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Abstract Objective  To construct a recombinant adenovirus vector for expressing the IL-18 binding protein (IL-18BP)/IL-4 fusion gene and confirm the anti-inflammatory effect of this gene. Materials and methods  The recombinant virus expressing IL-18BP/IL-4 fusion protein (AD-IL-18BP/IL-4) was constructed. AD-IL-18BP/IL-4 was used to infect synovial fibroblasts (SF). ELISA and Western blot analysis were used to determine the expressions of the proteins IL-4 and IL-18BP. To investigate the protective effects of this vector on rheumatoid arthritis, SF were infected with AD-IL-18BP/IL-4 and stimulated by LPS (1 μg/ml) 4 h later. The expression levels of TNF-α, IL-6, IL-8, and IL-18 in the culture supernatant were detected by ELISA and production of PGE2 and NO was estimated. The protein expression of COX-2, iNOS, and NF-κB p50 in treated SF was analyzed by Western blot. Results  AD-IL-18BP/IL-4 can effectively express the IL-18BP/IL-4 fusion protein. The expressions of TNF-α, IL-6, IL-8, and IL-18 were significantly inhibited in LPS-stimulated SF after treatment with AD-IL-18BP/IL-4. The production of PGE2 and NO was significantly decreased. Moreover, NF-κB p50, COX-2, and iNOS levels in SF were markedly suppressed by AD-IL-18BP/IL-4. Conclusion  AD-IL-18BP/IL-4 can suppress the production and expression of inflammatory cytokines such as COX-2, iNOS, and NF-κB in LPS-stimulated SF. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic immunological disease, the invasive monocytes/macrophages and lymphocytes present in synovial cells and synovial tissue produce many cytokines and inflammatory mediators by paracrine signaling and plays a role in the pathological progress in RA patients. Interleukin-18 (IL-18) is a representative proinflammatory factor and displays multiple biological functions. This study was designed to investigate the expression of IL-18 and its receptor (IL-18R) and IL-18 binding protein (IL-18BP) in serum, synovial fluid, and synovial tissue of patients with RA, and to identify the pathological role of IL-18 in RA. Serum, synovial fluid, and synovial tissue were obtained from RA patients. Samples from patients with osteoarthritis and healthy people were obtained as controls. Levels of IL-18, IL-18BP, and PGE2 in serum and synovial fluid were measured by enzyme-linked immunosorbent assay. The biological activity of IL-18 in serum and synovial fluid was detected on the basis of IFN-γ secretion from IL-18-responding human myelomonocytic KG-1 cells. NO in serum and synovial fluid was detected by Griess reaction. Expression of IL-18, IL-18BP, IL-18R, iNOS, and COX-2 mRNA and protein in synovial tissues was determined by quantitative reverse transcriptase polymerase chain reaction and Western blot. This study shows the expression levels of IL-18, IL-18R, iNOS, COX-2, and the biological activity of IL-18 in both serum and synovial fluid and tissue of patients with RA were significantly increased compared with the corresponding samples from the two control groups. In addition, expression of IL-18BP in patients with RA was decreased compared with samples from the two control groups. In conclusion, the overexpression of IL-18 and IL-18R may play an important role in the pathogenesis of RA. [ABSTRACT FROM AUTHOR]

Aim: The aim of the present study was to investigate the expression of Toll-like receptors (TLR) 7 and 9 in peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B virus (HBV) infection and related hepatocellular carcinoma. Methods: The study group was comprised of 52 patients: 41 with chronic hepatitis B and 11 healthy controls. The protein and mRNA levels of TLR7 and TLR9 were evaluated using real-time PCR, Western blot analysis, and flow cytometry. We also detected the serum viral load of HBV in the patients and analyzed the correlation between HBV-DNA copies and the TLR expression. Results: Our results demonstrated a lower TLR7 expression in all HBV infection groups compared to the controls. We found that HBV infection led to a decreased expression of TLR9 mRNA, but an increased expression of the TLR9 protein compared to the healthy group. The TLR protein levels are related to serum HBV-DNA ( P < 0.01). Conclusion: There are downregulations of TLR7 expression and TLR9 mRNA in PBMC of HBV-infected patients, but an increased TLR9 expression at the protein level. [ABSTRACT FROM AUTHOR]

Recepteur d'origine nantais (RON) belongs to a subfamily of receptor tyrosine kinases (RTK) with unique expression patterns and biological activities. RON is activated by a serum-derived growth factor macrophage stimulating protein (MSP). The RON gene transcription is essential for embryonic development and critical in regulating certain physiological processes. Recent studies have indicated that altered RON expression contributes significantly to cancer progression and malignancy. In primary tumors, such as colon and breast cancers, overexpression of RON exists in large numbers and is often accompanied by the generation of different splicing variants. These RON variants direct a unique program that controls cell transformation, growth, migration, and invasion, indicating that altered RON expression has the ability to regulate motile/invasive phenotypes. These activities were also seen in transgenic mice, in which targeted expression of RON in lung epithelial cells resulted in numerous tumors with pathological features of human bronchioloalveolar carcinoma. Thus, abnormal RON activation is a pathogenic factor that transduces oncogenic signals leading to uncontrolled cell growth and subsequent malignant transformation. Considering these facts, RON and its variants can be considered as potential targets for therapeutic intervention. Experiments using small interfering RNA and neutralizing monoclonal antibodies demonstrated that suppressing RON expression and activation decreases cancer cell proliferation, increases apoptotic death, prevents tumor formation in nude mice, and reduces malignant phenotypes. Thus, blocking RON expression and activation has clinical significance in reversing malignant phenotypes and controlling tumor growth. [ABSTRACT FROM AUTHOR]

Objective: To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. Methods: The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5′ end of the RNA transcript (SMART) technique and CDS III/3′ primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to λ TriplEx2 vector. Then λ phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. Results: The titers of unamplifed and amplified libraries were 1.94×106 pfu/ml and 1.49×109 pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1–2 kb in 64.29%, and 0.5–1.0 kb in 35.71%. Conclusion: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2. 15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency. [ABSTRACT FROM AUTHOR]