BACKGROUND: Defensins are small (3.5~5 kDa) cationic antimicrobial peptides that have a broad spectrum of activity that includes gram-negative bacterias, yeasts and enveloped viruses. The defensins contain six cysteine residues forming three disulfide bridges depending on the spacing of the cysteine residues and the connectivity of the disulfide bridge, defensins are classified into two families, the alpha-defensins (HNP) and beta-defensins (HBD). Recently two human epithelial beta defensins, HBD-1 and HBD-2 have been identified. HBD-1 has been detected in a number of normal mucosal sites, but HBD-2 is highly restricted in its expression by inflammatory stimulations. we invesigated the expression of hunam beta defensin in human male urogenital organs. METHODS: Specimens of normal human male testis, epididymis, prostate, seminal vesicles, vas deferens, urethra, bladder, ureter, kidney, pyelonephritis, epididymitis, clear renal cell carcinoma and transitional cell carcinoma of bladder were obtained as discarded material from urological surgery. Each sample was stored at snap frozen in liquid nitrogen subsequent to RNA extraction. Reverse transcription polymerase chain reaction (RT-PCR) was used to semiquantitate HBD-1 and HBD-2 mRNA using the housekeeping gene beta-actin as an internal control. Southern blotting and sequencing showed HBD-1, 2 expressions in male urogenital organs. RESULTS: We checked the expression of HBD-1, 2 mRNA in all specimen of normal human male urogenital organ, pyelonephritis, epididymitis, clear renal cell carcinoma and transitional cell carcinoma of bladder by RT-PCR and southern blotting analysis. We checked the homolgy of HBD-1, 2 by bands sequencing. CONCLUSION: Our study indicated that the normal male urogenital organs, infection and neoplasm in male urogenital organs expresses antimicrobial peptides. These may play an important role in the prevention of infections by bacterias, antimicrobial effects in infection and anticancer effects in neoplasm of male urogenital organs. These natural endogenous antibiotic peptides could be developed as novel therapeutic agents for fighting infections and neoplasms of the human male urogenital organs.
Actins ; alpha-Defensins ; Antimicrobial Cationic Peptides ; Bacteria ; beta-Defensins ; Blotting, Southern ; Carcinoma, Renal Cell ; Carcinoma, Transitional Cell ; Cysteine ; Defensins ; Epididymis ; Epididymitis ; Genes, Essential ; Gram-Negative Bacteria ; Humans* ; Kidney ; Male* ; Nitrogen ; Peptides ; Polymerase Chain Reaction ; Prostate ; Pyelonephritis ; Reverse Transcription ; RNA ; RNA, Messenger ; Seminal Vesicles ; Testis ; Ureter ; Urethra ; Urinary Bladder ; Vas Deferens ; Yeasts

OBJECTIVETo investigate the expression and the distribution of human beta defensin (hBD)-4 in healthy gingiva.

METHODSHealthy gingival specimens were collected. The expression of hBD-4 peptides in 18 gingival specimens were detected by immunohistochemistry. The hBD-4 mRNA were determined in freshly isolated gingival tissue by real time reverse transcription-polymerase chain reaction (real time RT-PCR) in 30 gingival specimens.

RESULTSIn 18 gingival specimens, hBD-4 peptides were expressed in 13 gingival specimens. In 30 gingival specimens, hBD-4 were detected in 4 gingival specimens by real time RT-PCR.

CONCLUSIONThe distribution and the expression levels of hBD-4 are different in healthy gingiva. This result may suggest that the hBD-4 play a role in maintaining the periodontal health.

Gingiva ; Humans ; RNA, Messenger ; beta-Defensins

BACKGROUND AND OBJECTIVES: It is believed that the innate immunity plays a critical role in protecting the tubotympanum from being infected because the middle ear cavity is normally sterile despite of a paucity of immune cells. Among known antibacterial molecules, defensins have been shown to contribute significantly to innate immunity. However, it is still unclear whether or not beta defensins are expressed in human middle ear mucosa. MATERIALS AND METHOD: Immunolabeling and RT-PCR were performed with the mucosal specimen from normal subjects and otitis media patients, respectively. Expression of beta defensin 2 mRNA was compared between the control group and experimental group that was treated by inflammatory stimuli in the animal models using RT-PCR. RESULTS: beta defensin 1 was expressed in both normal and inflamed middle ear mucosa of human, but beta defensin 2 and 3 were found only in the inflamed mucosa. The expression of beta defensin 2 mRNA was up-regulated when the interleukin-1alpha (IL-1alpha) or lipopolysaccharide (LPS) was treated in the middle ear mucosa of the experimental animals. CONCLUSION: We could show that beta defensins are expressed in the human middle ear mucosa and that beta defensin 2 is up-regulated by the inflammatory stimuli, IL-1alpha or LPS.
Animals ; beta-Defensins* ; Defensins ; Ear, Middle* ; Humans* ; Immunity, Innate ; Interleukin-1alpha ; Models, Animal ; Mucous Membrane* ; Otitis Media ; RNA, Messenger

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.
beta Catenin ; beta-Defensins ; Burns ; Defensins ; Humans ; Immune System ; Immunohistochemistry ; Keratinocytes ; Keratins ; Peptides ; Protein Precursors ; RNA, Messenger ; Skin ; Skin Diseases ; Wound Infection

OBJECTIVETo explore the role and potential mechanism of human α-defensin 1 (HNP-1) on low-density lipoprotein (LDL) oxidation ability of human endothelial cells (EVC304).

METHODSPost incubation with LDL for 3 h, the malondialdehyde (MDA) and protein carbonyl (PCO) were detected in untreated ECV304 (control) and in HNP-1 transfected ECV304 in the presence and absence of siRNA against HNP-1. Flow cytometry and fluorescence microscopy were used to detect the generation of oxygen free radical in the ECV304 which have been pretreated by LDL, LPS and HNP-1, respectively.

RESULTCompared with control group, MDA level was significantly increased in HNP-1 transfected [(4.21 ± 0.03) vs. (3.15 ± 0.02) nmol/mg · pro] or in HNP-1 stimulated ECV304 cells [(14.49 ± 1.10) vs. (9.47 ± 1.18) nmol/mg · pro], which could be significantly downregulated by siRNA [(3.76 ± 0.48) vs. (4.54 ± 0.28) nmol/mg·pro, all P < 0.05]. PCO was also significantly increased in HNP-1 transfected ECV304 cells. The levels of free radical were significantly increased in HNP-1 transfected or HNP-1 stimulated ECV304 cells.

CONCLUSIONHNP-1 can enhance the LDL oxidation ability of human endothelial cells via promoting the generation of free radicals.

Cell Line ; Endothelial Cells ; metabolism ; Humans ; Lipoproteins, LDL ; metabolism ; RNA, Small Interfering ; Transfection ; alpha-Defensins ; genetics ; metabolism

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.
Binding, Competitive ; Cell Survival ; Cells, Cultured ; Corneal Diseases/metabolism ; Electrophoretic Mobility Shift Assay ; Epithelium, Corneal/drug effects/*immunology/metabolism ; Gene Expression ; Humans ; Interferon Type II/metabolism/pharmacology ; Interleukin-1/*pharmacology ; Lipopolysaccharides/metabolism/pharmacology ; NF-kappa B/metabolism ; Promoter Regions (Genetics)/drug effects/genetics ; Research Support, Non-U.S. Gov't ; Tumor Necrosis Factor-alpha/metabolism/pharmacology ; beta-Defensins/*biosynthesis/genetics/metabolism

OBJECTIVES: To investigate the effect of icariin (ICA) on early β-defensin-2 and T cell subsets in rats after tracheotomy. METHODS: A total of 54 SPF male Sprague-Dawley rats were randomly divided into a normal control group (group A), a model group (group B), and a model+ICA treatment group (group C), with 18 rats in each group. A tracheotomy intubation model of the B and C group was prepared. After 6 h of surgery, ICA intervention was given to group C. Groups A and B were given the same amount of normal saline. Lung tissue, alveolar lavage fluid and peripheral blood were taken at 24 h, 72 h and 168 h, respectively. The expression of rat β-defensin-2 mRNA in lung tissue was detected by RT-PCR. The content of β-defensin-2 in alveolar lavage fluid and peripheral blood serum was detected by ELISA. The content of peripheral blood T cell subsets (CD3, CD4, CD8) was detected by flow cytometry, and the ratio of CD4/CD8 was calculated. RESULTS: After tracheotomy, the levels of β-defensin-2 mRNA and β-defensin-2 in lung tissue from the group B were increased significantly at 24 h, then they were decreased gradually, and decreased most significantly at 168 h (<0.05). The content of β-defensin-2 in peripheral blood of group B decreased gradually, and the content of β-defensin-2 in 168 h was significantly lower than that in 24 h (<0.05), but there was no significant difference between group B and group A (>0.05). The level of CD3 T cells in peripheral blood was significantly lower than that in the group A (<0.05), but their was no significant difference in CD4 and CD8 T cells compared with group A (>0.05). After ICA intervention in group C: lung tissue, alveolar lavage fluid, peripheral blood serum β-defensin-2 content, and peripheral blood CD3 and CD4 T cell levels were gradually increased, significantly higher than those in the group B (<0.05). CD8 T cell level was significantly lower than that in the group A at 24 h (<0.05), the CD4/CD8 ratio was significantly higher at 168 h than those in the group A or B (both <0.01). CONCLUSIONS ICA can improve the early lung immune function in rats with tracheotomy, which might be related to up-regulation of β-defensin-2 in lung tissue and alveolar lavage fluid, concomitant with increases in CD3 and CD4 T cells and CD4/CD8 ratio in peripheral blood while reduction in CD8 cells.
Animals ; Flavonoids ; Male ; Rats ; Rats, Sprague-Dawley ; T-Lymphocyte Subsets ; Tracheotomy ; beta-Defensins

The aim of this study was to screen the active regions and transcription factor binding sites in the promoter of the CBD103 gene related to Arctic fox coat color, and to provide a basis for revealing the molecular genetic mechanism of CBD103 gene regulating the coat color formation. The 5'-flanking region fragment 2 123 bp of Arctic fox CBD103 gene was cloned, and 4 truncated promoter reporter vectors of different lengths were constructed. The promoter activity was detected by the dual-luciferase reporter assay system. Point mutations were performed on the 3 predicted specificity protein 1 (Sp1) transcription factor binding sites in the highest promoter active region, and 3 mutant vectors were constructed. The activity was then detected by the dual-luciferase reporter assay system. The results showed that the region 1 656 (-1 604/+51) had the highest activity in the 4 truncated promoters of different lengths, and the promoter activity of the three mutant vectors constructed in this region were significantly lower than that of the wild type (fragment 1 656). The region of -1 604 /+51 was the core promoter region of CBD103 gene in Arctic fox and -1 552/-1 564, -1 439/-1 454 and -329/-339 regions were positive regulatory regions. This study successfully obtained the core promoter region and positive regulation regions of the Arctic fox CBD103 gene, which laid a foundation for further study on the molecular genetic mechanism of this gene regulating Arctic fox coat color.
Animals ; Binding Sites ; Foxes ; Luciferases ; Promoter Regions, Genetic ; Sp1 Transcription Factor ; beta-Defensins

OBJECTIVETo investigate the expression characteristics of human beta-defensin-2 (HBD-2) mRNA and protein in salivary gland benign and malignant tumor tissues, as well as in salivary gland inflammation.

METHODSThe expression of HBD-2 in salivary gland benign tumor, salivary gland cancer, inflammation tissues and normal salivary gland tissues were detected by polymerase chain reaction (PCR), real-time polymerase chain reaction (Real-Time PCR) and immunohistochemical. The differences expression of HBD-2 mRNA and protein were analyzed.

RESULTSRT-PCR results showed that HBD-2 mRNA expression in salivary gland benign tumors, salivary gland cancer, and inflammation tissues was 6.468-, 0.334-, and 10.563-fold higher than that in normal tissues, respectively (P < 0.05). HBD-2 was expressed in the nuclei of these organs and malignant tissues.

CONCLUSIONHBD-2 mRNA and protein expressions are significantly increased in salivary gland benign tumor tissues and inflammation tissues compared with those in normal salivary gland tissues, but are significantly decreased in salivary gland cancer. The protein nuclear transfer in salivary gland cancer tissues is also significantly increased.

Humans ; Inflammation ; Polymerase Chain Reaction ; RNA, Messenger ; Salivary Gland Neoplasms ; Salivary Glands ; beta-Defensins ; biosynthesis

OBJECTIVETo investigate the expression of human beta-defensin-2 (HBD-2) in laryngeal squamous cell carcinoma (LSCC), and reveal the immune significance of HBD-2 in LSCC.

METHODSThe expression of HBD-2 and the infiltration of CD1a(+) dendritic cells were verified by immunohistochemistry strept actividin-biotin complex (SABC) method in thirty-four cases of LSCC's paraffin sections, and the expression of HBD-2 mRNA was detected in eighteen cases of postoperative tumor specimens and five cases of normal paracarcinoma tissue using cDNA probe by in situ hybridization. Based on the type and characteristic of datas, SPSS13.0 program package was used for statistical calculation.

RESULTS(1) The difference of expression of HBD-2 was significant among the grades of malignant cell differentiation (F = 6.809, P < 0.05), the expression of HBD-2 in well differentiated group was significantly increased compared with moderately and poorly differentiated group (P < 0.05), there wasn't significant difference with the clinical type, T status and lymphatic metastasis in the expression of HBD-2. (2) The difference of expression of HBD-2 mRNA among different grades of malignant cell differentiation was significant (F = 16.391, P < 0.05), less or no positive signal was observed in poorly differentiated group. (3) The infiltration of CD1a(+) dendritic cells had positive correlation with the expression of HBD-2 (r = 0.343, P < 0.05).

CONCLUSIONSThe expression of HBD-2 was related to the grade of malignant cell differentiation in LSCC, which was regulated at the level of transcription and translation. HBD-2 might play a role in anti-tumor immunity by chemo-attractiving and activating of immature dendritic cells.

Carcinoma, Squamous Cell ; Dendritic Cells ; Humans ; Laryngeal Neoplasms ; Lymphatic Metastasis ; beta-Defensins