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@#Objective To develop and verify a method for detecting the activity of neutralizing antibodies in ELISA antibody positive serum of rats immunized with recombinant human interleukin-1 receptor antagonist(rhIL-1Ra). Methods The SD rats were subcutaneously immunized with 3,20 and 100 mg/kg rhIL-1Ra injection respectively,10 rats in each group,half male and half female,twice a day at an interval of at least 4 h between each dose for 13 consecutive weeks. The blood samples were collected from the jugular vein of rats during the administration period and the recovery period. The serum samples were isolated and detected for the antibody titers by ELISA,and the samples positive for rhIL-1Ra antibody were purified by Protein A chromatographic column. Based on,D10G4·1 cells biological activity assay,a method for the detection of neutralizing antibody activity was developed and verified for the specificity,sensitivity and precision. The neutralizing antibody activity of rhIL-1Ra antibody positive serum determined by ELISA was detected by using the developed method.Results With the increase of doses,the serum antibody titers of rats in various dose groups gradually increased,and there were still antibodies in the recovery period,and the titer was still high. Rabbit anti-rhIL-1Ra monoclonal antibody showed obvious neutralizing effect on rIL-1Ra,while rabbit anti-rIFN-2b monoclonal antibody had no dose-effect relationship with rIL-1Ra. The sensitivity of the method was 171. 93 μg/mL;The CVs of precision verification were not more than 20%. The positive antibody sera detected by ELISA all had neutralizing effect on rhIL-1Ra injection,which was consistent with the results detected by ELISA. Conclusion The method developed in this study has good specificity and high sensitivity in the detection of serum neutralizing antibody activity in rats immunized with rhIL-1Ra,which can be used to detect the serum neutralizing antibody activity of animals with rhIL-1Ra repeated administration.
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The interleukin-1 (IL-1) signaling pathway plays a vital role in multiple mechanisms associated with myocardial ischemia-reperfusion (I/R) injury, including inflammation and apoptosis. An IL-1 receptor antagonist (IL-1Ra) can block IL-1 by competitive binding to the IL-1 receptor type I (IL-1RI) and thus may provide a cardioprotective effect. In the present investigation, we determined whether exogenous administration of recombinant human IL-1Ra (rhIL-1Ra) provides a protective role against myocardial I/R injury. Sprague-Dawley rats underwent surgical coronary artery ligation (or sham operation) by occlusion of the left anterior descending artery (LAD) for 30 min followed by reperfusion. After 30 min of reperfusion, a 2 mg/kg dose of rhIL-1Ra was injected subcutaneously. This was followed up with once daily injections for seven days. Echocardiography revealed that ejection fraction (EF) values were significantly greater in the rhIL-1Ra-treated animals. RhIL-1Ra was found to reduce the severity of myocardial injury and increase the viability of the cardiac tissue. There was found to be less IL-1ß expression in rhIL-1Ra-treated animals than in controls. These results provide compelling data to suggest administration of IL-1Ra could have a significant cardioprotective effect against myocardial I/R injury and thus IL-1Ra could emerge as a potential clinical drug for the treatment of myocardial I/R injury.
Subject(s)
Cardiotonic Agents/administration & dosage , Interleukin 1 Receptor Antagonist Protein/administration & dosage , Myocardial Reperfusion Injury/diagnostic imaging , Myocardial Reperfusion Injury/prevention & control , Animals , Humans , Male , Rats , Rats, Sprague-Dawley , Recombinant Proteins/administration & dosageABSTRACT
Objective To investigate the effect of recombinant human interleukin 11(rhlL-11)in the treatment of idiopathic thrombocytopenic purpura(ITP)on the levels of Th1,Th2 and the expression of their transcription factors T-bet mRNA,GATA-3 mRNA.Methods Fifty-six cases adult ITP patients hospitalized in the department of hematology of the Second People's Hospital of Datong from May 2015 to December 2016 were collected,including 21 males and 35 females,aged 29~73 years; 10 healthy people in the same period were enrolled as control group,4 males and 6 females,aged 20~52 years.Th1 and Th2 cell ratio and Th1/Th2 ratio of ITP patients were detected by flow cytometry before and after treatment.The expression levels of transcription factor T-bet and GATA-3 were measured using real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR)before and after treatment.Results The effective rate of rhlL-11 in ITP treatment was 76.8%(43/56).For the effective patients,the median PLT after treatment increased(25.0(15. 0,36.0)×109/L vs.68.0(49.0,108.0)×109/L,Z=-5.712,P<0.001); Th1 cells decreased,compared with that before the treatment(14.8 %(12.6%,17.6%)vs.10.6 %(9.8%,12.6%),Z=-4.825,P<0.001);Th2 cell increased,compared with that before the treatment(0.4%(0.3%,0.5%)vs.1.2%(0.9%,1.4%), Z=-5.720,P<0.001); Th1/Th2 decreased,compared with that before the treatment(40(30,49)vs.10.6(7.8,12.0),Z=-5.711,P<0.001];the expression level of T-bet mRNA decreased(0.36(0.18,0.51)vs 0.09(0.05,0.13),Z=-2.668,P=0.008);the expression level of GATA-3 mRNA increased,compared with that before treatment(0.04(0.03,0.05)vs.0.12(0.09,0.15),Z=-2.366,P=0.018).For ineffective patients,the median PLT before treatment was(11.0(8.0,15.5)×109/L),and the median PLT after treatment was(15.0(10.0,19.5)×109/L)(Z=-3.027,P=0.002); there was no significant difference in Th1,Th2, ratio of Th1/Th2 and T-bet and GATA-3 mRNA expression level before and after treatment in patients with ITP (P>0.05).Conclusion rhIL-11 can effectively correct the imbalance in Th1 and Th2 cells and the imbalance of T-bet and GATA-3 in ITP patients,but it has no obvious therapeutic effect on a small number of patients
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Sepsis and septic shock are associated with high mortality rates and remain a serious menace for the critically ill patient. Concurrent activation of pro- and anti-inflammatory pathways and an excessive cytokine release represent initial key features in the deregulation of the humoral and cellular antimicrobial defense. Research of the last decades addressed both the ebullient inflammation as well as the resulting long-term failure of the host immunity. While the reestablishment of an adequate immune-competence is still under investigation, many promising experimental trials to limit the inflammatory response during sepsis were challenged by missing beneficial effects in clinical studies. Nevertheless, due to advanced knowledge about the complex regulation of inflammatory mediators and their overlapping involvement in other potentially life-threatening diseases, further evaluation of these approaches in relevant subgroups could help to identify critically ill patients with potential benefit from anti-inflammatory therapies.
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Objective To evaluate the effects of the recombinant human interleukin-1 receptor antagonist(rhIL-1ra)on a toluene-2, 4-diisocyanate (TDI)-induced guinea pig allergic rhinitis (AR)model. Methods An AR model was established via sensitization and challenge of two-step procedure using TDI in guinea pigs. Normal animals were treated only with the olive oil(TDI vehicle). Sixty adult guinea pigs were randomly divided into six groups (n=10): normal group, model group (rhIL-1ra vehicle), positive control group (budesonide, 25.6 µg/kg), rhIL-1ra treated groups (rhIL-1ra 50, 100 and 200 µg/kg, respectively). From day 8 after sensitization, animals of all the groups were treated respectively with the agents or vehicle once a day for 14 days. During the observation period, the index of clinic score was recorded for every animal. At day 14 of the dosing, guinea pigs were sacrificed 30 min after the last TDI challenge and observation. Blood samples were taken from the abdominal aorta to prepare the serum for detection of histamine, and the nasal mucosase were dissected for histamine detection and histopathological observation. Results Compared with the guinea pigs in normal group, those in the model group exerted the typical symptoms of AR. It was shown that rhIL-1ra could improve nasal symptoms and cause a significant decrease in the instances of nasal sneezing as well. In addition, rhIL-1ra significantly reduced the concentrations of histamine in the nasal mucosa and IgE in the blood compared with those in the model group (P<0.05). Moreover, the pathological results showed that less edema, vasodilation and inflammatory cell infiltration were found in the nasal mucosa after rhIL-1ra application. Budesonide also showed the above effects with no significant difference compared with rhIL-1ra. Conclusion A guinea pig allergic rhinitis model is successfully induced by TDI. The results indicated that rhIL-1ra(50-200 µg/kg)is effective in improving allergic rhinitis. Our findings indicated that rhIL-1ra might serve as a potential new drug for allergic rhinitis therapy.
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Objective To evaluate the effects of the recombinant human interleukin-1 receptor antagonist(rhIL-1ra)on a tol?uene-2,4-diisocyanate(TDI)-induced guinea pig allergic rhinitis (AR)model. Methods An AR model was established via sensiti?zation and challenge of two-step procedure using TDI in guinea pigs. Normal animals were treated only with the olive oil(TDI vehicle). Sixty adult guinea pigs were randomly divided into six groups(n=10):normal group,model group(rhIL-1ra vehicle),positive con?trol group(budesonide,25.6μg/kg),rhIL-1ra treated groups(rhIL-1ra 50,100 and 200μg/kg,respectively). From day 8 after sensi?tization,animals of all the groups were treated respectively with the agents or vehicle once a day for 14 days. During the observation pe?riod,the index of clinic score was recorded for every animal. At day 14 of the dosing,guinea pigs were sacrificed 30 min after the last TDI challenge and observation. Blood samples were taken from the abdominal aorta to prepare the serum for detection of histamine , and the nasal mucosase were dissected for histamine detection and histopathological observation. Results Compared with the guinea pigs in normal group,those in the model group exerted the typical symptoms of AR. It was shown that rhIL-1ra could improve nasal symptoms and cause a significant decrease in the instances of nasal sneezing as well. In addition,rhIL-1ra significantly reduced the concentrations of histamine in the nasal mucosa and IgE in the blood compared with those in the model group(P<0.05). Moreover, the pathological results showed that less edema,vasodilation and inflammatory cell infiltration were found in the nasal mucosa after rhIL-1ra application. Budesonide also showed the above effects with no significant difference compared with rhIL-1ra. Conclusion A guinea pig allergic rhinitis model is successfully induced by TDI. The results indicated that rhIL-1ra(50-200μg/kg)is effective in im?proving allergic rhinitis. Our findings indicated that rhIL-1ra might serve as a potential new drug for allergic rhinitis therapy.
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To determine the incidence of the positive neutralizing anti-human interleukin receptor antagonist (anti-IL-1Ra), a novel assay based on the proliferation of human melanoma A375.S2 cells was developed and validated. In the presence of a growth-limiting concentration of IL-1ß, A375.S2 cells were able to regain proliferation following the addition of IL-1Ra in a concentration-dependent manner. This dose-response effect enabled the validation of a standard curve for calculation of the concentration of IL-1Ra or, inversely, the concentration of neutralizing anti-IL-1Ra antibodies in cell culture medium or sera. The assay used CCK-8 as an indicator of proliferation. The dose-response relationship between rhIL-1Ra (dose range of 5-75 ng/ml rhIL-1Ra) and A375.S2 cell proliferation was sigmoidal and fitted a four-parameter logistic model. The percent coefficients of variation (%CVs) of quality control samples were 12.5 and 11.9% for intra-assay repeatability and 14.5 and 19.5% for inter-assay repeatability, while the total accuracy was in the range of 97.2-103.6%. For the neutralization assay, the optimal sample dilution factor was found to be 40-fold and the reasonable standard for positive and negative decision was calculated to be 59.4% neutralization rate. The %CVs of quality control samples were 12.7 and 24.0% for intra-assay repeatability and 11.6 and 30.0% for inter-assay repeatability. Analysis using the assay showed that rats could produce neutralizing anti-IL-1Ra antibodies after repeated intramuscular injection with rhIL-1Ra, and this response was not significantly dependent on the dose injected.
Subject(s)
Antibodies, Neutralizing/blood , Biological Assay/methods , Cell Proliferation/drug effects , Interleukin-1beta/blood , Receptors, Interleukin-1/antagonists & inhibitors , Animals , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Escherichia coli/genetics , Female , Humans , Logistic Models , Male , Neutralization Tests , Rats, Wistar , Recombinant Proteins , Sensitivity and SpecificityABSTRACT
Aim: To study the therapeutic effects of recombinant human interleukin 1 receptor antagonist (rhIL-1ra) on type II collagen-induced arthritis (CIA) rats. Methods: SD rats were divided randomly into six groups including normal, model, rhIL-1ra (7.5,30,120 mg·kg-1) and anakinra(120 mg· kg-1) groups. Collagen II emulsion was used to induce CIA model in rats. The body weight was observed once a week. Paw swelling of CIA rats was measured with volume meter. C II induced delayed-type hypersensitivity (DTH) was measured. Meanwhile, the level of anti-C II IgG antibody in serum was assayed by ELISA. Results: The onset of paw swelling was on dlO after injection of emulsion. The level of serum anti-C II IgG antibody was increased significantly in CIA. Pathological changes in joints of CIA rats showed hyperplastic synovium of CIA, inflammatory cells infiltration, pannus, destruction of cartilage and bone. rhIL-1ra(7.5,30,120 mg·kg-1·d-1 x 7 d) and anakinra (120 mg·kg-1·d-1 x 7 d) subcutaneous injection (sc) inhibited inflammatory swelling. rhIL-1ra(30, 120 mg·kg-1·d-1 x 7 d) significantly suppressed the DTH reaction induced with C II in CIA rats. Moreover, rhIL-1ra reduced the level of anti-C II IgG antibody in serum. Pathological examination showed rhIL-1ra(120 mg·kg-1·d-1 x 7 d) significantly improved subchondral inflammation, synovium hyperplasia, pannus and cartilage damage. Conclusion: rhIL-1ra has therapeutic effects on CIA rats.
