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Natural killer (NK) cells and cytolytic T lymphocytes (CTL) have been implicated as important effectors of antiviral defense. We previously isolated a novel antibacterial polypeptide, which was identified as high mobility group nucleosomal-binding domain 2 (HMGN2), from human mononuclear leukocytes. This study examined the antiviral activity of HMGN2 against human hepatitis virus B. HMGN2 was isolated and purified from the acid soluble proteins of the human THP-1 cell line, and identified by mass spectrum, Western blot and antibacterial assay. The hepatitis B virus (HBV)-transfected HepG2.2.15 cell line was used in the in vitro assay system. In the range of 1-100 microg/ml HMGN2, no cytotoxicity for HepG2.2.15 cells was detected by MTT assay. When incubated with HMGN2 at 1-100 microg/ml for 72 or 144 h, there was a significant reduction in hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) expression, which were detected by ELISA, and a significant reduction in HBV DNA copies, which was determined by the real time quantitative PCR, in the supernatant of HepG2.2.15 cells. Northern and Southern blot analysis also showed that the levels of the HBV 3.5 kb and the 2.4/2.1 kb mRNA species and HBV replicative intermediate DNA were significantly reduced in the HMGN2-treated HepG2.2.15 cells. These results indicated that HMGN2 protein could markedly inhibit HBV protein expression and replication in vitro.

This study was conducted to isolate and purify antimicrobial polypeptides HMGN2 (high mobility group nucleosomal-binding domain2) from human lymph node, to detect the antimicrobial activity of HMGN2, and to determine the subcellular location of HMGN2 in human lymph node. The antimicrobial polypeptides were purified by the Reverse Phase HPLC and identified by Tricine-SDS-PAGE. The antimicrobial activity was detected by agar diffusion test. Mass spectrum and Western-blot analysis indicated the individual character of protein. HMGN2 was isolated and purified from human lymph node, and it showed antimicrobial potency against the pathogenic strain E. coli 54,080. The immunocytochemistry staining indicated that HMGN2 was present both in human lymph node cells' nucleus and cytoplasm. In conclusion, HMGN2 protein is of antimicrobial activity and it is probably involved in the defence of innate immunity in vivo.

Interleukin-8 (IL-8) is an important activator and chemoattratant of neutrophils and has been implicated in airway inflammatory diseases. To explore the new gene therapeutic strategies for airway inflammation, plasmid expressing dominant negative myeloid differentiation protein (MyD88 DN) was constructed and transfected into human airway epithelial cell lines A549 and SPC-A-I. The cells were challenged with M. tuberculosis, P. aeruginosa or K. pneumoniae and the release of IL-8 was measured using ELISA. The results showed that the supernatants of M. tuberculosis and R. aeruginosa enhanced IL-8 release from the epithelial cells; and transfection of MyD88 DN diminished this effect. MyD88 DN also reduced IL-8 release from cells induced by live bacteria of P. aeruginosa or K. pneumoniae. These data suggest that MyD88 could be used as a target gene in the gene therapy of airway inflammation.

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.

This study sought to clarify the distribution of HMGN2 in HeLa cells. The recombinant eukaryotic expression vectors pcDNA3. 1-myc-his-HMGN2 and pEGFP-N1-HMGN2 were constructed, and then were transfected into HeLa cells. immunocytochemistry staining indicated that HMGN2 were present not only in HeLa nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant by ELISA with rabbit anti-serum against HMGN2 and mouse anti-His6 monoclonal antibodies. The confocal microscope observation showed the same subcellular localization as that of immunocytochemistry staining. There results suggested that HMGN2 could be present in the nucleus and cytoplasm of HeLa cell as well as in the extracellular environment.

Leukocytes are a central cellular ele- ment of innate-immune defense in mammals. In addition to the generation of toxic oxygen radicals and nitric oxide, leukocytes express and secrete a broad array of antimicrobial proteins and peptides. In the study, an antimicrobial polypeptide was iso- lated and purified from human peripheral blood mononuclear leukocytes in the presence of inter- leukin (IL)-2. Microsequencing provided that its N-terminal amino sequence was PKRKAEGDAK, which was identical to high mobility group nucleo- somal-binding domain 2 (HMGN2). Mass spectro- metric value and Western blot also indicated its individual character of HMGN2. The antimicrobial assays showed that the Escherichia coli-based pro- duction of HMGN2 had a potent antimicrobial ac- tivity against E. coli ML-35p, Pseudomonas aerugi- nosa ATCC 27853, and to some extent, against Candida albicans ATCC 10231. The HMGN2 -helical domain had the same antimicrobial activ- ity as HMGN2. The immunocytochemistry stain- ing, enzyme-linked immunosorbent assay, and Western blot revealed that HMGN2 was present in the cytoplasm of mononuclear leukocytes and re- leased to the extracellular environment when stim- ulated with IL-2. These results suggest that HMGN2 would be a novel antimicrobial effector molecule of human mononuclear leukocyte. J. Leukoc. Biol. 78: 1136-1141; 2005.

Total RNA was extracted from human LAK cell, and a cDNA encoding mature peptide HMG-17 and its alpha helix domain was amplified by RT-PCR. The recombinant prokaryotic expression vector pGEX-1lambdaT-HMG-17 and pGEX-1lambdaT HMG-17alpha helix was constructed. Using affinity chromatography, thrombin cleaving and AU-PAGE elution, we obtained the purified HMG-17. Analyses of MIC, MEC and MBC indicated that HMG-17 and HMG-17alpha had strong antibacterial activity. MIC of the alpha-helic domain was almost the same as that of HMG17, suggesting that the alpha-helic structure would be essential for the antibacterial activity of HMG-17.

The recombinant PSMA DNA vaccine for active immunotherapy of prostate cancer was investigated. Two DNA vaccine recombinant plasmids, pcDNA3.1/PSMA and pcDNA3.1/hBD-2-PSMA, were constructed by inserting the hBD-2 gene and PSMA gene into an eukarytoic expression vector pcDNA3.1. Expression of the two recombinants was detected in transfected COS-7 cells and inoculated mouse muscular cells by RT-PCR and immunohistochemical method. When immunized with pcDNA3.1/PSMA and pcDNA3.1/hBD-2-PSMA, the immunized BALB/c mice acquired specific antibody and T cell response to PSMA. The quantity of the spleen lymphocytes and their CTL activity against PSMA gene transfected-BALB/3T3 cells significantly increased in the immunized mice, and the CTL activity of lymphocytes from pcDNA3.1/hBD-2-PSMA immunized mice was significantly higher than that of pcDNA3.1/PSMA immunized mice. This result suggests that pcDNA3.1/hBD-2-PSMA would probably be developed as a DNA vaccine for the immunotherapy of prostate cancer.

This study was aimed at constructing a prokaryotic expression system to resolve the difficulties in acquiring antibacterial peptide and to meet the needs of research and drug development. Total RNA was extracted from human pulmonary gland epithelial cell line SPC-A-1, and a cDNA encoding mature FALL-39 peptide was amplified by RT-PCR. The recombinant prokaryotic expression vector pGEX-1 lambda T-FALL-39 was constructed. Using affinity chromatography, thrombin cleaving and AU-PAGE elution, we obtained the purified FALL-39. MIC, MEC, MBC analyses demonstrated that the FALL-39 had strong antibacterial activity.

To examine Bifidobacterium mediated induction of human defensin 2 mRNA expression in human intestinal epithelial cells and define the bioactive components of Bifidobacterium.Bifidobacterium was cultured in MRS medium in anaerobic incubator and its cell wall was isolated by sonication and centrifugation. The cell wall proteins of Bifidobacteria were extracted with 2% SDS. The level of hBD-2 mRNA in HT-29 cells was detected by reverse-transcription PCR and Northern blot analysis.There was no detectable signal of hBD-2 mRNA in the unstimulated HT-29 cells. However, the heat-killed Bifidobacteria, cell wall of Bifidobacteria, and cell wall proteins remarkably induced hBD-2 mRNA expression in HT-29.Bifidobacterium or its cell wall proteins can induce hBD-2 gene expression in human intestinal epithelial cells, which may play a role in the mechanisms of innate defense against pathogens in the intestine.

To define the regulation mechanism of the enhancement expression of human beta-defensin-1 (hBD-1) stimulated by bacille Calmette-Guerin (BCG) at transcription level.A series of 5'-flanking deletions of hBD-1 gene were ligated into pEGFP-1 and pCAT basic vector. pEGFP hBD-1 recombinants were transfected into SPC-A-1 cells and the green fluorescence protein (GFP) expression was observed by fluorescence microscopy. SPC-A-1 cells were co-transfected with pCAT hBD-1 constructs and pSV-beta-Galactosidase control vector and were stimulated with effective BCG cell wall components. CAT and beta-Gal protein expressions were determined by ELISA.The promoting activity of the -69 region was lower than that of the region -575 and -314. After being stimulated with the active fraction of BCG cell wall proteins, the relative CAT expression of pCAT hBD-1/-69 still showed the increasing tendency of -2161 construct. Computer consensus match analysis indicated that the nucleotides between -63 bp to -50 bp was the potential binding site for C/EBP beta.The nucleotides from -69 to bp is essential to the enhancement of hBD-1 gene transcription by BCG.

To disclose whether BCG can enhance Fall-39 gene expression in human pulmonary gland epithelial cells, and to isolate and identify the bioactive proteins that stimulate Fall-39 gene expression in human pulmonary gland epithelial cells.Fall-39 mRNA expression in SPC-A-1 cells was detected by using RT-PCR. The cell wall proteins were isolated by sucrose density-gradient centrifugation and fractionated by Sephadex G-75 column chromatography. The antibacterial activity of the cell culture supernatant was determined by agarose radial diffusion assay.The enhanced expression of Fall-39 mRNA was dose- and time-dependent. BCG cell wall proteins fraction 4 had an activity that remarkably enhanced Fall-39 mRNA expression in SPC-A-1 cells and augmented antibacterial activity of the cell culture supernatant.These results indicated that BCG could stimulate Fall-39 mRNA expression in human pulmonary gland epithelial cells, thus providing an evidence base for shaping well a new strategy to enhance mucosa antibiotic peptide expression for the prevention and treatment of mucosal infections.

Human beta-defensins (HBDs) are small, cationic endogenous antimicrobial peptides isolated from the hemodialates of patients with chronic renal failure (HBD-1) and Psoriasic skin (HBD-2). They may play an important role in mucosal innate host defense against infections. In this study, we examined their mRNAs expression in normal and pregnant women genital tract.Reverse transcription-polymerase chain reaction (RT-PCR) was performed in the detection of HBD-1 and HBD-2 mRNA expression in the samples from normal and pregnant women genital tract. beta-actin was taken as positive control.RT-PCR detection revealed that HBD-1 mRNA expressed in all samples of normal women taken from vagina, cervix, endometrium, fallopian tube, and ovary, and HBD-2 mRNA widely expressed in genital tract except vagina and ovary. In pregnant women, HBD-1 mRNA widely expressed in chorion, villus, placenta, and umbilical cord, but not in amnion, and HBD-2 mRNA were detected in chorion, villus and placenta tissues.The findings of this study on the wide expression of HBD-1 and HBD-2 in both the normal and the pregnant women genital tract suggest that HBDs may play an important role in human genital tract defense mechanisms.

This study was made to isolate and purify antibacterial polypeptides from human cervical mucus.Human cervical mucus was collected from human healthy subjects, and the acid-soluble extract was obtained by solving the mucus with 5% acetic acid in the presence of protease inhibitor. By using gel overlay technique, one protein band that was potently antibacterial against E. coli ML-35P was identified. To purify the antibacterial polypeptide, the preparative acid-urea gel electrophoresis and Reverse Phase HPLC were performed.A purified antibacterial polypeptide was obtained and named HCP-1. Tricine-SDS-PAGE indicated that the relative molecular mass of HCP-1 was around 14 x 10(3). Agar radial diffusion assay showed that the purified polypeptide had antibacterial activities against E. coli ML-35P and Pseudomanas aeruginosa ATCC65922.HCP-1 may be a new effector molecule of human cervical mucus.

Constructing a point-directed mutant of FALL-39 and prokaryotic expressive vector, and determining its antibacterial activity.A two-step polymerase chain reaction (PCR) was used for the site-directed mutagenesis. Two sets of primers (P1, P2, P3, P4) were designed according to FALL-39 gene sequence and mismatch was introduced into P2 for a substitution of AAG for AAT at position 32. Mutagenesis was performed in a two-step PCR, the amplified fragments from the second PCR, which contain the mutation site, were subcloned into the vector PGEX lambda 1T and verified by sequencing analysis. Antibacterial activities of the E. coli-based product against E. coli and P. aeruginosa were detected by using minimal inhibitory concentration (MIC), minimal effective concentration (MEC) and minimal bactericidal concentration (MBC).A mutant of FALL-39, whose AAT at position 32 is substituted by AAG, was obtained by the two-step PCR. The antibacterial activities of the E. coli-based product of the recombinant FALL-39-Lys32 against E. coli ML-35p and P. aeruginosa ATCC27853 were much stronger than those of FALL-39. MIC and MBC of the FALL-39 against E. coli were 18.75 micrograms/ml and 37.50 micrograms/ml, whereas those of FALL-39-Lys32, 10.94 micrograms/ml and 21.88 micrograms/ml respectively.A point-directed FALL-39-Lys32 mutant was obtained by using two-step PCR and its antibacterial activity was increased, suggesting that increased cationic FALL-39 mutant might enhance its antibacterial activity.

The aim of this study is to explore the possibility and technical itinerary of establishing an mammal engineering cell line in which hBD-2 can be effectively expressed, secreted, detected, separated and purified. The full hBD2 cDNA was inserted into the multi clone site of a eukaryotic expressive plasmid pcDNA3.1/Myc-His(+) and located closely at the upstream of two tag gene (myc and 6 Poly-histidines) so as to construct another recombinant eukaryotic expressive vector of hBD-2 gene: rpcDNA3.1/Myc-His/hBD-2. By the use of RT-PCR with special primers, a band of 240 bp was amplified from COS-7 cells transfected by this recombinant plasmid, which matched full length of cDNA coding hBD2 plus myc epitope and 6 poly-histidines tags. Western blot analysis with specific anti-histidines antibody revealed that the lysate of COS-7 cells transfected by rpcDNA3.1/Myc-His/hBD-2 had a strong band with molecular weight of about 10 Kd that was approximate to the size of chiasmic peptide. Antibacterial activity assay showed that obvious bacterial inhibition occurred in both lysate and supernatant of COS-7 cells transfected by rpcDNA3.1/Myc-His/hBD-2.

To examine interleukin-8 (IL-8) mRNA expression induced by flow shear stress in human umbilical vein endothelial cells (HUVECs) and investigate its transcriptional activation.Flow laminar shear stress 4.2 dyne/cm(2) was used for the stimulating experiments. The flow shear stress-induced IL-8 mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). pEGFP1 was used to construct IL-8 reporter gene pEGFP1-IL8USCS for determining IL-8 gene transcriptional activation through gene transfer and flow cytometric analysis. NF-kappaB nuclear translocation was observed by immunocytofluorescent staining. Western blot was used to examine IkappaB phosphorylation and degradation. RT-PCR, Northern blot, immunocytofluorescent staining and laser confocal microscopy were used to determine Toll-like receptor-4 (TLR-4) expression at mRNA and protein levels in the cells.There was a marked increase in IL-8 mRNA expression in HUVECs after 120 min of exposure to laminar flow shear stress. When exposed to shear stress for 180 min, there was an increase in enhanced green fluorescent protein expression in pEGFP1-IL8USCS-transfected endothelial cells. NF-kappaB p65 immunocytofluorescent staining of HUVECs showed that when exposed to the same flow shear stress for 30 or 60 min, the cell nuclei became stained; after 90 or 120 min, the staining became much more pronounced. A significant increase in P-IkappaB in the cell lysates occurred after 10 min of exposure while blot density dramatically dropped after 60 min of exposure. The density of the IkappaB blot dropped with increasing exposure time after 30 min. TLR-4 was present on the surface of HUVECs. HUVECs constitutively expressed TLR-2 and TLR-4 mRNA; when exposed to flow shear stress for 60 min, TLR-4 mRNA expression increased.NF-kappaB activation is involved in flow shear stress-induced IL-8 mRNA expression in human umbilical vein endothelial cells. TLR-4 receptor for innate immunity most likely mediate these events.

This study is to evaluate the role of TLR-4 in lower laminar shear stress-induced interleukin-8 gene transcription activation in vascular endothelial cells by using gene mutation and transfection techniques. RT-PCR, Northern hybridization and immunocytochemical fluorescent staining showed that TLR-4 was expressed in human umbilical vein endothelial cells(HUVEC). When stimulated with 4.2 dyne/cm2 shear stress for 1 hour, an increase of TLR-4 mRNA expression was observed in HUVECs detected by RT-PCR and Northern hybridization. The intracellular domain deletion mutant TLR-4 cDNA (lacking the 155 COOH terminal amino acids of the wild type (TLR-4) and -102 -61 bp DNA sequence in 5'-flanking region of IL-8 gene (IL-8USCS) were isolated from endothelial cells by RT-PCR and PCR. These two DNA fragments were cloned into pcDNA3 and pEGFP1 respectively to construct TLR-4 dominant negative mutant pcDNA3-mTLR4 and IL-8 reporter gene pEGFP1-IL8USCS. ECV304 cells were transfected with the pEGFP1-IL8USCS or co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 by Dosper liposome transfectional reagent and selected by G418, then stimulated with 4.2 dyne/cm2 shear stress for 3 hours. Flow cytometric analysis showed that when exposed to 4.2 dyne/cm2 shear stress for 3 hours, there was a marked increase in the green fluorescent protein expression in pEGFP1-IL8USCS-transfected ECV304 cells. In contrast, there was almost no change in the green fluorescent protein expression in the cells co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 after the stimulation, suggesting the TLR-4 mutant depressed TLR-4 signaling. These experiments suggest that the inflammatory TLR-4/NF-kappa B signaling pathway would probably be involved in flow shear stress-induced IL-8 gene expression in human vascular endothelial cells.

In order to demonstrate that IL-8 mRNA expression in endothelial cells is not only regulated by chemical factors, but also by mechanical factors, in this article, after pretreating cultured human umbilical vein endothelial cells (HUVECs) with shear stress for different time, we employed both RT-PCR to assay IL-8 mRNA expression and immunocytochemical staining to detect NF-kappaB activation in HUVECs. We found that: (i) IL-8 mRNA expressed little in HUVECs untreated or pretreated with low laminar shear stress for 0.5 hour; IL-8 mRNA expression was increased when HUVECs were pretreated with low laminar shear stress for 1 hour, and increased further when pretreated for 2 hours; (ii) the immunoreactivity of NF-kappaB p65 in the nuclei of HUVECs untreated or pretreated with low laminar shear stress for 0.5 hour was negative, while it became weak positive in the nuclei of HUVECs pretreated with shear stress for 1 hour and positive in the nuclei of HUVECs pretreated for 2 hours. The results imply that low laminar shear stress was capable of inducing IL-8 gene expression and activating NF-kappaB, which were both time-dependent. The induction of IL-8 gene expression by laminar shear stress is probably due to the activation of NF-kappaB. We suggest that IL-8 mRNA expression in endothelial cells induced by low shear stress may play a key role in the pathogenesis and development of both inflammation and arterioatherosclerosis.