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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.

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.

experimental pancreatitis and explore the mechanism of bacterial translocation at the molecular level in mice with severe acute pancreatitis.Thirty-six ICR mice were randomly divided into 6 groups with 6 mice in each group. The animals in the experimental groups (from A to E) received seven intraperitoneal injections of caerulein (50 microg/kg body weight) at hourly intervals over 6 hours, and were killed at 9, 18, 24, 48 and 72 hours after the first injection. The control group (F) received intraperitoneal injection of the same volume of saline, and the animals were killed at the 18th hour after the first injection. Blood and pancreatic tissue samples were obtained after the animals were killed. Amylase and pancreatic pathological alterations were observed. The ileum sequential segments were removed from each mouse. The amplification products of RT-PCR were electrophoresed and the images were analyzed by UVI software.Acute necrotizing pancreatitis in ICR mice were induced by the intraperitoneal injection of caerulein at large doses. Markedly pathological lesions were observed at the 18th hour after the first injection of caerulein. And, intestinal cryptdin-4 mRNA expression was down regulated slightly at the 9th hour and most markedly at the 18th hour (P0.05). After 24 hours, the cryptdin-4 mRNA expression recovered to the normal level gradually.Acquired cryptdin-4 deficiency may play an important role in the pathogenesis of bacterial tanslocation in acute necrotizing pancreatits.

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.

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.

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.