DEFB1 gene polymorphisms and tuberculosis in a Northeastern Brazilian population

Abstract β-Defensin-1, an antimicrobial peptide encoded by the DEFB1 gene, is known to play an important role in lung mucosal immunity. In our association study we analyzed three DEFB1 functional polymorphisms -52G>A (rs1799946), -44C>G (rs1800972) and -20G>A (rs11362) in 92 tuberculosis patients and 286 healthy controls, both from Northeast Brazil: no association was found between the studied DEFB1 polymorphisms and the disease. However we cannot exclude that this lack of association could be due to the low number of subjects analyzed, as suggested by the low statistical power achieved for the three analyzed SNPs (values between 0.16 and 0.50).

DEFB1 polymorphisms are involved in susceptibility to human papillomavirus infection in Brazilian gynaecological patients

The human beta defensin 1 (hBD-1) antimicrobial peptide is a member of the innate immune system known to act in the first line of defence against microorganisms, including viruses such as human papillomavirus (HPV). In this study, five functional polymorphisms (namely g-52G>A, g-44C>G and g-20G>A in the 5’UTR and c.*5G>A and c.*87A>G in the 3’UTR) in the DEFB1 gene encoding for hBD-1 were analysed to investigate the possible involvement of these genetic variants in susceptibility to HPV infection and in the development of HPV-associated lesions in a population of Brazilian women. The DEFB1 g-52G>A and c.*5G>A single-nucleotide polymorphisms (SNPs) and the GCAAA haplotype showed associations with HPV-negative status; in particular, the c.*5G>A SNP was significantly associated after multiple test corrections. These findings suggest a possible role for the constitutively expressed beta defensin-1 peptide as a natural defence against HPV in the genital tract mucosa.

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. .

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.

Study of rat beta-defensin-2 gene and protein expression in ventilator-associated pneumonia / 中华危重病急救医学

OBJECTIVE: To study the changes in the expression of rat beta-defensin-2 (RBD-2) gene in the lung tissue with P. aeruginosa (PA) pneumonia following tracheal mechanical ventilation (MV), and to evaluate the pathogenesis of ventilator-associated pneumonia (VAP). METHODS: A total of 58 normal healthy Sprague-Dawley rats, weighing between 280 and 320 g, were randomly divided into the control group and the conventional MV group (CMV). A tracheal catheter was inserted via mouth in every rat under urethane anesthesia. PA (1 MIC, 0.2 ml) was instilled into the tracheal in the control group. Rats of CMV group received MV (V(T)=12 ml/kg) through tracheal tube for 24 hours, and then were challenged intra-tracheally with PA (1 MIC, 0.2 ml). Fluid loss was replenished through intravenous infusion. The arterial catheter was used for hemodynamics, parameters were monitored, and arterial blood gases were determined. Samples of lung were harvested at 0 hours, 15 hours, 3 hours, 6 hours, 12 hours, 1 day, 3 days and 5 days, respectively, after bacterial challenge. The mRNA of RBD-2 was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the protein levels were analyzed by Western blotting. RESULTS: Expression of RBD-2 mRNA and protein was lower in CMV group compared with the control 3 hours before instillation of bacteria. RBD-2 mRNA increased 3 hours after bacteria instillation, reaching the peak at 12-24 hours. No significant difference in RBD-2 expression between the control group and the CMV group within 3 hours, but it was significantly higher at 3 hours, 6 hours, 12 hours, 1 day, 3 days and 5 days in the control group than in the CMV group. The number of inflammatory cells infiltrating the bronchial submucous layer was significantly higher in the control group than in the CMV group (P<0.05). There was milder interstitial pulmonary edema and less red blood cells in the alveoli in the control group than in the CMV group. The mortality rate of the CMV group was 60%, which was significantly higher than that of the control group (20%, P<0.05). The positive rates of blood culture and bronchoalveolar lavage fluid (BALF) bacterial culture were also higher in the CMV group (P<0.05). The survival rate in CMV group (40%) was lower than that of the control group (P<0.05). CONCLUSION: The lowering of BD-2 gene and protein expression in the CMV group 3 hours after bacteria challenge might be one of the contributory factors in causing VAP.