Association of IgG1 Antibody Clearance with FcγRIIA Polymorphism and Platelet Count in Infliximab-Treated Patients

The FcγRIIA/CD32A is mainly expressed on platelets, myeloid and several endothelial cells. Its affinity is considered insufficient for allowing significant binding of monomeric IgG, while its H131R polymorphism (histidine > arginine at position 131) influences affinity for multimeric IgG2. Platelet FcγRIIA has been reported to contribute to IgG-containing immune-complexe clearance. Given our finding that platelet FcγRIIA actually binds monomeric IgG, we investigated the role of platelets and FcγRIIA in IgG antibody elimination. We used pharmacokinetics analysis of infliximab (IgG1) in individuals with controlled Crohn’s disease. The influence of platelet count and FcγRIIA polymorphism was quantified by multivariate linear modelling. The infliximab half-life increased with R allele number (13.2, 14.4 and 15.6 days for HH, HR and RR patients, respectively). It decreased with increasing platelet count in R carriers: from ≈20 days (RR) and ≈17 days (HR) at 150 × 10⁹/L, respectively, to ≈13 days (both HR and RR) at 350 × 10⁹/L. Moreover, a flow cytometry assay showed that infliximab and monomeric IgG1 bound efficiently to platelet FcγRIIA H and R allotypes, whereas panitumumab and IgG2 bound poorly to the latter. We propose that infliximab (and presumably any IgG1 antibody) elimination is partly due to an unappreciated mechanism dependent on binding to platelet FcγRIIA, which is probably tuned by its affinity for IgG2.     

免疫磁性微球的制备及对IgG的分离

采用反相悬浮包埋法制备SPA-Sepharose免疫磁性微球,并对产物的结构、粒径、磁性能进行了表征,所得产物粒径大约为5μm,表面包裹葡萄球菌蛋白A(Staphylococcal Protein A,简称SPA),分散性较好,具有超顺磁性。利用SPA对免疫球蛋白G(IgG)的特异性吸附反应及磁性微球的超顺磁性,从猪血清中分离IgG,试验结果表明:SPA免疫磁性微球对猪血清的最大吸附效率是70.12%,可反复使用29次。     

β-乳球蛋白IgG抗原决定簇的识别

以β-乳球蛋白氨基酸序列为模板,错位合成β-乳球蛋白多肽,以收集到的牛乳过敏患者血清为抗体,鉴定β-乳球蛋白IgG抗原决定簇,探讨牛乳过敏机理．结果表明,β-乳球蛋白IgG抗原决定簇有2条,它们在β-乳球蛋白中的氨基酸序列定位分别为aa22—36和aa127—141．结果表明通过特异性水解抗原决定簇实现牛奶脱敏的方法是可行的．     

Application of N-succinimidyl 4-[(18)~F](fluoromethyl) benzoate to protein labeling

N-succinimidyl 4-[18F](fluoromethyl) benzoate for protein labeling was prepared (57%, EOB) in about 30min. Reaction conditions of S18FMB with IgG including pH of solutions, protein concentration, reaction temperature and time were studied. The optimal labeling conditions were: 0.2mg/mL IgG, pH = 7.8-8.5, 25℃, and reaction time 5min.Under these conditions the yield was about 80%. The 18F-labeled protein was purified by size exclusion chromatography.     

Application of an ultrahigh-resolution scanning electron microscope (UHS-T1) to biological specimens

In 1985 we developed an ultrahigh-resolution scanning electron microscope with a resolution of 0.5 nm. It is equipped with a field emission gun and an objective lens with a very short focal length. In this study we report a survey of some different preparation techniques and biological specimens using the new scanning electron microscope. Intracellular structures such as cell organelles were observed surprisingly sharper than those observed by ordinary scanning electron microscopes. However, at magnifications over 250,000 X, platinum particles could be discerned as scattered pebbles on the surface of all structures in coated materials. Using an uncoated but conductively stained specimen, we successfully observed ribosomes on a rough endoplasmic reticulum at a direct magnification of 1 million. In these images some protrusions were recognized on the ribosomes. Ferritin and immunoglobulin G were used as samples of biological macromolecules. These samples were observed without metal coating and conductive staining. The ferritin particles appeared as rounded bodies without any substructure on the surface and immunoglobulin G as complexes of three-unit bodies. In the latter the central body might correspond to the Fc fragment and two side ones to Fab fragments. We assume that ultrahigh-resolution scanning electron microscopy is an effective means for observation of the cell fine structures and biological macromolecules. It will open a new research field in biomedicine.     

IgG4相关性硬化性胆管炎:肝胆胰外科医生应该重视的内科疾病

IgG4相关性疾病(IgG4-related disease,IRD)是一类近10余年逐渐被认识的以血清IgG4升高、席纹状纤维化以及大量IgG4阳性浆细胞浸润为特征的多器官和系统受累的自身免疫性疾病。胆道是IRD常受累的器官之一。IgG4相关性硬化性胆管炎(IgG4 related sclerosing cholangitis,IRSC)是IRD在胆道的临床表现形式。IRSC多有胆管壁局限性增厚、胆管扩张以及梗阻性黄疸等与胆胰恶性肿瘤相类似的临床表现。IRSC对激素敏感,预后良好,合并恶性肿瘤罕见,绝大多数无需手术治疗,但近几年笔者临床工作中仍不时见到因"胆管扩张、梗阻性黄疸"行手术治疗,术后病理证实为IRSC的病例,回顾这些病例的诊治过程,鲜有术前考虑IRSC的情况。临床上也可见到术前因血清IgG4升高,起初误诊为IRSC,错过最佳手术时机,而术后病理证实为胆管癌的病例。故加强对IRSC的宣讲,特别是加深肝胆胰外科医生对IRSC的认识具有重要临床意义。     

ASGPR‐Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes

Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N‐acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes.     

Activation of Mast-Cell-Derived Chymase in the Lacrimal Glands of Patients with IgG4-Related Ophthalmic Disease

The purpose of this present study was to investigate the distribution and expression of chymase in the lacrimal glands (LGs) of patients afflicted with IgG4-related ophthalmic disease (IgG4-ROD). LGs from patients with severe canalicular obstruction were considered the control group. Toluidine blue staining confirmed a significant increase in the number of mast cells in the LGs obtained from the IgG4-ROD patients. In addition, immunostaining of serial sections from the LGs showed a significant increase in the number of chymase-positive cells and tryptase-positive cells in the IgG4-ROD LGs compared to the normal control LGs. The mRNA expression of chymase, tryptase, TGF-β1, and collagen-I tended to increase in the IgG4-ROD LGs. Immunostaining of vimentin and α-smooth muscle actin (α-SMA) showed that myofibroblasts were the main cellular components in severely fibrotic regions of LGs in patients with IgG4-ROD. Linear regression analyses on the number of mast cells, chymase-positive cells, and tryptase-positive cells revealed significant positive correlations between those respective cells. Our findings suggest that chymase may play a role in the fibrotic disorder of IgG4-ROD LGs through the regulation of TGF-β1 activation and collagen-I deposition, and that it may be a therapeutic target for patients afflicted with IgG4-ROD.