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Objective To establish a high throughput screening assay for identifying human small molecular antagonists targeted IL-6R.Methods The full length gene of the human IL-6R extracellular region was amplified by PCR and cloned into a eukaryotic expression vector to construct recombination expression plasmid pABHis -IL6R that was then transfected transiently into HEK293T cells to prepare recombination protein IL-6R.Western blotting assay and receptor-ligand binding experiment were used to analyze the bioactivity of IL-6R.A new screening method based on ELISA was established using the function of IL-6R binding to its ligand and the characteristics of Fc fragment binding to IgG-HRP.Then Z′-factor was calculated and a known antagonist ab 47215 was used to assess the stability and reliability of the new assay .Results Recombination plasmid pABHis-IL6R was constructed and soluble IL-6R was prepared.IL-6R reported herein could be recognized by an anti-IL-6R antibody and specifically bind to its ligand in a dose response manner .A Z′-factor of 0.53 was obtained that could serve high throughput screening assay .Ab47215 , as a known specific antagonist , was able to block rhIL-6 from binding to the receptor in a dose-dependent manner in the new screening assay , the IC50 of which was (0.55 ± 0.11)μg/ml.Conclusion An innovative and easy screening assay for identifying human IL-6R antagonists is established , which might help discover potent and specific antagonists .
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Objetivos: Comparar níveis plasmáticos do receptor solúvel da interleucina-6 (IL-6sR) entre gestantes normotensas e com pré-eclâmpsia.Métodos: Realizou-se coleta de sangue materno no período pré-parto de 21 pacientes com pré-eclâmpsia e 39 controles normotensas. As amostras foram armazenadas a menos 80°C até a análise laboratorial. Os níveis séricos de IL-6sR foram mensurados através do teste imunoenzimático ELISA. Para comparar os grupos foi utilizado teste t de Student. Consideraram-se significantes os resultados com P menor do que 0,05.Resultados: Os dados de gestantes com pré-eclâmpsia e gestantes normotensas, respectivamente, foram: idade materna 22,3±4,8 vs 26,0±3,7 anos (P=0,06); idade gestacional 32,7±5,8 vs 40,1±0,8 semanas (P=0,01); pressão arterial sistólica 143,0±2,2 vs 118,8±3,1 mmHg (P=0,01); pressão arterial diastólica 112,5±4,0 vs 77,2±10,2 mmHg (P=0,01); ácido úrico 5,87±1,10 vs 4,57±0,12 mg/dL (P=0,02); creatinina 0,82±0,12 vs 0,73±0,09 mg/dL (P=0,01); peso do recém-nascido 2.130,7±839,3 vs 3.555,0±261,0 gramas (P=0.01) e peso da placenta 621,3±167,0 vs 796,3±154,2 gramas (P=0,05).A relação proteinúria/creatininúria no grupo das pacientes com pré-eclâmpsia foi de 2,40±1,31. O valor de IL-6sR(ng/dL) na pré-eclâmpsia foi 28,7±10,8 vs 16,5±6,4 na gestante normotensa (P=0,01).Conclusões: Estes resultados mostram o aumento dos níveis plasmáticos do IL-6sR em pacientes com pré-eclâmpsia, em relação a gestantes normotensas. Mais estudos se mostram necessários para o esclarecimento da fisiopatologia desta entidade, como a análise de outras citocinas ligadas a esse receptor, visto que elas podem ser a chave para a resposta inflamatória sistêmica que ocorre nestas pacientes e, portanto, para o seu tratamento...
Aims: To compare Interleukin-6 soluble receptor (IL-6sR) plasmatic levels between normotensive pregnant controls and preeclamptic women.Methods: Maternal blood samples were collected before delivery from 21 patients with preeclampsia and 39 normotensive pregnant controls. Samples were stored at -80°C until laboratory assay. IL-6sR was measured by ELISA enzyme immunoassay. To compare groups Student's t test was used. Results with P less than 0.05 were considered significant.Results: Data from preeclampsia and normotensive pregnant controls were respectively: maternal age 22.3±4.8 vs 26.0±3.7 years (P=0.06); gestational age 32.7±5.8 vs 40.1±0.8 weeks (P=0.01); systolic blood pressure 143.0±2.2 vs 118.8±3.1 mmHg (P=0.01); diastolic blood pressure 112.5±4.0 vs 77.2±10.2 mmHg (P=0.01); uric acid 5.87±1.10 vs4.57±0.12 mg/dL (P=0.02); creatinine 0.82±0.12 vs 0.73±0.09 mg/dL (P=0.01); birth weight 2130.7±839.3 vs 3555.0±261.0 g (P=0.01); placental weight 621.3±167.0 vs 796.3±154.2 g (P=0,05). Proteinuria over creatininuria ratio in the preeclampsia group was 2.40±1.31. The concentration of IL-6sR (ng/dL) was 28.7±10.8 in preeclampsia vs 16.5±6.4 in normotensive pregnant controls (P=0.01).Conclusions: These results show an increased plasma levels of IL-6sRin patients with preeclampsia compared to normotensive pregnant women. More studies are necessary to clarify the pathophysiology of this entity, including the analysis of other cytokines linked to this receptor, due to the fact that they can be the key for the systemic inflammatory response that occurs in these patients and therefore for their treatment....
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Humans , Female , Inflammation , Pre-EclampsiaABSTRACT
Objective To study the correlation between single nucleotide polymorphism (SNP) of downstream mononucleotide in signal transduction of Toll-like receptors and predisposing genes of rheumatoid arthritis (RA).Methods One hundred and sixty-two RA patients were selected from northern part of Han people in China,and 188 healthy subjects were enrolled as normal controls.Rs7744 of Myd88,rs5030445 of TRAF6,rs11265618 and rs4845626 of IL-6R were analyzed.The data of genotypic frequency and allele genotypic frequency were statistical analyzed by x2 test between the two groups.Results The difference of allele frequency of TRAF6 rs5030445 between the two groups were remarkable (x2=5.716,P＜0.05),and so did the IL-6R rs11265618 (x2=5.704,P＜0.05).But the difference of genotypic frequency was not statistically significant for locus of Myd88 rs7744,TRAF6 rs5030445 and IL-6R rs11265618 (P＞0.05).And the differenceof allele frequency and genotypic frequency between the two groups had no statistical significance in locus of Myd88 rs7744 and IL-6R rs4845626 (P＞0.05).Conclusion There is significant correlation between single nucleotide polymorphism and predisposing genes of RA.The G allele of TRAF6 rs5030445 gene locus is a possible predisposing gene for RA.The T allele of IL-6R rs11265618 is a possible predisposing gene of RA.
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Objective To investigate the levels of IL-2,IL-6and their receptors in patients with systemic lupus erythematosus(SLE)before and after treatment.Methods The levels of IL-2,IL-6and their receptors were detected by ELISA,immunofluorescence labelling technique and flow cytometry analysis,respectively,in peripheral blood taken from SLE patients before and after treatment and normal controls.Results①IL-2was significantly decreased(P
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Objective To establish the sensitive,specific,stable and convenient immunoradio assay for detecting human soluble IL-6R?.Methods The hybridoma cell lines were obtained by fusing spleen cells of BALB/c mice that had been immunized with soluble IL-6R? protein to mouse myeloma cells sp2/0. Ascites were used to produce the monoclonal antibodies (mAbs). The mAbs were purified by protein G immunoaffinity method. The mAb SI10 was used as coating antibody, the other mAb H126 recognized different epitope from SI10 was labeled by 125I. Results The immunoradio assay for detecting soluble IL-6R? was set up. It has high stability and accuracy. The detecting limit is 10 ng/ml. The serum concentration of soluble IL-6R? is (81.96 ? 7.23) ng/ml in healthy donors and (237.58?70.96) ng/ml in patients with multiple myeloma. Significant difference was founded between two groups (P
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Aim To study the change of the expression of the gp80 and gp130 subunits of the IL- 6 receptor (IL- 6R)in the osteoporotic bone marrow, to explore the role of IL- 6 receptor system in the osteoporosis resulted from the loss of estrogen.Methods Twenty female 12- week old SD rats [weight (200± 20) g] were randomly divided into 2 groups: the osteoporosis model group (10 rats),the control group (10 rats).Respectively in the 0th ,12th week after ovariectomy to analyze the levels of gp80 and gp130.Results In the 0th,12th week after ovariectomy,the level of gp130 (pg/μ g total RNA): the control group's level had no significant change (80± 3 vs 82± 4,t=0.85,P >0.05), while the osteoporosis group's level became significant higher (80± 3 vs 290± 40,t=6.46,P< 0.01).In the 0th ,12th week after ovariectomy, the level of gp80(pg/μ g total RNA): the control group's level had no significant change (85.4± 2.7 vs 85.6± 2.8,t=0.35,P >0.05), while the osteoporosis group's level became significant higher (85.4± 2.7 vs 210± 40,t=6.32,P< 0.01).Conclusion There was a significant increase of the expressions of gp130 and gp80 in the bone marrow of osteoporotic rat.
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Objective To detect the expression of IL-6 in peripheral blood of patients with gastric cancer,as well as the expressions of IL-6,IL-6 receptor(IL-6R) and proliferating cell nuclear antigen(PCNA) in the neoplastic tissue in order to investigate the clinical significance of such expressions in looking for a convenient and effective way for the diagnosis and therapy of gastric cancer.Methods Enzyme-linked immunosorbent assay(ELISA) was employed for the parallel semi-determination of IL-6 levels in the peripheral blood of 30 patients with gastric cancer,as well as the levels of IL-6 and IL-6R in the neoplastic and adjacent tissues.The expression of PCNA in the neoplastic tissues was determined with immuno-histochemistry technique.Moreover,the correlation was comprehensively analyzed among IL-6,IL-6R,PCNA and the clinicopathologic findings of gastric cancers.Results The IL-6 levels in both peripheral blood and neoplastic tissues of 30 patients were significantly higher than those of the normal controls.The IL-6 levels in peripheral blood decreased significantly after operation;the IL-6 and IL-6R levels in cancer tissues were significantly higher than those in their adjacent tissues with a positive correlation between IL-6 and IL-6R levels.The expressions of IL-6,IL-6R and PCNA in both peripheral blood and neoplastic tissues of patients were positively related to the clinical manifestation of gastric cancer,including the differentiation degree of cancer cells,tumor size,infiltration extent and the metastasis of cancer cells.Conclusions IL-6,IL-6R and PCNA are the important factors which affect the degree of malignancy of the gastric cancer concerned,and they also,reflect the possibility of infiltration and metastasis of cancer cells.The IL-6 level in peripheral blood and the levels of IL-6,IL-6R and PCNA in tumor tissues of patients with gastric cancer are obviously high.The expression levels of all these factors are correlated with the clinicopathologic findings of gastric cancer.An adequate determination of IL-6 and PCNA is of benefit to the diagnosis and treatment of gastric cancer,and to the judgment of the course and prognosis of gastric cancer.
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Gene expressions of interleukin-6 receptor subunit gp130 and gp80 were investigated by competitive RT-PCR in the rat model of osteoporosis. A significant increase in expressions of gp130 and gp80 was found in bone marrow of osteoporotic rat, this change may be a possible mechanism in osteoporosis due to loss of estrogen.
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Objective To investigate the role of IL-6 and IL-6R in the pathogenesis of systemic lupus erythematosus (SLE) and its clinical significance. Methods RT-PCR technique was used to semiquantitatively analyze IL-6R mRNA expression in peripheral blood mononuclear cells (PBMC), and double antibody sandwich ELISA was applied to determine the serum level of IL-6 from 89 SLE patients . Results The positive IL-6R mRNA expression was found in each individuals among active SLE group (groupⅠ), inactive SLE group (groupⅡ) and control group (group Ⅲ). The mean levels of IL-6R mRNA expressions in groupⅠ,Ⅱ or Ⅲ were 0.902 ? 0.273, 0.519?0.11 and 0.573?0.24, respectively. Compared with group Ⅱand group Ⅲ, the mean level of IL-6R mRNA expression in groupⅠ was markedly increased (P = 0.00), while there was no statistical difference between group Ⅱ and group Ⅲ(P = 0.289). The mean serum levels of IL-6 in groupⅠ,Ⅱ and Ⅲ were 71.89 ? 20.02,17.96 ? 6.93, and 0.035 ? 0.035, respectively. Compared with group Ⅱand group Ⅲ, the mean level of IL-6 in groupⅠ was significantly higher than those in groupⅡ and groupⅢ (P = 0.00), while that in groupⅡwas higher than group Ⅲ (P = 0.00). There was a positive correlation between IL-6 and IL-6R in active SLE group and inactive SLE group(r = 0.887, and r = 0.615 P
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Serum concentrations of interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R) were determined in patients with Graves′ ophthalmopathy (GO), Graves′disease without GO (NGO) and controls. Patients with GO treated by corciosteroids were assessed by the clinical activity score (CAS). The results suggested that serum levels of both IL-6, sIL-6R and CAS could reflect the activity of GO and predict the outcome of corcicosteroid therapy.