Bactericidal/permeability-increasing protein (BPI) can bind to and specifically neutralize lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria. In order to evaluate potent LPS-neutralizing activity of bovine BPI, the full-length coding sequence (1 449bp) or 714 bp N-terminal coding sequence (BPI714) of bovine BPI was transfected into mHEK293 cells and the expression of LPS-induced inflammatory cytokines was studied. First, we constructed the lentiviral expression vectors and generated mHEK293 cells stably expressing recombinant bovine BPI or BPI714. Then, we detected the expression of IL-8, IL-1β, TNF-α, NF-κB-1 and NF-κB-2 genes by real-time PCR at 0, 1, 3, 6, 12, 24, 36 and 48 h post of LPS induction in cells with or without recombinant bovine BPI or BPI714 ectopic expression, respectively. In response to LPS, the robust abundance of inflammatory cytokines including IL-8, IL-1β, TNF-α and NF-κB-2 was observed in wild type mHEK293 cells at eachtime point. On the contrary, mRNA abundance of IL-8, TNF-α and NF-κB-2 in transfected mHEK293 cells showed no significant changes at each indicated time point. Our results demonstrated that recombinant bovine full length BPI or BPI714 down-regulated the expression of inflammatory cytokines and revealed that either of bovine BPI or BPI714 was able to inhibit the immune respond stimulated by LPS. This study provides evidence for further investigating the mechanisms and application of BPI/LPS-neutralizing activity and also documents a reliable approach for analysis of the efficacy of antibacterial proteins.
Animals ; Antimicrobial Cationic Peptides ; chemistry ; Blood Proteins ; chemistry ; Cattle ; Cytokines ; biosynthesis ; HEK293 Cells ; Humans ; Interleukins ; biosynthesis ; Lipopolysaccharides ; chemistry ; NF-kappa B ; biosynthesis ; Transfection ; Tumor Necrosis Factor-alpha ; biosynthesis

OBJECTIVETo study the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) mRNA in mouse osteoblasts.

METHODSCalvariae derived from CD-1 neonatal mouse (after born 24 h). Bone samples were processed by the collagenase/trypsin digestion method. Mouse osteoblasts were cultured in vitro. After 48 hours of addition of 1,25(OH)2D3 (0, 10(-8), 10(-9), 10(-11) mol/L) to the culture medium of mouse osteoblasts, the content of the OPG protein in culture medium was estimated with enzyme linked immunosorbent assay. Total RNA was prepared from mouse osteoblasts. mRNA expression of OPG and RANKL were detected by reverse transcription-polymerase chain reaction.

RESULTSThe mRNA expression of OPG in osteoblasts added with 1,25(OH)2D3 significantly decreased compared with the controls, which was markedly dose-dependent. OPG protein production in the medium decreased after treatment with 1,25(OH)2D3. In contrast, RANKL mRNA expression levels in osteoblasts significantly increased after 48 h of culture with 1,25(OH)2D3.

CONCLUSION1,25 (OH)2D3 can stimulate RANKL mRNA expression, but decrease OPG mRNA levels in vitro in mouse osteoblasts.

Animals ; Animals, Newborn ; Calcitriol ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; Glycoproteins ; biosynthesis ; genetics ; physiology ; Ligands ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; NF-kappa B ; biosynthesis ; genetics ; Osteoclasts ; metabolism ; physiology ; Osteoprotegerin ; RANK Ligand ; RNA, Messenger ; biosynthesis ; genetics ; Receptor Activator of Nuclear Factor-kappa B ; Receptors, Cytoplasmic and Nuclear ; analysis ; biosynthesis ; genetics ; physiology ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

In order to get soluble TNF receptor (sTNFR) II with good neutralizing activity against TNFalpha, we constructed the fusion gene sTNFRII-gAD, which encoded human sTNFR II and the globular domain of adiponectin (gAD), and then expressed it in mammalian cells and analyzed its anti-TNFalpha activity. First, sTNFRII cDNA was obtained by RT-PCR from the total RNA of human peripheral blood lymphocytes, and fused in frame with gAD gene. Then, the fusion gene sTNFRII-gAD was cloned into the expression vector pAAV2neo to result in the plasmid pAAV2neo-sTNFRII-gAD. By immunofluorescent staining with monoclonal antibody either against TNFRII or against adiponectin, we demonstrated that the pAAV2neo-s7NFRII-gAD-transiently-transfected BHK-21S cells were positive. To obtain G418-resistant BHK-21S/pAAV2neo-sTNFRII-gAD cells, we cultured the transfected BHK-21S cells above in 10% FBS containing DMEM media with 800 microg/mL G418 for 15 days, and changed the serum-containing culture media to a serum-free chemically defined media so as to change the cells culturing style from adhesion to suspension. 24 hours later, we harvested the supernatant of the culture for sTNFRII-gAD fusion protein characterization and anti-TNFalpha activity analysis. With monoclonal antibody either against TNFRII or against adiponectin, the Western blotting analysis showed that the sTNFRII-gAD fusion protein was expressed and existed as monomer, trimer and multimer forms in the supernatant. The bioactivity assay demonstrated that the sTNFRII-gAD fusion protein had the ability to neutralize TNFalpha so as to inhibit the cytotoxicity of TNFalpha on L929 cells. Put together, this study has laid the groundwork for large-scale preparation of sTNFRII-gAD fusion protein.
Adiponectin ; biosynthesis ; genetics ; Animals ; Cells, Cultured ; Cricetinae ; Humans ; Protein Structure, Tertiary ; genetics ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Solubility ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors

OBJECTIVETo investigate the effects of LPS and/or TNF-alpha on periodontal ligament cell (PDLC) proliferation and OPG secretion.

METHODSHealthy premolars extracted for orthodontic reasons from a 12 years old boy were obtained, and periodontal tissues were collected and cultured to obtain PDLCs. Cloned PDLCs were obtained by means of limited dilutions, and were characterized as follows: alkaline phosphatase activity, collagen III production and bone-like nodules formation. LPS and rhTNF-alpha were added into culture media and their effects on PDLC proliferation and OPG secretion were observed. The OPG concentrations in cell culture supernatants were detected by sandwich ELISA. Living cell numbers were demonstrated by MTT test. The average levels of OPG secretion by a single cell were calculated by dividing OPG concentration with MTT result.

RESULTSrhTNF-alpha above 10 micro g/L decreased the mtt and opg detecting results, but increased the opg/mtt values (P < 0.05). However, LPS had no effect on mtt, opg or opg/mtt values. Neither it had any interaction with rhTNF-alpha (P > 0.05).

CONCLUSIONSTNF-alpha prohibits the proliferation of PDLCs but enhances their OPG secretion. However, LPS has no effect on neither side. Our works support the hypothesis that there may be an inverse feedback regulation pattern of increasing periodontal OPG production against local bone resorption activity. PDLCs might not be the natural target cells of LPS' direct cytotoxic effect.

Child ; Glycoproteins ; biosynthesis ; drug effects ; Humans ; In Vitro Techniques ; Lipopolysaccharides ; pharmacology ; Male ; Osteoprotegerin ; Periodontal Ligament ; metabolism ; pathology ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; drug effects ; Receptors, Tumor Necrosis Factor ; biosynthesis ; drug effects ; Tumor Necrosis Factor-alpha ; pharmacology

In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.
Adiponectin ; biosynthesis ; genetics ; Animals ; Bioreactors ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

In order to investigate the effects of LPS on the TACE gene transcription and expression and its regulating effect on the TM-TNF secretion, in vitro studies were carried out on HL-60 cells stimulated by LPS. TACE, TNF-alpha mRNA levels were detected by Dot-Elisa and the distribution of membrane molecules determined by flow cytometry assay and indirect immunofluorescence. The results showed that: (1) TACE was detected in or on HL-60 cells and it is predominantly localized on cell surface and to a perinuclear compartment. (2) LPS induced a time dependent increasement of TNF-alpha mRNA and enhanced TNF conversion with decreasing distribution of TNF in cell surface and increasing secretion of TNF protein. Such conversion could be inhibited by TACE ODN. (3) LPS also induced time-dependently increased expression of TACE gene and activation of its function. On the other hand, TACE protein in cell lysate and on cell surface was decreased. It was suggested that TACE molecular structure might change following its mediating membrane-anchored molecular secretion.
ADAM Proteins ; ADAM17 Protein ; Gene Expression ; HL-60 Cells ; Humans ; Lipopolysaccharides ; pharmacology ; Metalloendopeptidases ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transcription, Genetic ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.
Animals ; Antibodies, Bispecific ; biosynthesis ; immunology ; Antibodies, Neutralizing ; biosynthesis ; immunology ; Escherichia coli ; Fibronectins ; chemistry ; immunology ; Humans ; Mice ; Single-Chain Antibodies ; biosynthesis ; immunology ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo study the purification, refolding and bioactivity of streptavidin-tagged human tumor necrosis factor-alpha (SA-TNF-alpha) bi-functional fusion protein.

METHODSSA-TNF-alpha fusion protein was expressed in BL21(DE3) host bacteria, purified using Ni-NTA affinity chromatography and refolded by dilution and dialysis followed by identification using Western blotting. The effect of SA-TNF-alpha fusion protein against L929 cells was evaluated by MTT assay. Flow cytometry was used to analyze the surface modification of biotinylated MB49 tumor cells by SA-TNF-alpha fusion protein.

RESULTSRecombinant SA- TNF-alpha fusion protein was expressed in BL21(DE3) at about 30% of total bacterial protein, with a purity of about 95% after purification. The SA-TNF-alpha fusion protein existed as dimmers, tetramers and higher order structures after refolding. The fusion protein exhibited a bi-functionality by inhibiting L929 cells and SA-mediated high-affinity binding to biotinylated cell surfaces, with an anchor modification rate of above 90%.

CONCLUSIONThe dimmers, tetramers and higher order structures of the obtained SA-TNF-alpha fusion protein all exhibit a bi-functionality, and may serve as a potential candidate therapeutic agent for tumors.

Chromatography, Affinity ; methods ; Escherichia coli ; genetics ; metabolism ; Humans ; Nickel ; Protein Folding ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Streptavidin ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

This paper is aimed to present a research on fusion protein of human tumor necrosis factor-alpha (hTNF-alpha), matrix metalloproteinase 1 (MMP1), and foldon sequence using the methord of gene engineering. We transformed the recombinant plasmid, which contains the DNA sequences of hTNF-alpha, MMP1, and foldon sequence, into Rosetta2, and successfully induced the fusion protein to express under given conditions by isopropyl beta-D-1-Thiogalactopyranoside (IPTG). Then we purified the expression product through a glutathione S-transferase (GST) resin and collected the interested protein. This research may lay the groundwork for scientific research and clinical application.
Base Sequence ; Escherichia coli ; genetics ; metabolism ; Humans ; Matrix Metalloproteinase 1 ; biosynthesis ; genetics ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

Animals ; Interleukin-1 ; biosynthesis ; genetics ; Liver ; blood supply ; Lung ; metabolism ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics