ABSTRACT
【Objective】 To evaluate the efficacy and safety of human coagulation factor Ⅷ developed by Shenzhen Weiguang Biological products Co, Ltd in the treatment of patients with hemophilia A. 【Methods】 A prospective, multi-center, open, single-group clinical study was conducted. A total of 65 subjects with hemophilia A were enrolled, and human coagulation factor Ⅷ(FⅧ) was injected according to the patients’ bleeding severity. The improvement score of bleeding symptoms and signs after the first infusion of the first bleeding event and the transfusion efficiency of FⅧ activity at 10 min and 1 hour after infusion were taken as the main efficacy indexes. The improvement scores of bleeding symptoms and signs after the first infusion and the increase of FⅧ activity at 10 min and 1 hour after infusion were the secondary efficacy indexes. 【Results】 The 65 subjects were enrolled in safety analysis set (SS) and full analysis set (FAS), and 58 of them were enrolled in protocol analysis set (PPS). Ten minutes and one hour after the first infusion, the level of factor Ⅷ activity in the subjects increased significantly, and the FⅧ activity increased by 100% or more in more than 79% of the subjects. The average infusion efficiency of FⅧ activity in all subjects was more than 100%. In 70% of the subjects, the pain was relieved rapidly and /or the bleeding symptoms were significantly improved 8 hours after each bleeding infusion, and the improvement rate of bleeding symptoms and signs reached 100% 72 hours after infusion. 【Conclusion】 After infusion of human coagulation factor Ⅷ, the activity level of factor Ⅷ in patients with hemophilia A significantly increased. The infusion efficiency can reach a optimal level, and the bleeding symptoms can be significantly improved.
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【Objective】 To research the effect of the Fc, Fab and F(ab′)2 fragments of immunoglobulin G, the main components of Human Immunoglobulin(pH4) for Intravenous Injection(IVIG), on the phagocytic function of macrophages derived from THP-1 cells. 【Methods】 First of all, IVIG was digested with papain and pepsin to obtain Fc, Fab and F(ab′)2, and these components were then identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards, propylene glycol monomethyl ether acetate (PMA) was used to induce THP-1 cells to differentiate into M0 macrophages. Finally, the sensitized erythrocytes were labeled with carboxy fluorescein succinimidyl ester (CFSE), and the effect of the above components on the phagocytic ability of M0 macrophages to engulf sensitized erythrocytes was detected by flow cytometry. 【Results】 The identification results of SDS-PAGE showed that the prepared IgG fragments met the requirements of subsequent experiments. Flow cytometry performs showed that the phagocytosis model of M0 macrophages had been successfully established. When the concentration of Fc increased from 0.1μg/ mL to 10μg/ mL, the phagocytosis rate of erythrocytes sensitized by M0 macrophages decreased from (24.21±0.58) % to (12.27±0.19) %. When the concentration of IVIG protein increased from 0.1 μg/ml to 10 μg/ml, the phagocytosis rate decreased from (20.57±0.39) % to (0.20±0.03) %. Meanwhile, at the same protein concentration (10 μg/ml), the inhibitory effect of Fc on phagocytosis was only half that of IVIG. In addition, Fab, F(ab′)2, and human serum albumin could not inhibit phagocytosis of M0 macrophages. 【Conclusion】 IVIG can effectively inhibit the phagocytosis of THP-1 derived M0 macrophages, which is mainly dependent on the Fc, but not related to the Fab of IgG and F (ab′)2.
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【Objective】 To establish and evaluate a nephelometric assay for the determination of immunoglobulin A (IgA) residues in human intravenous immunoglobulin(IVIG). 【Methods】 BN ProSpec© automatic protein analyzer and its supporting immunoglobulin A determination kit (nephelometry) produced by German Siemens and the national standard of human IgA were used to establish the nephelometric assay to determine IgA residue in test products and verify the methodology. The test products include IVIG (pH4) prepared by low-temperature ethanol protein separation process and a novel IVIG prepared by chromatography. 【Results】 The average deviation of three calibration curves for IgA residues determination by the nephelometric assay were 1.08%, 0.95% and 1.54%,, and the three deviations of the quality control were 4.00%, -2.30% and -0.20%, respectively, which indicated good calibration and quality control. In the specificity test, the average recovery rates of IgA for reference substance 1 containing 100g/L maltose and reference substance 2 containing 20g/L glycine were 102.7% and 105.8%, respectively. The relative standard deviation (RSD) values of the repeatability tests of the two test products were 3.9% and 1.9%, and the RSD values of the intermediate precision test were 3.6% and 2.3%, respectively.The difference values at each time point in the durability test of test products′ storage time were all less than 10%, and the RSD values of the two test products in the durability test of kits of different batches were 2.8% and 2.2%, respectively. In the accuracy test, the average recovery rates of IVIG (pH4) added to the standard were 94.2%, 101.7% and 96.2%, respectively, and the average recovery rates of the novel IVIG added to the standard were 102.8%, 106.3% and 99.7%, respectively. The average recovery rate of the limit quantification test was 101.0%, and the RSD was 4.0%. 【Conclusion】 Nephelometric assay has the advantages of strong specificity, high precision and accuracy, good repeatability, simple and rapid operation, and automation, and can be used for the determination of IgA residue in IVIG (pH4) and novel IVIG products.
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Objective To investigate the protective effect of Carbachol on hyperoxia-induced acute lung injury (HALI) in mice and its related mechanisms. Methods Thirty-two healthy male ICR mice were randomly divided into four groups:control group, hyperoxia exposure three days group (HO3d group), hyperoxia exposure three days + Carbachol group (HO3d+Carba group), and Carbachol group (Carba group), eight mice in each group. The pathological changes of lung tissue in each group were observed under light microscope after the models were completed in each group.The expression of TLR4 and NF-κB protein in lung tissues were detected by Western blot, and the expression of HMGB-1 and TNF-α mRNA in lung tissues by RT-PCR. LSD-t test was used for sample pairwise comparison, and one-way ANOVA for intergroup comparison. P<0.05 was considered statistically significant. Results There was no statistical difference between the control group and the Carba group (P> 0.05), and no obvious abnormal changes in lung tissue structure. The expression of TLR4, NF-κB protein and HMGB-1 and TNF-α mRNA in the HO3d group were significantly higher than those in the control group (P<0.01), and there were obvious bleeding on the surface of the lung tissue and severe pathological damage. The expression of TLR4,NF-κB protein and HMGB-1 and TNF-α mRNA in the HO3d+Carba group were significantly lower than those in the HO3d group(P<0.01), while lung tissue damage degree was also lower than that in the HO3d group. Conclusions Hyperoxia can increase the expression of TLR4 and NF-κB in lung tissues, and cause inflammatory injury in lung tissue. Carbachol can reduce the release of HMGB-1 and TNF-α inflammatory factors in hyperoxia-induced acute lung injury, and its mechanism is related to the inhibition of TLR4/NF-κB signal pathway, which has a protective effect on HALI.
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Objective To investigate both in mechanism of hyperoxia-induced acute lung injury (HALI) by vivo experiment,to observe the Bruton' s tyrosine kinase (Btk) and nuclear factor kappa B (NF-κB) signals expression level.Methods Total of 72 healthy male Kunming mice were randomly (random number) divided into four groups:air control group,hyperoxia exposure 3 days group (H3d group),hyperoxia exposure 3 days + inhibitor group (H3d + Ⅰ group) and inhibitor groups.Then the pathological changes of lung tissues were observed under light microscope;The total protein content (TP) of bronchoalveolar lavage fluid (BALF) and wet/dry weight ratio (W/D) of lung were detected;The protein expression of Btk,p-Btk,pNF-κB p65 were mersured by Western blot;tlhe mRNA level of IL-6 was determined by real-time polymerase chain reaction (qRT-PCR);the level of monocyte chemoattractant protein-1 (MCP-1) in serum was detected by enzyme-linked immunosorbent assay (ELISA).Statistcal significance was determined by 1-way ANOVA.Results There were no significant difference in the data between the control group and the inhibitor group (P ＞ 0.05).The pathological injury in light microscope,content of total protein in BALF,W/D ratio of lung tissues in H3d group were significantly higher than H3d + Ⅰ group (Respectively P =O.002,P =0.000).Western blot analysis showed that expression of Btk,p-Btk,pNF-κB p65 in H3d group were significantly higher than those in H3d + Ⅰ group (Respectively P =0.002,P =0.013,P =0.000).RT-qPCR results showed that the expression of IL-6 mRNA in H3d group were significantly higher than control group (P =0.004),inhibitor group (P =0.000) and H3d + Ⅰ group (P =0.021).In addition,The serum MCP-1 levels in H3d group were higher markely than the control group (P =0.002),inhibitor group (P =0.000) and H3d + Ⅰ group (P =0.009).The correlation analysis showed that pNF-κB p65 were positively correlated wiht Btk and p-Btk (r =0.902 and 0.954,P ＜ 0.01).Conclusions Btk may trigger the release of IL-6 and MCP-1 by mediating the signaling pathway of NF-κB in vivo study,which was most important in the occurrence of HALI.Therefore,inhibiting the Btk activity would alleviate the severity of lung injury effectively.
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<p><b>OBJECTIVE</b>To correlate sperm nucleoprotein transition (SNT) with sperm morphology, DNA damage and embryo development, and assess its value for assisted reproductive technology (ART).</p><p><b>METHODS</b>The SNT of 437 infertile men underwent ART were assayed, and its correlation with sperm morphology, DNA damage, fertilization rate, normal fertilization rate, cleavage rate, available embryo rate, D3 high quality embryo rate, blastocyst formation rate and high quality blastocyst rate were analyzed.</p><p><b>RESULTS</b>The normal morphology rate of sperms, DNA damage, fertilization rate, normal fertilization rate, cleavage rate, embryo transfer rate (ETR), D3 high quality embryo rate, blastocyst formation rate (BFR) and high quality blastocyst in normal males (Group A, abnormal rate≤30%, 135 subjects) did not significantly differ from those with an abnormal rate between 30% and 60% (Group B, 170 subjects) (P>0.05). For those with an abnormal rate of above 60% (Group C, 132 subjects), the sperm normal morphology rate, DNA damage, normal fertilization rate, ETR, D3 high quality embryo rate, high quality blastocyst rate were significantly lower compared with Group A (P<0.01), while no significant difference was found in fertilization rate, cleavage rate and BFR between groups A and C (P>0.05).</p><p><b>CONCLUSION</b>SNT is related with sperm morphology rate, DNA damage and embryo development, and should be assessed before ART.</p>