OBJECTIVETo construct pPICZalphaA-soluble interleukin-1 receptor type I (sIL-1RI) recombinant expression vector containing the gene fragment encoding the extracellular domain of sIL-1RI for its expression in Pichia pastoris.

METHODSsIL-1RI gene was amplified by RT-PCR and inserted into the yeast expression vector pPICZalphaA by digestion ligation. The recombinant plasmid pPICZalphaA-sIL1RI was transformed into E.coli Stb13, and the positive clones were analyzed by PCR and DNA sequencing. The pPICZalphaA-sIL1RI recombinant plasmid was electroporated into GS115 cells and the transformants were analyzed by PCR. After phenotype identification, the recombinant strains were induced by methanol to express the target protein, which was analyzed by Western blotting of the cell extract and supernatant.

RESULTSThe recombinant plasmid pPICZalphaA-sIL-1RI was constructed successfully, and the results of Western blotting showed that yeast induced by methanol expressed a protein of about 39 kD.

CONCLUSIONsIL-1RI protein has been successfully expressed in P.pastoris expression system, which provides the basis for further study of sIL-1RI.

Escherichia coli ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Plasmids ; Receptors, Interleukin-1 Type I ; biosynthesis ; genetics

OBJECTIVETo investigate the association of two single-nucleotide polymorphisms (SNPs) in IL1R1 gene (rs1558641 and rs949963) with the susceptibility to asthma in children from Central China.

METHODSA case-control study was performed in the asthma group and the control group, consisting of 208 children with asthma and 223 normal children from Central China, respectively. The genotypes of two SNPs in IL1R1 gene, rs1558641 and rs949963, were identified using polymerase chain reaction-restriction fragment length polymorphism. The serum level of IL1R1 was determined by enzyme-linked immunosorbent assay.

RESULTSThere were no significant differences in genotype and allele frequencies of rs1558641 between the asthma and control groups. In terms of rs949963, the frequencies of GG genotype and alleles were significantly higher in the asthma group than in the control group (P<0.05). The asthma group had a significantly higher serum level of IL1R1 than the control group (P=0.011). Moreover, the serum level of IL1R1 was significantly higher in patients with GG genotype than in those with AA or AG genotype for rs949963 (P=0.028).

CONCLUSIONSIL1R1 SNP rs949963 is associated with the susceptibility to asthma in children from Central China and may increase the serum expression of IL1R1.

Alleles ; Asthma ; genetics ; Case-Control Studies ; Child ; Child, Preschool ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Infant ; Male ; Polymorphism, Single Nucleotide ; Receptors, Interleukin-1 Type I ; genetics

OBJECTIVETo investigate the effect of berberine on expressions of tumor necrosis factor alpha (TNF-alpha) and receptor type I (TNFR1) in Abeta25-35-induced inflammatory reaction in SH-SYSY cell lines.

METHODThe 5 micromol . L-1 Abeta25-35 was used to treat SH-SY5Y cells for 24 hours, in order to establish the Alzheimer's disease (AD) model. Before modeling, berberine was given for pretreatment for 2 hours. The experiment included the normal control group, the AD model group, and indometacin low dose and high dose groups. Spectrophotometry was adopted to detect the activity of LDH. Meanwhile, the level of TNF-alpha was determined by ELISA, and the expression of TNFR1 genes was detected by RT-PCR.

RESULTCompared with the normal control group, the AD cell model group showed significant increase in LDH, TNF-alpha, and TNFR1 gene and protein expressions in the culture media. After intervention with berberine, the activity of LDH and TNF-alpha reduced in cell supernatant. The intervention with berberine could down-regulate TNFR1 gene and protein expressions, particularly 1, 10 x 10(-6) mol . L-l berberine showed a more notable effect in regulating TNFR1.

CONCLUSIONBerberine has the protective effect in Abeta-induced inflammatory injury in SH-SY5Y cells. Its mechanism may be related to the expression of its anti inflammatory factor TNF-alpha and its type I receptor TNFR1. Specifically, its regulation to TNFR1 shows dose dependence.

Amyloid beta-Peptides ; toxicity ; Berberine ; pharmacology ; Cell Line ; Humans ; Inflammation ; chemically induced ; metabolism ; Peptide Fragments ; toxicity ; Receptors, Interleukin-1 Type I ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo isolate and characterize human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs).

METHODSThe synovial membrane tissues were obtained from 4 RA patients, 1 chondroma patient and 1 healthy subject and FLS were isolated by means of tissue culture. The cell morphology was observed by phase-contrast microscope and the cell surface markers were detected by flow cytometry.

RESULTSThe FLSs were successfully cultured from the synovial membrane tissues with good cell homogeneity after the third passage. The FLSs of the 3rd to 7th passages were stable and proliferated actively, followed by slow proliferation and aging since the 8th passage. Flow cytometry showed that the 4th-passage FLSs from the RA patients contained 99.04% CD90(+) cells, 2.73% CD3(+) cells, 0.29% CD3(-)CD19(+) cells, 2.81% CD3(-)CD16(+)CD56(+) cells, 5.89% CD14(+) cells, and 54.17% CD55(+) cells. The presence of interleukin-1 receptor type I (IL-1RI, 158.63-/+20.32 pg/ml) and IL-1beta (4.67-/+0.82 pg/ml) were detected in the cell culture supernatant of the 4th-passage FLSs from the RA patients by enzyme-linked immunosorbent assay ELISA.

CONCLUSIONFLSs from RA patients can be effectively culture by means of tissue culture, and the cultured FLSs show high expressions of CD90, IL-1RI and IL-1beta.

Adult ; Aged ; Arthritis, Rheumatoid ; pathology ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Female ; Fibroblasts ; pathology ; Humans ; Interleukin-1beta ; metabolism ; Male ; Middle Aged ; Receptors, Interleukin-1 Type I ; metabolism ; Synovial Membrane ; cytology ; pathology ; Thy-1 Antigens ; metabolism

OBJECTIVETo investigate the effects of local injections of recombinant soluble human receptors on experimental orthodontic tooth movement in the rat.

METHODS64 male Sprague-Dawley rats were observed. Starting at day 1, three groups of animals each received local injections of soluble interleukin-1 receptor II (sIL-1-R II ), soluble' tumor necrosis factor receptor I (sTNF-R I ) and their combinations. One group served as the control. The amount of tooth movement was recorded and selected tissue sections were stained with hematoxylin-eosin (HE) to observe the histological morphologic alterations of the periodontal tissues and also were stained with tartrate-resistant acid phosphatase (TRAP) histochemistry to analyze the changes of the amount and distribution of osteoclasts and odontoclasts.

RESULTSThe time-depended histomorphology changes in each receptor group were similar to those in the control group, but the resorption of alveolar bone was slighter at each time point and the surface of root appeared no or a few cement resorption signs. On day 14, the TRAP-positive cells on the surface of alveolar bone and root were reduced by approximately 50% relative to those in the control group (P<0.05). Whereas statistical tests revealed there were no significant differences among the experimental groups (P>0.05).

CONCLUSIONLocal injections of recombinant human sIL-1-R II and sTNF-R I in the orthodontic tooth of rats could reduce the amount and velocity of orthodontic tooth movement and reduce the incidence of root resorption.

Animals ; Dental Cementum ; Interleukin-1 ; Male ; Osteoclasts ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Receptors, Tumor Necrosis Factor ; Receptors, Tumor Necrosis Factor, Type I ; Root Resorption ; Tooth Movement Techniques ; Tooth Root

OBJECTIVETo assess the value of rheumatoid factors IgM-RF, IgA-RF, IgG-RF, interleukin-1 receptor type I (IL-1RI) and cyclin-dependent kinase 2 (CDK2) in the diagnosis of rheumatoid arthritis (RA).

METHODSIgM-RF, IgA-RF, IgG-RF, IL-1RI and CDK2 were detected in 47 patients with active RA, 47 with inactive RA, 20 with active systemic lupus erythematosus (SLE), 20 with inactive SLE, 20 with acute upper respiratory tract infection (AURTI), and 20 healthy controls using enzyme-linked immunosorbent assay (IgM-RF, IgA-RF, IgG-RF, IL-1RI) and time-resolved fluoroimmunoassay (CDK2).

RESULTSPatients with active and inactive RA showed significant differences in peripheral serum CDK2 and IgA-RF levels (P<0.05). Between active and inactive RA patients, RA patients and SLE patients, RA patients and AURTI patients, and RA patients and the control subjects, the area under curve (AUC) of the receiver operating characteristic curve (ROC) was 0.561, 0.814, 0.799, and 0.888 for IgM-RF, 0.596, 0.678, 0.729, and 0.850 for IgA-RF, 0.614, 0.718, 0.692, and 0.791 for IgG-RF, 0.646, 0.691, 0.762, and 0.835 for IL-1RI, 0.803, 0.753, 0.741, and 0.840 for CDK2, respectively.

CONCLUSIONSIgM-RF may have high value in the diagnosis and differential diagnosis of RA, and CDK2 can be useful for differential diagnosis between active and inactive RA.

Adult ; Area Under Curve ; Arthritis, Rheumatoid ; blood ; diagnosis ; Cyclin-Dependent Kinase 2 ; blood ; Female ; Humans ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Male ; Middle Aged ; ROC Curve ; Receptors, Interleukin-1 Type I ; blood ; Rheumatoid Factor ; blood

Interleukin-1 receptor antagonist (IL-1ra), tumor necrosis factor soluble receptors (sTNF-R) type I and II, and regulated upon activation, normal T-cell expressed and secreted (RANTES) play an important role in the modulation of primary glomerulonephritis (GN) course. The aim of the study was to assess whether pre-treatment measurements of IL-1ra, sTNF-R, and RANTES assessed conjointly may be useful as predicting factors in patients with GN. In 84 patients (45 males and 39 female) serum concentration (pg/mL) and urinary excretion (pg/mgCr) of cytokines were measured. After 12 months of therapy with steroids and cyclophosphamide the patients were divided into two subgroups: Responders (R) and Non-Responders (NR) according to the treatment results. The urinary IL-1ra, TNF-RI and RII were significantly higher in R than NR (1,732 vs 646 with P < 0.001, 13.1 vs 6.3 with P = 0.005, and 33.6 vs 14.4 with P = 0.012). The urinary RANTES excretion was increased in NR (79.6 vs 28.5; P < 0.001). The multivariable analysis showed that if conjointly assessed, only urinary IL-1ra, TNF-R I and R II, RANTES with 85% probability pointed the feature remission (R). In conclusion, the urinary excretion of IL-1ra, TNF-R I and R II, and RANTES examined conjointly are effective in predicting favorable response to immunosuppressive treatment in patients with GN.
Adult ; Cyclophosphamide/therapeutic use ; Female ; Glomerulonephritis/drug therapy/*metabolism/pathology ; Humans ; Immunosuppressive Agents/therapeutic use ; Interleukin 1 Receptor Antagonist Protein/*analysis/blood/urine ; Lymphocyte Activation ; Male ; Middle Aged ; Multivariate Analysis ; Predictive Value of Tests ; Receptors, Tumor Necrosis Factor, Type I/*analysis/blood/urine ; Receptors, Tumor Necrosis Factor, Type II/*analysis/blood/urine ; Steroids/therapeutic use ; T-Lymphocytes/immunology/metabolism

OBJECTIVETo investigate the spectrum of β -thalassemia mutations in Guizhou Province.

METHODSFor 542 individuals suspected to have β -thalassemia by decreased mean corpuscular volume (MCV) and corpuscle hemoglobin (MCH) by routine blood test and hemoglobin electrophoresis, reverse dot blot hybridization (RDB) was performed to detect 17 known β -thalassemia mutations, including 8 common and 9 rare mutations. For cases where no mutation was identified, the entire human β -globin gene was screened to find other rare mutations. The distribution and frequencies of detected β -thalassemia mutations were then analyzed.

RESULTSA total of 460 individuals were diagnosed as β -thalassemia by DNA analysis, which included 352 heterozygotes, 67 compound heterozygotes and 41 mutant homozygotes. A total of 12 β -thalassemia mutations were detected in these individuals. The mutations have ranked from high to low frequency as: CD17 (40.74%), CD41-42 (33.69%), IVS-II-654 (13.76%), -28 (3.70%), β E (3.35%), CD71-72(1.94%), CD43 (1.06%), IVS-I-1 (0.71%), CD27-28 (0.35%), -29(0.35%), CAP (0.18%), and CD121 (0.18%). The former six mutations have accounted for 97.18% of all. CD121 (GAA> TAA) detected from a heterozygote, as a dominant mutation, has been firstly found in the Chinese population.

CONCLUSIONThe spectrum of β -thalassemia in Guizhou Province showed certain distinct characteristics, with CD17 being the most common mutation. The newly discovered mutation of CD121 has expanded the spectrum of β -thalassemia in Chinese population. Our result may provide valuable information for the prevention and control of β -thalassemia in Guizhou.

Adolescent ; Adult ; Asian Continental Ancestry Group ; genetics ; Child ; Child, Preschool ; China ; DNA Mutational Analysis ; Female ; Humans ; Infant ; Leukosialin ; genetics ; Male ; Middle Aged ; Mutation ; Platelet Membrane Glycoprotein IIb ; genetics ; Receptors, Interleukin-1 Type I ; genetics ; Young Adult ; beta-Globins ; genetics ; beta-Thalassemia ; diagnosis ; ethnology ; genetics

BACKGROUNDOsteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-alpha receptor type I (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis.

METHODSAdenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 x 10(8) plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 x 10(8) pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 x 10(8) pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The samples were examined by light microscopy and quantitatively evaluated.

RESULTSIntra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF-alpha blockade has little effect on joint destruction.

CONCLUSIONThe enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.

Adenoviridae ; genetics ; Animals ; Arthritis, Experimental ; genetics ; therapy ; Cartilage ; metabolism ; pathology ; Cartilage, Articular ; metabolism ; pathology ; Cell Line ; Cells, Cultured ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Interleukin 1 Receptor Antagonist Protein ; Osteoarthritis ; genetics ; therapy ; Rabbits ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; physiology ; Sialoglycoproteins ; genetics ; physiology ; Synovial Fluid ; metabolism ; Synovial Membrane ; cytology ; metabolism ; Transfection ; methods

OBJECTIVETo identify the single nucleotide polymorphisms (SNPs) in the regulatory and coding regions of human interleukin-1 receptor type I (IL-1R1) gene and to assess their potential effect on the function of IL-1R1.

METHODSThe 5' flank region, exons, parts of the introns, as well as 3' flank region of IL-1R1 gene were sequenced to identify and characterize the SNPs in Chinese population. Effects of the SNP on the structure and function of IL-1R1 were analyzed by computational methods.

RESULTSSixteen SNPs were identified through a 9643 bp sequencing of IL-1R1 gene. Among them, four were in 5' flank region, four in intron region, one in coding region, and seven in 3' untranslated region. A novel SNP in Chinese population was involved in a structural change in IL-1R1, which may influence the signal transduction of IL-1R1.

CONCLUSIONThe SNP in the IL-1R1 gene might influence its function as an important receptor of IL-1 family.

Amino Acid Sequence ; Asian Continental Ancestry Group ; Cell Membrane ; metabolism ; Computational Biology ; Exons ; genetics ; Humans ; Hydrophobic and Hydrophilic Interactions ; Introns ; genetics ; Molecular Sequence Data ; Polymorphism, Single Nucleotide ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Receptors, Interleukin-1 Type I ; chemistry ; genetics ; metabolism ; Sequence Homology, Amino Acid