Analysis of factors affecting stability of high concentration VHH-Fc fusion protein / 中国生物制品学杂志

@#Objective To explore the factors affecting the stability of high concentration variable domain of heavy-chain antibody-Fc(VHH-Fc) fusion protein.Methods Three groups of forced degradation experiments,shaking,light and 40℃ high temperature were set up.Differential scanning fluorimetry,dynamic light scattering(DLS) and ultra performance liquid chromatography-mass spectrometry(UPLC-MS) were used to detect the effects of the three forced degradation conditions on the conformational stability,colloidal stability,average hydrodynamic diameter and post-translational modifications of high concentration VHH-Fc fusion protein.Results Under the light condition,the onset temperature of unfolding(T_(onset)),melting temperature(T_m) and aggregation onset temperature(T_(agg)) of high concentration VHH-Fc fusion protein decreased the most,and the oxidation ratio of Met160 and Met266 increased significantly.Under the condition of shaking,the variation of the diffusion interaction parameter(k_D) and the average hydrodynamic diameter was the largest.Conclusion Light can significantly reduce the conformational stability of high concentration VHH-Fc fusion protein and induce methionine oxidation.Shaking has the most significant effect on its colloidal stability and promotes aggregation.

FcγRIIa and Fcγ RIIIa genes polymorphism in Egyptian children with primary immune thrombocytopenia

Abstract Introduction Phagocytosis of autoantibody-sensitized coated platelets through Fc gamma receptors on phagocytic cells is an important mechanism of thrombocytopenia in primary immune thrombocytopenia (ITP). Objective We aimed to investigate the contribution of the FcγRIIa and FcγRIIIa genes polymorphism to the risk of ITP and their association with disease characteristics in Egyptian children. Methods A case control study was conducted on eighty children with primary ITP and eighty age and sex healthy matched subjects as a control group. The FcγRIIa and FcγRIIIa genes polymorphism was detected using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results We found that the FcγRIIa‐131H and ‐131R allele frequencies were 51.3 % and 48.7%, respectively, in children with ITP, versus 75% and 25%, respectively, in controls (p= 0.002). The compound heterozygous HR genotype was significantly higher in ITP patients (p < 0.05). The FcγRIIIa-158F and ‐158V allele frequencies were 46.3% and 53.7%, respectively, in children with ITP, versus 70% and 30%, respectively, in controls (p= 0.002). The compound heterozygous VF genotype was significantly higher in ITP patients (p < 0.05). The combined HR/FV genotype was 47.5% in ITP patients, versus 10% in controls (p < 0.001). No significant difference was found between children with newly diagnosed ITP and those who developed chronic ITP, regarding the frequency distribution of the FcγRIIa and FcγRIIIa alleles and genotypes (p > 0.05). Conclusion There is a possible association of the FcγRIIa and FcγRIIIa genes polymorphism with the risk for, and genetic susceptibility to ITP in Egyptian children, but large-scale studies are still needed to support our findings.

Development and verification of whole-column imaging capillary isoelectric focusing electrophoresis analysis method for charge heterogeneity of human growth hormone Fc fusion protein / 中国生物制品学杂志

@#Objective To develop and verify a whole-column image capillary isoelectric focusing(iCIEF) electrophoresis method to analyze the charge heterogeneity of recombinant human growth hormone Fc fusion protein(Fc-rhGH).Methods The iCIEF analysis method of Fc-rhGH was developed by optimizing the target protein concentration,cosolvent(urea)concentration and focusing time.The target protein was simultaneously analyzed by this method and traditional flat plate isoelectric focusing(IEF) electrophoresis,and the results were compared;The specificity,accuracy,precision,limit of quantitation(LOQ) and durability of the developed method were verified.Results The optimized method was using the mixed solution of 8 mol/L urea,0.35% methyl cellulose(MC),4% amphoteric electrolyte and 0.5% isoelectric point marker as the sample buffer,and the focusing condition was 1 500 V 1 min,3 000 V 5.5 min.IEF was not suitable for analyzing the charge heterogeneity of Fc-rhGH solution.Using the optimized iCIEF for analysis,the target protein was significantly different from the unrelated protein,and the baseline of blank reagent was stable;The recovery rate of accuracy verification was within 90%～110%,and the linear range was 0.25～0.75 mg/mL(50%～150% of the target loading volume);The RSD of each isomer pI in the repeatability verification was less than 0.3%,and the RSD of peak area percentage was less than 5%;The LOQ was 0.04 mg/ml.The sample storage time durability,amphoteric electrolyte pharmalyte 3-10 durability and MC durability of this method were good.Using this method to analyze the charge heterogeneity of Fc-rhGH physicochemical reference substance,eight charge heterogeneities of the reference substance were effectively separated,and the pI ranged from 5.9 to 6.4.Conclusion The developed iCIEF method had good specificity,accuracy,precision and durability,and was more suitable for efficient analysis of charge heterogeneity of Fc-rhGH than traditional flat plate IEF,which was of great significance for the quality control of Fc-rhGH and other Fc fusion proteins.

Expression of recombinant human interleukin-29-Fc fusion protein in HEK293F cells and analysis of its anti-tumor activity in vitro / 中国生物制品学杂志

@#Objective To express recombinant human interleukin-29-Fc(rhIL-29-Fc) fusion protein in human embryonic kidney 293-F(HEK293F) cells and analyze its anti-tumor activity in vitro.Methods The recombinant expression plasmid UCOE-IL-29-Fc was constructed and transiently transfected into HEK-293F cells.After expression and purification,rhIL-29-Fc fusion protein was obtained and identified by SDS-PAGE and Western blot;Female Japanese white rabbits were immunized with rhIL-29 and rhIL-29-Fc protein subcutaneously in the left ear respectively,2 rabbits in each group,0.5 mg per rabbit.Blood samples were collected from the vein of right ear,and the serum was separated.The half-life was measured by ELISA and the anti-proliferation effect of rhIL-29-Fc protein on human colon cancer HT-29,human colon cancer HCT-116,human Burkkit lymphoma Daudi,human non-small cell lung cancer NCI-H1975,human small cell lung cancer NCI-H209,human esophageal cancer EC109 and human pancreatic cancer PANC-1 cells in vitro was detected by CCK-8 assay,and the inhibitory concentration 50(IC_(50)) was calculated.Results The recombinant expression plasmid UCOE-IL-29-Fc was constructed correctly as identified by double digestion and sequencing.After transient transfection into HEK-293 cells for 6 d,the culture supernatant was harvested.The relative molecular mass of the purified rhIL-29-Fc fusion protein was consistent with the expectation.The protein showed a specific binding reaction with mouse anti-human IL-29 monoclonal antibody with a concentration of 1.5 mg/ml and a purity of 93%.RhIL-29-Fc protein had a half-life of 25 h and showed different inhibitory effects on the proliferation of 7 kinds of tumor cells,and the IC_(50) on different cells was also different.Conclusion The rhIL-29-Fc fusion protein was successfully expressed in HEK-293F cells,and the half-life of the fusion protein was 20 h longer than that of rhIL-29.According to the different anti-tumor proliferation activity in vitro and IC_(50) results on 7 kinds of tumor cells,it was found that the anti-tumor activity of rhIL-29-Fc fusion protein was higher than that of rhIL-29.This study laid a foundation of the development of IL-29 protein in the treatment of tumors.

The serum levels of FCGBP, SAA1, CXCL10 in children with mycoplasma pneumoniae pneumonia and their relationship with prognosis / 中国医师杂志

Objective:To investigate the serum levels and clinical significance of Fc fragment of the IgG-binding protein (FCGBP), serum amyloid protein A1 (SAA1), and CXC chemokine ligand 10 (CXCL10) in children with mycoplasma pneumoniae pneumonia (MPP) and their relationship with prognosis.Methods:A prospective study was conducted on 122 children with MPP admitted to the department of pediatrics of the 970th Hospital of the Joint Logistics Support Force of the Chinese People′s Liberation Army from January 2019 to December 2021. According to the severity and prognosis of MPP, they were divided into mild and severe groups, good prognosis group, and poor prognosis group. Forty healthy children who underwent physical examination during the same period were set as the control group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of FCGBP, SAA1, and CXCL10 in each subject, and to compare the differences in serum levels of FCGBP, SAA1, and CXCL10 among different groups. Multivariate logistic regression analysis was used to investigate the influencing factors of poor prognosis in MPP patients. The diagnostic value of individual and combined detection of serum procalcitonin (PCT), FCGBP, SAA1, and CXCL10 for poor prognosis in MPP children by analyzing the receiver operating characteristic (ROC) curve.Results:The levels of serum FCGBP [(115.68±10.57)ng/ml, (78.41±6.73)ng/ml, (12.55±3.25)ng/ml], SAA1 [(34.18±3.72)mg/L, (25.54±2.63)mg/L, (6.74±0.82)mg/L], and CXCL10 [(714.26±55.64)ng/L, (353.74±42.67)ng/L, (106.25±12.92)ng/L] in the severe MPP group were significant higher than those in the mild MPP group and the control group, with statistical significance (all P<0.05). The white blood cell (WBC), neutrophil percentage, C reactive protein (CRP), erythrocyte sedimentation rate (ESR), PCT, lactate dehydrogenase (LDH), D-dimer (D-D), FCGBP, SAA1, CXCL10 of the children in the poor prognosis group were significantly higher than those in the good prognosis group, and the differences were statistically significant (all P<0.05). Multivariate logistic regression analysis showed that increased PCT ( OR=1.603, 95% CI: 1.190-2.160), FCGBP ( OR=1.757, 95% CI: 1.115-2.770), SAA1 ( OR=1.900, 95% CI: 1.327-2.720) and CXCL10 ( OR=1.704, 95% CI: 1.212-2.397) were independent risk factors for poor prognosis of MPP children (all P<0.05). The combined detection of serum PCT, FCGBP, SAA1, and CXCL10 had a significantly higher diagnostic value for the risk of poor prognosis in children with MPP than a single indicator. Conclusions:The elevated levels of serum FCGBP, SAA1, and CXCL10 in children with MPP are associated with the severity of MPP and are independent risk factors for poor prognosis in MPP patients.

Effect of IgG dimer concentration on the binding ability of IgG Fc fragment in IVIG to THP-1 cell surface receptors / 中国输血杂志

【Objective】 To investigate the effect of immunoglobulin G (IgG) dimer concentration of intravenous immunoglobulin (IVIG) on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Methods】 Firstly, protein purification and high performance liquid chromatography (HPLC) were used to prepare different concentrations of IgG dimers. After that, IgG dimer was added to IVIG to prepare IVIG containing different concentrations of IgG dimer. Finally, based on the method established in our laboratory, we analyzed the effect of IgG dimer concentration in IVIG on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Results】 When the concentration of IgG dimer in IVIG was 1.11%-10.30%, its binding ability to Fc receptors on the surface of THP-1 cell was 97.67%-135.33%, and this binding ability was positively correlated with the concentration of IgG dimer. When the IgG dimer concentration exceeded 13.22%, the binding ability had no correlation with the IgG dimer concentration. 【Conclusion】 A certain concentration of IgG dimer can promote the binding ability of the IgG Fc fragment in IVIG to receptors on the surface of THP-1 cells, which needs further verification from animal experiments and clinical data.

Development of the method for determinating ADCC biological activity of IVIG / 中国输血杂志

【Objective】 To establish a method for determinating the antigen-dependent cell-mediated cytotoxicity (ADCC) of human immunoglobulin (pH4)for intravenous injection (IVIG) on luciferase reporter gene-modified cell assay. 【Methods】 As effector cells, Jurkat-NFAT-Luc-CD16 cells were used in the assay, and PLC/PRF/5 cells were used as target cells. After incubation of effector cells and target cells with IVIG, the method for determinating ADCC biological activity of IVIG was established by detecting luciferase released by activated T nuclear factor after binding of IVIG Fc fragment to effector cells. Meanwhile, the experimental assay conditions were optimized, and the methodology was verified subsequently. 【Results】 IVIG had a dose-response relationship in this method, which was consistent with four parameter logistic model. And the PLC/PRF/5 cells were finally determined as the target cells. The initial dilution concentration of antibody was 20 mg/mL, and the ratio dilution was 1∶2, and the effector to target ratio was 1∶3, and co-incubation time of two cells and IVIG was 24 hours. Within-run and between-run analysis including three independent tests, initial working concentration relative light unit (RLU) and the relative standard deviation (RSD) of the concentration for 50% of maximal effect(EC50) were less than 11%. The relative titers of the recovery samples of the two different dilution groups were (23.50±1.69)% and (49.30±2.97)%, respectively, and the corresponding recovery rates were (93.50±6.30)% and (96.24±5.43)%, respectively, with RSD less than 11%. 【Conclusion】 The method for determinating ADCC biological activity of IVIG based on luciferase reporter gene-modified cell assay was successfully established. It could be applied in determinating the ADCC biological activity of IVIG, and has the advantages of satisfactory linearity, accuracy, precision and specificity.

Eukaryotic expression and biological activity identification of fusion protein ABD-Fc-IL-2 / 中国生物制品学杂志

@#Objective To express the fusion protein ABD-Fc-IL-2 in eukaryotic cells and detect its biological activity. Methods The target gene SP-ABD-Fc-IL-2 was amplified by direct and overlapping PCR,and then ligated to vector pcDNA3. 1(+).The obtained recombinant plasmid pcDNA3. 1/SP-ABD-Fc-IL-2 was transiently transfected into CHO-S cells to express the fusion protein ABD-Fc-IL-2,which was purified by Protein A beads affinity chromatography. The specificity of the purified fusion protein was detected by Western blot,the biological activity was detected by CTLL-2/MTT cell proliferation colourimetry,and the interaction between ABD fragment and human serum albumin(HSA)was detected by pull down/Western blot. Results The recombinant plasmid pcDNA3. 1/SP-ABD-Fc-IL-2 was constructed correctly as identified by restriction analysis and sequencing. The purified fusion protein ABD-Fc-IL-2 showed a purity of 90% and bound specifically to mouse anti-IL-2 monoclonal antibody with the biological activity of 3. 29 × 108IU/mL. The ABD of fusion protein and HSA bound to each other. Conclusion The eukaryotic fusion protein ABD-Fc-IL-2 had high biological activity,which promoted the proliferation of CTLL-2 cells and maintained the binding ability of ABD fragment to HSA.

Application of Design of Experiment in development of purification process of recombinant human growth hormone-Fc fusion protein / 中国生物制品学杂志

@#ObjectiveTo optimize the condition for anion exchange chromatography in purification process of recombinant human growth hormone-Fc(rhGH-Fc)fusion protein by Design of Experiment(DOE)so as to decrease the content of host cell protein(HCP)in bulk of protein.MethodsA complete factorial experimental design with four factors at two levels was established by the DOE in Minitab 19 software.The condition(flow rate,sample load,pH value of buffer and salt concentration of eluent)for anion exchange chromatography in purification process of rhGH-Fc was optimized by DOE to find out the operating space.ResultsThe experimental results were predicted accurately by the established DOE model.The sample load,pH value of buffer,salt concentration of eluent as well as the interaction of pH value of buffer and salt concentration of eluent showed significant influence on the HCP content in the harvest.The optimal sample load,flow rate as well as pH value and salt concentration of eluent were 15 mg/mL,120 cm/h,7.0 ～ 8.0 and 0.1 mol/L respectively.The HCP contents in eluents under the optimal condition were less than 400 ng/mg,which met the requirements for verification within the range of results in determined operating space.ConclusionThe condition for removal of HCP by anion exchange chromatography was successfully optimized by DOE,which provided a reference for further application of DOE in the biopharmaceutical field.

Optimization of glycosylation type of recombinant human growth hormone-Fc fusion protein expressed in CHO cells / 中国生物制品学杂志

@#Objective To optimize the expression of recombinant human growth hormone-Fc(rhGH-Fc)fusion protein in CHO cells in order to obtain better glycosylation ratio and lower content of highmannose.Methods CHO cells expressing rhGH-Fc were cultured in a 7 L bioreactor.The glycosylation modifications of rhGH-Fc were adjusted by improving the composition of feeding media(using three commercial media:Gly-1:EX-CELL Glycosylation Adjust,Gly-2:SHEFF-CHO PLUS PG ACF and Gly-3:EfficientFeed C + AGT Supplement & GlycanTune C + Total Feed),and the glycosylation type and proportion of the target proteins were analyzed by mass spectrometry.Results The G0F(main glycosylation types:G0,G1 and G2;F:fucose)of Gly-1,Gly-2 and Gly-3 were 32.89%,58.66% and 33.28%,the G1F were 31.39%,18.03%and 34.90%,and the G2F were 31.39%,18.03% and 34.90%,respectively.Gly-1 and Gly-3 made the target protein contain less G0F while more G2F;Gly-3 feeding scheme-showed less high mannose modification than the other two schemes.Conclusion Gly-1 medium changed the glycosylation modification from G0F to G1F and G2F,while Gly-2 medium changed that from G2F and G1F to G0F.However,Gly-3 medium changed the glycosylation modification from G0F to G1F and G2F,and the contentof high mannose was less than 5%,which may have a better effect on modifying glycosylation type and proportion of the target protein.

Research progress of novel drug therapy for antibody-mediated rejection in kidney transplantation / 中华泌尿外科杂志

Antibody-mediated rejection (AMR) is the primary factor affecting the long-term prognosis of kidney transplant recipients and kidney allograft. Currently, there is no universally recognized or approved drug for the treatment of AMR. Therefore, more novel drug studies and clinical trials are urgently needed in order to change the long-term prognosis of kidney transplant recipients. Based on the core principles of prevention and treatment of AMR, this paper discusses the mechanism and efficacy of several new types of drugs of most concern in the treatment of AMR from three aspects: removing donor specific antibody, blocking antibody-mediated and complement-mediated tissue damage, and inhibiting the proliferation and activation of antibody-producing cells. These emerging drugs have shown potential in preventing and treating AMR and improving the prognosis of recipients, which is expected to change the dilemma of AMR treatment in the future and provide more effective treatment options for improving the long-term prognosis of kidney transplant recipients.

Research advances on the relationship between infectious diseases and programmed cell death / 中华临床感染病杂志

In recent years, with the progress of research on the molecular mechanism of cell death, it has been discovered that there are many new types of programmed cell death, including non-apoptotic (10 types) and apoptotic (2 types), which are widely involved in the pathogenesis of infectious diseases and tumors. It is also a frontier research topic and provides new ideas for disease prevention and treatment. This article reviews the published literature on programmed cell death, focusing on the characteristics of cell necrosis, apoptosis, pyroptosis, ferroptosis, neutrophil inflammatory cell death (NETosis), cuproptosis, and widespread apoptosis (PANoptosis), as well as their relationship with infectious diseases.

Method for the detection of human immunoglobulin Fc function based on surface plasmon resonance: preliminary establishment and validation / 中国输血杂志

【Objective】 To establish a method for detecting human immunoglobulin Fc function based on surface plasmon resonance technology. 【Methods】 Based on the characteristic that FcγRI can be binded to the Fc segment of IgG, the affinity constant of the sample was detected by surface plasmon resonance, and its Fc function was the KD ratio of the sample to the standard. The method was validated for specificity/specificity, precision and robustness. The method and the pharmacopoeia method were used to detect the Fc function of 30 human immunoglobulins, and the correlation and consistency of the detection results were analyzed. 【Results】 The method validation results showed that this method has strong specificity/specificity (t values were 0.15, 0.22, both P>0.05), good precision (CV value 5.37%~10.69%) and good robustness (CV value 10.06%). The detection results of this method and the pharmacopoeia method have high correlation (r=0.96, P<0.05) and high consistency (Bias-2.060, 95% Limits of Agreement-5.628~1.508). 【Conclusion】 A method for detecting human immunoglobulin Fc function based on surface plasmon resonance has been successfully established.

Effect of IgG fragments in IVIG(pH4)on phagocytosis of sensitized erythrocytes by macrophage / 中国输血杂志

【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.

The Capping Protein β-Subunit Mediates Fusicoccin-induced Stomatal Movement in Arabidopsis / 中国生物化学与分子生物学报

Actin dynamics in guard cells play a critical role in stomatal movement. Remodeling of actin arrays is triggered by different biotic and abiotic stimuli, which requires precise control. However, the molecular mechanism underlying this process is not well understood. Here we investigated whether and how the capping protein (CP) regulates actin filaments during fusicoccin (FC) -induced stomatal opening. We found that both stomatal opening and F-actin rearrangement are sensitive in the Capping Protein β-subunit (CPB) cpb-3 mutants, which resulted in its hypersensitivity to drought stress. The leaves detached from cpb-3 had a higher water loss rate (63. 45%) than from the wild type (48. 99%), and the stomatal aperture of cpb-3 was about 20% greater than in the wild type. After 1 h of FC treatment, the proportion of cpb-3 guard cells with radial actin arrays increased to 65. 5% dramatically, while only approximately 47. 2% guard cells in WT exhibited transversely oriented actin filaments. Moreover, the record of transmembrane Ca

Therapy of myasthenia gravis: looking forward to the era of targeted immunotherapy / 中华神经科杂志

Myasthenia gravis is mainly acetylcholine receptor antibody-mediated, T cells-dependent and complement participated acquired autoimmune disease characterized by impairment of the neuromuscular transmission. The main clinical feature of the disease is the presence of fatigability or muscle weakness. Most patients can be successfully managed with nonspecific immunotherapies such as corticosteroid and non-steroidal immunosuppressants. However, the side effects caused by long-term corticosteroid therapy are still a hurdle in the treatment of myasthenia gravis (MG). Oral non-steroidal immunosuppressants, as add-on therapy, can greatly reduce the relapse of the disease, but some drugs have a slow onset of action and the potential for significant toxicity, and even increase the risk of infection and neoplasms with long-term treatment. Despite these therapies, a minority of patients can be refractory because of incompletely responding or not well tolerated to available therapies. Thus, the need to avoid the use of corticosteroids, or at least reduce their use as much as possible should concern all patients with MG. Targeted immunotherapy is a therapeutic monoclonal antibody or antibody fragment targeting immune cells, complement, neonatal Fc receptor and cytokines. Recently, targeted immunotherapy has completed phase Ⅱ and Ⅲ clinical trials in patients with MG, and some of them have been approved by Food and Drug Administration. These promising biologics showed efficacy in symptoms persistent improvement, steroids reduction and were well tolerated, now evolving into powerful tools changing the algorithm of MG. This paper summarizes the results of clinical trials of new biologics in MG and looks forward to the prospect of MG treatment.

Proteomics study of Mycoplasma pneumoniae pneumonia reveals the Fc fragment of the IgG-binding protein as a serum biomarker and implicates potential therapeutic targets / 医学前沿

Macrolide and corticosteroid resistance has been reported in patients with Mycoplasma pneumoniae (MP) pneumonia (MPP). MP clearance is difficult to achieve through antibiotic treatment in sensitive patients with severe MPP (SMPP). SMPP in children might progress to airway remodeling and even bronchiolitis/bronchitis obliterans. Therefore, identifying serum biomarkers that indicate MPP progression and exploring new targeted drugs for SMPP treatment require urgency. In this study, serum samples were collected from patients with general MPP (GMPP) and SMPP to conduct proteomics profiling. The Fc fragment of the IgG-binding protein (FCGBP) was identified as the most promising indicator of SMPP. Biological enrichment analysis indicated uncontrolled inflammation in SMPP. ELISA results proved that the FCGBP level in patients with SMPP was substantially higher than that in patients with GMPP. Furthermore, the FCGBP levels showed a decreasing trend in patients with GMPP but the opposite trend in patients with SMPP during disease progression. Connectivity map analyses identified 25 possible targeted drugs for SMPP treatment. Among them, a mechanistic target of rapamycin kinase (mTOR) inhibitor, which is a macrolide compound and a cell proliferation inhibitor, was the most promising candidate for targeting SMPP. To our knowledge, this study was the first proteomics-based characterization of patients with SMPP and GMPP.

Recent advances in neonatal Fc receptor targeted drug delivery systems / 药学学报

The neonatal Fc receptor (FcRn) was first found to be a membrane protein that maternal antibodies transmitted to fetuses and newborns, and also expressed in multiple organs and tissues for whole life in adults. It plays a significant role to central regulate the lifespan of immunoglobulin G and serum albumin, as well as its involvement in innate and adaptive immune responses. In modern biopharmaceuticals, FcRn is a great potential drug delivery target and a highlighted subject for current research. This paper briefly describes the basic biological properties and action mechanism of FcRn, as well as the commonly used drug carrier design strategies of FcRn, especially the functional applications of prolonging half-life, targeted drug delivery, transmembrane and antigen presentation and so on. We propose that these distribution in different tissues and the diverse biological activities may have significant implications of targeting FcRn for novel drug delivery systems and immunotherapy.

The value of baseline Doppler ultrasonography in the prediction of treatment response with recombinant human tumor necrosis factor-α receptor Ⅱ immunoglobulin G Fc fusion protein in patients with ankylosing spondylitis / 中华风湿病学杂志

Objective:To predict the inflammatory activity of patients with ankylosing spondylitis (AS) after 12 weeks treatment with recombinant human tumor necrosis factor-α receptor Ⅱ immunoglobulinG Fc fusion protein (rhTNFR:Fc) by Doppler ultrasonography at baseline.Methods:A total of 60 patients with AS were selected, and their general clinical characteristics before and after treatment were compared. Meanwhile, Doppler ultrasonography of the sacroiliac joint was performed to compare the Doppler parameters before and after treatment, and the correlation between baseline Doppler ultrasonography and clinical characteristics was analyzed, along with its diagnostic performance. The pre-treatment and post-treatment parameters were compared to the measured data followed by paired t-test for normal distribution, and the counting data were paired with Chi- square test. Pearson correlation test was used to analyze the correlation between pretreatment ultrasound parameters and pre-treatment disease activity. All statistical tests were bilateral, with a statistically significant difference of P<0.05. Results:After treatment, the overall score [(1.4±1.0) points vs (6.0±1.8) points, t=17.80, P<0.001], night pain score [(1.6±1.2) points vs (5.7±1.5) points, t=15.80, P<0.001], back pain score [(1.9±1.3) points vs (5.5±1.2) points, t=16.39, P<0.001], morning stiffness [(12±6) min vs (38±21) min points, t=8.93, P<0.001], Bath ankylosing spondylitis disease activity index (BASDAI) [(1.1±0.6) vs (4.6±1.3), t=12.41, P<0.001], ankylosing spondylitis disease activity score-C-reactive protein (ASDAS-CRP) [(1.0±0.4) points vs (3.7±0.9) points, t=22.01, P<0.001] and ASDAS-erythrocyte sedimentation rate (ESR) [(1.0±0.7) points vs (4.0±0.8) points, t=20.10, P<0.001] of patients with ankylosing spondylitis were lower than those before treatment, and the differences were statistically significant ( P<0.001). Compared with AS patients before treatment, the color blood flow grading score was significantly lower after treatment [(1.7±0.8) points vs (3.9±1.1) points, t= 12.86, P<0.001). The post-treatment proportion of AS patients with bilateral sacroiliac joint blood flow signal was 67% (40/60), which was lower than 87% (52/60) before treatment, but the difference was not statistically significant ( P=0.251). After treatment, the peak systolic velocity (PSV), pulsatile index (PI) and resistance index (RI) were significantly higher than those before treatment [(30±17) cm/s vs (19±8) cm/s, t=-5.42, P<0.001; (1.55±0.69) vs (1.00±0.45), t=0.45, P<0.001; (0.81±0.11) vs (0.55±0.14), t=11.20, P<0.001)]. The end diastolic velocity (EDV) before and after treatment had no statistical significant differences [(6.7±2.5) cm/s vs (6.3±1.9) cm/s, t=0.80, P=0.428]. Baseline Doppler ultrasound parameters and pre-treatment clinical indicators showed that PI and RI were negatively correlated with BASDAI ( r=-0.49, P=0.005; r=-0.51, P<0.001) , and blood flow grades were positively correlated with BASDAI ( r=0.46, P=0.028). However, there were no significant correlation between PSV, EDV and BASDAI ( r=-0.12, P=0.176; r=0.03, P=0.756). Baseline Doppler ultrasound parameters were correlated with ASDAS-CRP ( r=-0.45, P=0.012; r=0.29, P<0.048; r=-0.52, P<0.035; r=-0.76, P<0.001; r=0.61, P<0.001). There was no correlation between EDV and ASDAS-ESR ( r=0.30, P=0.110), the other ultrasound Doppler parameters were correlated with ASDAS-ESR ( r=-0.36, P<0.001; r=-0.54, P<0.001; r=-0.61, P=0.021; r=0.41, P=0.028). The receiver operating characteristic curve was drawn with the baseline RI value as a variable. According to the ASDAS-CRP value, the diagnostic threshold for determining the presence or absence of AS activity after 12 weeks of treatment was 0.49, with an area under the curve of 0.817, sensitivity of 88.1%, specificity of 61.1%, positive predictive value of 66.7%, and negative predictive value of 86.1%. Conclusion:Baseline Doppler ultrasound correlates well with clinical indicators, among which baseline RI values is a good predictor of inflammatory activity status after rhTNFR:Fc treatment.

Preparation and characterization of Ad-ERα-36-Fc-GFP / 生物工程学报

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.