lymphoma / 天津医药

Objective To investigate the expression of humanβ-defensin 2 (HBD-2) in gastric mucosa of Helico-bacter pylori (H. pylori) associated gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and the role of HBD-2 in gastric MALT lymphoma. Methods Forty gastric mucosa specimens from patients with H. pylori associated gastric MALT lymphoma were collected. And 36 gastric mucosa specimens from chronic superficial gastritis without H. pylori infection were included as control group. The expression of HBD-2 was detected by immunohistochemistry staining. Results The ex-pression of HBD-2 was significantly higher in H. pylori associated gastric MALT lymphoma than that of control group. (P＜0.01). The expression of HBD-2 was significantly decreased after the eradication of H. pylori (P＜0.01). The expression of HBD-2 was significantly higher in H. pylori associated gastric MALT lymphoma than that of lymphoma cells (P＜0.01). There was no expression of HBD-2 in lymphoma cells. Conclusion HBD-2 is possibly involved in the pathogenesis of H. pylori associated gastric MALT lymphoma. But whether it has anti-tumor effect is not clear.

In vitro effects of human umbilical cord mesenchymal stem cells on airway secretions of ventilator associated pneumonia in newborns / 中华围产医学杂志

Objective To explore the effect and its possible mechanism of human umbilical cord mesenchymal stem cells (hUCMSCs) on bacterial growth in bronchoalveolar lavage fluid (BALF) of newborns with ventilator associated pneumonia (VAP).Methods Newborns admitted to neonatal intensive care unit (NICU) in Guangdong Women and Children Hospital Affiliated to Guangzhou Medical University from June 1,2012 to December 31,2012 were reviewed.The inclusion criteria were:(1)Positive BALF culture results.(2) Requirement of mechanical ventilation with tracheal intubation.(3) Diagnosed as ventilator-associated pneumonia.Two pieces of BALF samples of newborn were collected and randornaly divided into experimental and control group.hUCMSCs were added into the experimental group,while the same volume of conditioned medium was added into the control group.Both groups were incubated for six hours in humidified CO2 incubator at 37 ℃,then,bacterial growth was assessed by colony forming unit (CFU) counts.Levels of the antimicrobial peptides (Cathelicidin/LL-37 and human HBD-2) were determined by enzyme-linked immunosorbent assay and Western blot.Paired t-test was used for statistical analysis.Results Among the culture results of 31 newborns,there were Klebsiella pneumoniae (6 cases,19.3％),Stenotrophomonas narrow food aeromonas (6 cases,19.3％),Hemolytic staphylococci (5 cases,16.1％),Escherichia coli (3 cases,9.7％),Bacterial meningitis septicemia Elizabeth Platinum (3 cases,9.7％),Acinetobacter baumannii (3 cases,9.7％),Pseudomonas putida (2 cases,6.4％),Pseudomonas aeruginosa (1 case,3.2％),Staphylococcus aureus (1 case,3.2％) and Enterobacter cloacae (1 ease,3.2％).The CFU counts in experimental group were much less than those in control group [(2.60±0.67) ×104] CFU/ml vs [(1.18±0.32) ×105] CFU/ml,(t=-20.19,P＜0.01).Levels of Cathelicidin/LL-37 and HBD-2 in experimental group were higher than those in control group [Cathelicidin/LL-37:(8.98 ± 3.22) ng/ml vs (3.18 ± 1.57) ng/ml,t =17.79,P ＜ 0.01 ;HBD-2:(379.87±11.74) pg/ml vs (39.89±2.86) pg/ml,t=37.62,P＜0.01].Conclusions hUCMSCs had antimicrobial effect on bacterial growth in BALFs from ventilator associated pneumonia possibly by the secretion of antimicrobial peptides (Cathelicidin/LL-37 and HBD-2).

The Expression of Human Beta Defensins by Thermal Injury

PURPOSE: Defensins are important components of innate immune system. These peptides have antimicrobial activity against a wise variety of pathogens that associated with burn wound infection. In particular, human beta-defensins are expressed in normal epidermal region and showed differential expression of some skin disease. We investigated that expression of human beta-defensin by in vitro and ex-vivo by thermal condition. METHODS: To investigate the expression of human beta-defensins in acute burn condition, we cultured keratinocytes and used to rat's skin at this experiment. After thermal condition, we showed the expression of beta-defensins-2 (hBD-2), -3 (hBD-3), keratins, keratinocyte differentiation and junction protein levels by RT-PCR and immunohistochemistry (IHC). RESULTS: HBD-2 & involucrin were down-regulated from 1 hr to 8 hrs in mRNA level. But others were not changed in mRNA level. In protein level, hBD-3 was decreased but pan-cytokeratin and beta-catenin were not changed. CONCLUSION: HBD-2 was down-regulated in thermal injury. Because thermal injury could induce the influence of keratinocyte differentiation and the decrease of skin protection ability. Our results suggested that human beta-defensins plays an important role in protection by several injury.

Correlation of human β-defensin 1 gene polymorphism with fungal susceptibility to severe sepsis / 中华创伤杂志

Objective To investigate the correlation between gene polymorphism within human β defensin 1 (DEFB1) and fungal susceptibility to severe sepsis through case-control association study.Methods A total of211 patients with severe sepsis in ICU were enrolled in the present case control study. Sepsis in this study was diagnosed according to the definition of American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference in 1992 and 2002. Based on the development of fungal infection during ICU stay, all 211 patients were divided into fungal infection group (Group Ⅰ) and control group (Group C). Alleles and genotypes of-1816A/G, -390A/T, -52A/G, -44C/G and-20A/G within DEFB1 gene were assayed in all 211 patients by means of DNA direct sequencing, Allele-specific PCR amplifications or high-throughput site-specific TaqMan assay. Genetic analysis was employed to calculate the distribution frequency of haplotypes. The correlation between the genomic variations (allele,genotype and haplotype) and fungal infection was analyzed by Chi-square test or Fisher's exact test.Odds ratio (OR) was employed to reflect the correlation degree of genetic factor with fungal susceptibility to severe sepsis. Results Group Ⅰ enrolled 80 patients, of whom 43 pstients were male, at age of (60.81 ± 18.30) years. Group C enrolled 131 patients, of whom 80 patients were male, at mean age of (60.42 ± 17.03) years. No significant difference was found between two groups in aspect of gender and age (P＞0.05). The genetic locus of -1816A/G, -390A/T, -52A/G, -44C/G and -20A/G of both groups were in agreement with Hardy Weinberg equilibrium. No significant difference was found between two groups in the distribution of allelic frequencies and genotype frequencies (P ＞0.05). No significant difference was found in the distribution frequency of four common haplotypes of the above five genetic locus such as AAACG, ATGCA, GTGGG and ATACG (all P ＞ 0.05). Conclusions Genetic locus of -1816A/G, -390A/T, -52A/G, -44C/G and-20A/G within DEFB1 gene have no correction with fungal infections in severe sepsis, suggesting that DEFB1 gene polymorphism may not serve as a key genetic marker for the predisposition to fungal infection in severe sepsis.

Effects of hyperoxia on expression of lung β-defensin-2 mRNA in rats / 中华麻醉学杂志

Objective To investigate the effects of hyperoxia on the expression of β-defensin-2 mRNA in the lungs in rats. Methods Forty 21-day old male SD rats weighing 50-60 g were randomly divided into 5 groups ( n = 8 each): control group breathing room air (group C) and 4 hyperoxia groups breathing high concentration of oxygen (92%-94% O2 ) for 12, 24, 48 and 72 h respectively (group H1-4 ). The animals were sarificed at the end of O2 breathing. The lungs were removed for microscopic examination and determination of wet/dry lung weight ratio and expression of β-defensin-2 mRNA (by RT-PCR), total NF-κB p65 protein and NF-κB p65 protein in cell nucleus (by Western blot analysis). The level of activation of NF-κB was calculated. Results The W/D lung weight ratio was significantly higher in group H3 and H4than in group C. The lung injury scores were significantly increased and β-defensin-2 mRNA expression was significantly down-regulated in group H1-4 as compared with groups in a duration of hyperoxia dependent manner. The level of activation of NF-κB was up-regulated in group H1-4 compared with group C and peaked at 48 h of hyperoxia (group H3 ). Conclusion Inhalation of high concentration of O2 can induce lung injury by down-regulating lung β-defensin 2 mRNA expression. NF-κB signal transductinn pathway may not be involved in the underlying mechanism.

Expression and significance of mucosal β-defensin-2,TNFα and IL-1β in ulcerative colitis CHANG / 中华内科杂志

Objective To investigate the expression and significance of human β-defensin-2 (HBD2),TNFα and IL-1βin ulcerative colitis(UC).Methods Thirty-five patients with active UC diagnosed by the department of gastroenterology in West China Hospital were included in this study.Ulcerative colitis disease activity index(UCAI)was assessed and the pathological grades of UC were classified.Immunohistochemistry assay and real-time quantitative PCR were used for the expression of HBD2,TNFα,IL-1β in colonic mucosa of UC.Results Among the 35 patients with UC,10 cases were mild.13 moderate and 12 severe.Of the 35 cases.there were 11 with grade Ⅰ.13 grade Ⅱ and 11 grade Ⅲ lesion according to Truelove criteria.The score of UCAI had positive correlation with pathological grading (r=0.890,P＜0.01).The expressions of HBD2,TNFα,IL-1β in colonic mucosa of UC with immunohistochemistry and real-time quantitative PCR were significantly higher than those in healthy control (P＜0.05);the expressions increased gradually with the severity of pathological grade and there was a higher expression of them in inflamed area than in non-inflamed(P＜0.05).A good positive correlation was also found between HBD2 and other inflammatory cytokines.Conclusions It is shown that there is a higher expression of HBD2 in colonic mucosa as compared with healthy control.a higher expression of it in inflamed area than in non-inflamed area and a positive correlation of expression between HBD2 and pro-inflammatory cytokines such as TNFα and IL-1 β,implying that HBD2 and pro-inflammatory cytokines are interdependent and interactive playing an important role in magnifying and aggravating inflammatory injury in UC.

Relationship of genetic polymorphisms of heme oxygenase-1, tumor necrosis factor-α, human β-defensin -1 with chronic obstructive pulmonary disease / 中华老年医学杂志

Objective To investigate the relationship of smoking, the gene polymorphisms of heine oxygenase-1, tumor necrosis factor-α, and human β-defensin-1 with chronic obstructive pulmonary disease (COPD). Methods A case-control study was held to compare the genotype frequency distribution of heine oxygenase-1, tumor necrosis factor-α, human β-defensin-1 by PCR -RFLP method and to analyze the relationship among them. Results The L allele gene frequency of heine oxygenase-1 was 23.44% in COPD group and 10.71% in control group with significant difference(P＜0.05). The heterogenesis gene frequency of tumor necrosis factor-α in COPD group and control group were 18. 24% and 7.53% (P＜0.01). The heterogenesis gene frequency of human β-defensin-1 was 14.28% in COPD group and 5.00% in control group with significant difference(P＜0. 01). Conclusions Smoking is a risk factor for COPD. Genetic polymorphisms of heme oxygenase-1, tumor necrosis factor-α, human β-defensin-1 are related with the development of COPD.

Effects of mechanical stretch on the expression of human beta-defensin-3 in alveolar epithelial cells elicited by interferon- gamma / 中华急诊医学杂志

Objective To study the effect of mechanical stretch on the expression of human beta-defensin-3 (HBD-3) in alveolar epithelial cells(A549 cells) elicited by interferon-gamma(IFN-γ) and to investigate the role of HBD-3 in the pathogenesis of ventilator-associated pneumonia (VAP) . Method A549 cells cultured in vitro were treated with mechanical stretch (group S), 10 ng/ml IFN-γ (group I) ,and 10 ng/ml IFN-γ with mechanical stretch (group IS), respectively. Cells without treatment served as controls (group C). Cells were stretched by 20% amplitude of stretch at 30 cycles/mm by Flexercell-4000[TM]Unit for 2 h, 4 h, and 6 hours. The HBD-3 mRNA expression was determined by real-time RT-PCR after treatment. After 6 hours, treatment, cells were cultured for 24 hours and the expression of HBD-3 was examined by laser scanning confocal microscope. The experimental data were statistically analyzed by using one-way ANOVA analysis and q-test. Results The expression of HBD-3 mRNA in A549 cells could not significantly be changed by mechanical stretch alone. Compared with group C,the HBD-3 mRNA expression after treatment with 10 ng/ml IFN-γ for 2 hours,4 hours and 6 hours increased significantly by (2.63 C,the HBD-3 mRNA expression after treatment with 10 ng/ml IFN-γ and mechanical stretch for 2 hours,4 hours and 6 hours increased by (1.54 were significantly lower than those in group I (P < 0.01). The HBD-3 expression in group IS after mechanical stretch for 6 significantly different from than in group C. Conclusions Mechanical stretch can significantly suppress the up-regulation of HBD-3 in alveolar epithelial cells elicited by IFN-γ, and this may be one of the explaina-tions that patients under mechanical ventilatiori(MV) have a higher risk of VAP.

Human beta-defensin 2 is induced by interleukin-1b in the cornealepithelial cells

Mammalian epithelia produce the various antimicrobial peptides against the bacterial or viral infection, thereby acting as the active immune modulators in the innate immunity. In this study, we examined the effects of the various proinflammatory cytokines or LPS on cell viability and antimicrobial beta-defensin gene expressions in human corneal epithelial cells. Results showed that the cytokines or LPS did not exert severe cytotoxic effects on the cells, and that beta-defensin 1 was constitutively expressed, while beta-defensin 2 was specifically induced by IL-1beta, supporting the idea that these cytokines or LPS involve the defense mechanism in the cornea. Furthermore, the reporter and gel shift assay to define the induction mechanism of beta-defensin 2 by IL-1beta demonstrated that the most proximal NF-kB site on the promoter region of beta-defensin 2 was not critical for the process. Data obtained from the normal or patients with the varying ocular diseases showed that our in vitro results were relevant in the clinical settings. Our results clearly demonstrated that beta-defensin 1 and 2 are important antimicrobial peptides in the corneal tissues, and that the mechanistic induction process of beta-defensin 2 by IL-1beta is not solely dependent on proximal NF-kB site activation, thus suggesting that the long distal portion of the promoter is needed for the full responsiveness toward IL-1beta.