OBJECTIVETo study the expression of human beta-defensin-3 (hBD-3) induced by lipopolysaccharide (LPS) in human bronchial epithelial (HBE) cells, and explore the role of hBD-3 in respiratory infection.

METHODSHBE cells were stimulated with different concentrations of LPS (0.01, 0.1, 1 and 10 microg/mL). hBD-3 mRNA expression was detected by RT-PCR 2 hrs later. hBD-3 protein expression was detected by Western blot 4 hrs later.

RESULTShBD-3 mRNA and protein was weakly expressed in normal HBE cells. LPS stimulation resulted in a significant increase of hBD-3 mRNA and protein expression (p<0.01). hBD-3 mRNA and protein expression increased with increasing LPS concentrations. There were significant differences in the hBD-3 mRNA and protein expression in cells stimulated by different concentrations of LPS (p<0.05).

CONCLUSIONSLPS can induce hBD-3 expression in a dose-dependent manner. hBD-3 might play a role in initial defensive reaction against bacterial invasion.

Bronchi ; drug effects ; metabolism ; Dose-Response Relationship, Drug ; Humans ; Lipopolysaccharides ; toxicity ; RNA, Messenger ; analysis ; beta-Defensins ; analysis ; genetics

BACKGROUNDAntimicrobial peptides, including cathelicidin LL-37, human beta defensin (HBD)-2, and HBD-3, are important elements of the innate immune response and involved in modulation of the adaptive immunity, and they also play an important role in cutaneous defense against Mycobacterium tuberculosis.

METHODSThe fresh skin tissues and paraffin-embedded biopsy samples from three cutaneous tuberculosis, two tuberculids, and ten healthy individuals were collected. The expressions of LL-37, HBD-2, and HBD-3 mRNA in the lesions of three cutaneous tuberculosis and two tuberculids were detected by quantitative real-time polymerase chain reaction; the protein expressions were detected by immunohistochemistry and Western blotting methods.

RESULTSThe expressions of LL-37 mRNA and protein in the lesions of cutaneous tuberculosis and tuberculids were similar to that of normal skin. The expression of HBD-2 mRNA had an increasing trend in the lesions of cutaneous tuberculosis and tuberculids compared with that of normal skin; however, the expression of HBD-2 protein in the lesions of cutaneous tuberculosis had a decreasing trend compared with that of normal skin, and the expression of HBD-2 protein in the lesions of tuberculids was similar to that of normal skin. The expressions of HBD-3 mRNA and protein in lesions of cutaneous tuberculosis and tuberculids were similar to that of normal skin.

CONCLUSIONSOur study indicated that the expression of HBD-2 and HBD-3 mRNA and protein in lesions of cutaneous tuberculosis may be not consistent with that of tuberculids. However, an inherent limitation of the present study was that the sample size was small, and the roles and regulation mechanisms of LL-37, HBD-2, and HBD-3 in cutaneous tuberculosis and tuberculids need to be further investigated.

Adult ; Aged ; Antimicrobial Cationic Peptides ; genetics ; Female ; Humans ; Male ; Middle Aged ; RNA, Messenger ; analysis ; Tuberculosis, Cutaneous ; metabolism ; beta-Defensins ; genetics

Human beta defensin 2 (HBD-2) may play an important role in human defense against infection. Its antimicrobial capacity has been fully documented in in vitro study. In order to evaluae its in vivo effects, we developed an HBD-2 transgenic mouse model. The HBD-2 minigene containing CMV promoter, full length of HBD-2 cDNA and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57 X ICR hybridized mouse by microinjection, and offspring were produced. DNA was isolated from the tails of the mouse pups, and the HBD-2 minigene incorporation was analyzed by PCR using HBD-2 specific primers. The HBD-2 gene expression in the multi-tissues of transgenic mice was determined at mRNA level by RT-PCR and at peptide level by immunohistological staining with the use of HBD-2 monoclonal antibody. The results showed that among 17 F0 transgenic mice, HBD-2 positive signal was determined by PCR in 4 mice, suggesting that HBD-2 minigene has been incorporated into the offspring mice. Meanwhile, a widespread expression of HBD-2 mRNA and peptide was detected in the F1 transgenic mice's multi-tissues such as trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium and brain.
Animals ; Anti-Infective Agents ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Transgenic ; Models, Animal ; Polymerase Chain Reaction ; RNA, Messenger ; analysis ; biosynthesis ; genetics ; beta-Defensins ; biosynthesis ; genetics

OBJECTIVETo explore the protective effect of recombinant beta-defensin-2 (BD2) on acute lung injury induced by sepsis in rats.

METHODSRecombinant adenovirus carrying an expression cassette of rat BD2 (rBD2) or control adenovirus carrying empty vector was administered intratracheally to Sprague-Dawley rats 48 h before acute lung injury, which was induced by cecal ligation and double puncture (CLP). Histological changes of the lungs and 7-day survival rate were determined.

RESULTThe alveolar damage, interstitial edema, and infiltration of neutrophils were reduced and the survival rate was increased significantly in BD2 group than those in control group (P < 0.05). The CFU of abdominal bacteria was comparable to that in the control rats (P > 0.05).

CONCLUSIONThe administration of recombinant BD2 protects lung injury and improves the survival of rats with induced sepsis.

Acute Disease ; Adenoviridae ; genetics ; Animals ; Genetic Therapy ; methods ; Genetic Vectors ; Lung Diseases ; etiology ; therapy ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; genetics ; physiology ; Sepsis ; complications ; Survival Analysis ; beta-Defensins ; genetics ; physiology

OBJECTIVETo study the effect of Bifidobacterium on the expression of β-defensin-2 (BD-2) in intestinal tissue of neonatal rats with necrotizing enterocolitis (NEC).

METHODSA total of 40 rats were randomly divided into four groups: normal control, Bifidobacterium control, NEC model, and Bifidobacterium treatment, with 10 rats in each group. A rat model of NEC was induced by hypoxia, cold stimulation, and artificial feeding. The rats in the Bifidobacterium control and Bifidobacterium treatment groups were given Bifidobacterium via the gastric tube after cold stimulation once a day for three consecutive days. The morphological changes of the terminal ileum were observed under a light microscope and the intestinal injury score was determined. Immunohistochemistry and qRT-PCR were used to measure the protein and mRNA expression of BD-2 in the ileal mucosal tissue.

RESULTSThe NEC model group had a significantly higher intestinal injury score than the normal control, Bifidobacterium control, and Bifidobacterium treatment groups (P<0.05). The Bifidobacterium treatment group had a significantly higher intestinal injury score than the normal control and Bifidobacterium control groups (P<0.05). The mRNA and protein expression of BD-2 in the normal control group was significantly lower than in the Bifidobacterium control, NEC model, and Bifidobacterium treatment groups (P<0.05). The Bifidobacterium control group had significantly higher mRNA and protein expression of BD-2 than the NEC model and Bifidobacterium treatment groups (P<0.05). The Bifidobacterium treatment group had significantly higher mRNA and protein expression of BD-2 than the NEC model group (P<0.05).

CONCLUSIONSBifidobacterium can induce the expression of BD-2 in intestinal tissue of rats and reduce inflammatory response by increasing the expression of BD-2. This provides a protective effect on neonatal rats with NEC.

Animals ; Bifidobacterium ; Disease Models, Animal ; Enterocolitis, Necrotizing ; therapy ; Humans ; Infant, Newborn ; Intestinal Mucosa ; metabolism ; NF-kappa B ; physiology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology ; beta-Defensins ; analysis ; genetics ; physiology

Defensins and cathelicidins (LL-37) are major antimicrobial peptides (AMPs) of the innate immune system of the human skin. In normal non-inflamed skin these peptides are negligible, but their expression can be markedly increased in inflammatory skin disease such as psoriasis. We designed this study to identify the expressions of LL-37 in normal human keratinocyte (NHK) and HaCaT cells after exposure to stimulants and to investigate difference of LL-37 expression accompanied with cell differentiation status, and come to understand difference of susceptibility to infection in atopic dermatitis and psoriasis. Expressions of LL-37 in NHKs and HaCaT cells were evaluated by using RT-PCR, Western blotting, and immunohistochemical (IHC) staining at 6, 12, and 24 hr post stimulation after exposure to Ultraviolet B irradiation and lipopolysaccharide. And expression of LL-37 in skin biopsy specimens from patients with atopic dermatitis and psoriasis was determined by immunohistochemical analysis. In time-sequential analyses of LL-37 expression revealed that LL-37 was expressed in NHKs, but not in HaCaT cells. IHC analysis confirmed the presence of abundant LL-37 in the epidermis of psoriasis. Therefore we deduced that expression of LL-37 is affected by UV irradiation, bacterial infection, and status of cell differentiation.
Antimicrobial Cationic Peptides/analysis/*genetics ; Blotting, Western ; Cell Line ; Cells, Cultured ; Comparative Study ; Defensins/analysis/genetics ; Dose-Response Relationship, Drug ; Gene Expression/drug effects/radiation effects ; Humans ; Immunohistochemistry ; Keratinocytes/cytology/*metabolism ; Lipopolysaccharides/pharmacology ; Male ; RNA, Messenger/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/cytology/metabolism ; Skin Diseases/*genetics/metabolism/pathology

BACKGROUNDHuman beta-defensin-3 (HBD(3)) is an epithelial peptide that has been demonstrated to have a salt-insensitive broad spectrum of potent antimicrobial activity. Expressing antimicrobial peptides in Escherichia coli (E. coli) is very difficult for it can result in death of the bacterial host cells. Our aim was to establish a prokaryotic system expressing soluble HBD(3) protein and demonstrate the antimicrobial activity of the expressed protein. We then studied whether the host cells would activate the suicide pathways.

METHODSWe first cloned the complementary DNA coding for the mature chain of HBD(3), inserted it into the vector PGEX-KG then transformed E. coli BL21 (DE3) with the appropriate recombinant plasmid. After induction with 0.5 mmol/L isopropyl-1-thio-beta-D-galactopyranoside (IPTG) the transformed E. coli produced a recombinant glutathione S-transferase and HBD(3) (GST-HBD(3)) fusion protein. The fusion protein was treated with thrombin to produce pure HBD(3) protein then the antimicrobial activity of HBD(3) was evaluated in a liquid microdilution assay.

RESULTSThe fusion protein GST-HBD(3) was efficiently cleaved by thrombin and yielded HBD(3) that had anti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 microg/ml. The E. coli strain expressing the recombinant protein did not grow slower than the empty vector strain.

CONCLUSIONActive HBD(3) in E. coli by expressing the recombinant protein GST-HBD(3) could be produced, and suicide did not occur in the E. coli strain expressing the recombinant protein.

Amino Acid Sequence ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; growth & development ; Glutathione Transferase ; genetics ; metabolism ; Humans ; Molecular Sequence Data ; Plasmids ; genetics ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Staphylococcus aureus ; drug effects ; Thrombin ; metabolism ; beta-Defensins ; genetics ; metabolism ; pharmacology

AIMIdentification of the rodent counterparts of human and canine epididymal cDNAs HE3, HE4 and Ce8/Ly6G5C by sequence homology and analysis of their expression patterns and regulation level in the rat.

METHODS"Electronic screening" of Expressed Sequence Tag (EST) and genomic databases, followed by RT-PCR and Northern blot analysis.

RESULTSRodent ESTs and genomic sequences homologous to HE3, HE4 and Ce8/Ly6G5C were identified in the public databases and the "full-length" rat cDNAs cloned. To emphasise their homology to the human and canine genes, they were named Me3/Re3, Me4/Re4 and Re8 for mouse and rat counterparts, respectively. mRNA expression patterns were analysed in rats, including rat HE1 and HE5/CD52 counterparts as controls. Re3 and Re8 mRNAs were only found in the rat epididymis, while Re4 showed a broader tissue distribution. Within the epididymis, Re3 and Re4 mRNAs were detected in all regions; Re8, on the other hand, was restricted to the caput. During postnatal development, Re3 and control mRNAs were found from the earliest stages investigated, while Re8 mRNA was observed only from day 24 postnatum, corresponding to the onset of spermatogenesis in the prepubertal testis. Castration and testosterone supplementation of adult male rats suggested that none of the cloned mRNAs was directly androgen-regulated. Efferent duct ligation, however, showed that Re8 mRNA levels depended on testicular factors other than androgens.

CONCLUSIONThe novel rodent cDNAs can now be used to monitor epididymal gene expression more closely and to set up various regulatory and functional studies.

Amino Acid Sequence ; Animals ; Blotting, Northern ; Carrier Proteins ; chemistry ; genetics ; DNA, Complementary ; chemistry ; Dogs ; Epididymal Secretory Proteins ; chemistry ; genetics ; Epididymis ; chemistry ; Glycoproteins ; chemistry ; genetics ; Humans ; Male ; Molecular Sequence Data ; Proteins ; chemistry ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Homology ; Tissue Distribution ; beta-Defensins