静脉注射用人免疫球蛋白细菌内毒素检测

探索静脉注射用人免疫球蛋白(IVIG)细菌内毒素含量的鲎试验检测方法。根据《中国药典》(2000版)细菌内毒素含量的检测方法进行,将待检品用NaOH调pH至中性,稀释至2.4倍可用标示灵敏度为0.25EU/m l的特异性鲎试剂进行细菌内毒素检测,结果与家兔法进行比较,并且在样品中加入定量内毒素0.6 EU/m l用两种方法进行对比试验。结果表明用细菌内毒素检测法(鲎试验法)检测静脉注射用人免疫球蛋白是可行的。     

静注人免疫球蛋白IgG含量测定中的影响因素

为了进一步分析静注人免疫球蛋白(IVIG)IgG含量测定中的影响因素,分别以不同的稀释缓冲液、不同的稀释度、不同的稳定剂对IVIG的IgG含量进行测定。结果表明以不同的稀释缓冲液、不同的稳定剂测定的同批IgG含量无显著性差异;而用不同的稀释度测定的同批IgG含量有差异。因此可见,IVIG制品中采用的不同稳定剂对IgG含量测定影响不大;可以用不同的稀释度测定的均值作为该批IVIG制品的IgG含量。     

绵羊红细胞的质量对静脉注射用人免疫球蛋白抗补体活性测定的影响

为了分析IVIG抗补体活性测定出现负值与绵羊红细胞质量的关系。采用CH50试验测定IVIG ACA时,在被检样品和其他试验条件相同的情况下,比较了被污染的和未污染的绵羊红细胞对IVIG抗补体活性测定的影响。结果显示,使用被污染的绵羊红细胞(作为试验材料时所得的)检测10份样品ACA(%)均为负值;使用未污染的绵羊红细胞未出现负值。提示被污染的绵羊红细胞干扰了抗补体活性测定,从而引起CH50试验结果出现偏差。     

Systematic fractionation of oligosaccharides of human immunoglobulin G by serial affinity chromatography on immobilized lectin columns

Human immunoglobulin G is known to contain 16 different biantennary complex-type asparagine-linked sugar chains, each of which occurs in a nonsialylated, monosialylated, or disialylated form. These oligosaccharides can be separated into 14 fractions by sequential affinity chromatography with Aleuria aurantia lectin (AAL)-Sepharose, RCA120-WG003, and E4-phytohemagglutinin-agarose columns. Twelve of them were found to contain a single oligosaccharide, while the fraction which passed through all three columns was shown to contain two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. The fraction, which bound to the AAL-Sepharose column and passed through the remaining two lectin columns, also contained two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (Fuc alpha 1----6)GlcNAcOT. These results indicated that serial affinity chromatography with the three lectin columns can be used effectively to detect changes in the sugar chains of IgG resulting from diseases such as rheumatoid arthritis.     

Influence of high dose intravenous immunoglobulins (IVIG) on HSV-1 infection in immunosuppressed mice]

Influence of low and high doses of intravenous immunoglobulin (IVIG) was compared on the basis of survival studies of mice infected with temperature sensitive (ts 28 degrees C) clone of Herpes simplex virus (HSV-1) after previous treatment with cyclophosphamide (CY), Sandoglobulin containing intact IgG and Gamma-Venin containing only fragments of (Fab)2 IgG were used. It was found that 4 and 16 mg doses protected animals in contrast to the 32 mg dose of Sandoglobulin which protected mice in only small degree.     

Anti-inflammatory intravenous immunoglobulin (IVIg) suppresses homeostatic proliferation of B cells

An intravenous injection of plasma-derived immunoglobulins is used for the treatment of severe infectious and autoimmune disorders. Despite of its clinical efficacy, precise mechanisms by which intravenous immunoglobulin (IVIg) suppresses proinflammatory immune response are still enigmatic. Here, we provide in vitro evidence that IVIg inhibits homeostatic proliferation of B cells accompanied by induction of their cell aggregation. The IVIg-driven suppression of B cell proliferation and induction of cell aggregation are both unaffected by treatment with a neutralizing antibody against low-affinity Fc receptors for IgG (CD16/FcγRIII and CD32/FcγRII), known cell surface ligands for IVIg. Our observations propose a new immunosuppressive action of IVIg, which directly acts on steady-state B cells to suppress their homeostatic expansion.     

家蝇C-型凝集素的克隆与功能分析

【目的】从家蝇Musca domestica中克隆一种C-型凝集素(C-type lectin)基因,并分析其在家蝇免疫过程中的功能。【方法】根据家蝇转录组信息,克隆了一条C型凝集素基因,将其命名为Mdlectin-C1。构建Mdlectin-C1的原核表达载体,在大肠杆菌Escherichia coli中表达并纯化r Mdlectin-C1蛋白,制备多克隆抗体。利用实时定量PCR(quantitative real-time PCR,qRT-PCR)及Western blot技术对家蝇不同发育阶段和细菌感染前后Mdlectin-C1的表达水平进行检测。运用RNAi技术敲低家蝇1龄幼虫Mdlectin-C1基因表达,然后进行细菌刺激并观察幼虫存活率变化。【结果】Mdlectin-C1 c DNA包含一个546 bp完整开放阅读框,编码181个氨基酸残基,所推导多肽N端包含由21个氨基酸残基组成的信号肽,成熟肽中含有一个保守的碳水化合物识别结构域(carbohydrate-recognition domain,CRD)。qRT-PCR及Western blot结果显示,家蝇从1龄幼虫到蛹的发育过程中,Mdlectin-C1表达量逐步上升,蛹期达到最大值。在革兰氏阴性菌大肠杆菌E.coli和革兰氏阳性菌金黄色葡萄球菌Staphylococcus aureus刺激后,家蝇2龄幼虫Mdlectin-C1均上调表达,并分别在36和3 h时达到最高值。利用RNAi技术成功敲低Mdlectin-C1表达。对Mdlectin-C1基因敲低的1龄幼虫进行细菌刺激,敲低实验组幼虫存活率显著低于正常对照组。【结论】Mdlectin-C1可能参与家蝇抗菌免疫应答,并在此过程中具有重要作用。     

Selective aromatic-hydrophobic binding and fractionation of immunoglobulin by means of phenyl-(CH2)n-NH-substituted agaroses.

Under specified conditions the major portions of immunoglobulin preparations (“Fraction II”, IgG) are selectively bound by agaroses substituted with phenyl-(CH₂)_n-NH-groups as compared to agaroses carrying aliphatic n-alkyl-NH-groups of similar hydrophobicity and ligand density. These immunoglobulin preparations, even the most highly purified ones, were found to be inhomogeneous with respect to the aromatic-hydrophobic mode of binding and fractionation through differential adsorption on the basis of the hydrocarbon chain-length of the ligands has been attained.     

Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family

A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages.     

Enrichment of sialylated IgG by lectin fractionation does not enhance the efficacy of immunoglobulin G in a murine model of immune thrombocytopenia

Intravenous immunoglobulin G (IVIg) is widely used against a range of clinical symptoms. For its use in immune modulating therapies such as treatment of immune thrombocytopenic purpura high doses of IVIg are required. It has been suggested that only a fraction of IVIg causes this anti immune modulating effect. Recent studies indicated that this fraction is the Fc-sialylated IgG fraction. The aim of our study was to determine the efficacy of IVIg enriched for sialylated IgG (IVIg-SA (+)) in a murine model of passive immune thrombocytopenia (PIT). We enriched IVIg for sialylated IgG by Sambucus nigra agglutinin (SNA) lectin fractionation and determined the degree of sialylation. Analysis of IVIg-SA (+) using a lectin-based ELISA revealed that we enriched predominantly for Fab-sialylated IgG, whereas we did not find an increase in Fc-sialylated IgG. Mass spectrometric analysis confirmed that Fc sialylation did not change after SNA lectin fractionation. The efficacy of sialylated IgG was measured by administering IVIg or IVIg-SA (+) 24 hours prior to an injection of a rat anti-mouse platelet mAb. We found an 85% decrease in platelet count after injection of an anti-platelet mAb, which was reduced to a 70% decrease by injecting IVIg (p<0.01). In contrast, IVIg-SA (+) had no effect on the platelet count. Serum levels of IVIg and IVIg-SA (+) were similar, ruling out enhanced IgG clearance as a possible explanation. Our results indicate that SNA lectin fractionation is not a suitable method to enrich IVIg for Fc-sialylated IgG. The use of IVIg enriched for Fab-sialylated IgG abolishes the efficacy of IVIg in the murine PIT model.     

Glycosylation of Eag1 (Kv10.1) potassium channels: intracellular trafficking and functional consequences

N-Linked glycosylation is a common post-translational modification of membrane proteins. Here we report that mature Eag1 potassium channels carry sugar moieties linked to asparagines at positions 388 and 406. Asn-388 seems to undergo only core glycosylation, but complex sugars are bound to Asn-406. Correct complex glycosylation is required for proper trafficking of Eag1 to the plasma membrane but is also crucial for the correct function of channels already inserted in the membrane.     

Neurochemical and functional consequences following 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and methamphetamine

The neurochemical and functional consequences following MPTP administration to the rat were evaluated and compared to similar effects following methamphetamine administration. It was observed that MPTP induced long lasting depletions of striatal dopamine concentrations and this neurotoxic effect could be prevented by pargyline pretreatment. The MPTP-induced neuronal damage produced a tolerance to the disruptive effects of amphetamine and a supersensitivity to the disruptive effects of apomorphine in rats responding in a schedule controlled paradigm. Methamphetamine, like MPTP, produced depletions of striatal dopamine but these actions were potentiated by pargyline pretreatment. These observations are discussed in reference to possible deleterious effects following the administration of pargyline to patients with Parkinson's Disease.     

Structural and functional consequences of an N-glycosylation mutation (HEMPAS) affecting human erythrocyte membrane glycoproteins.

Band 3, the human erythrocyte anion exchanger (AE1), and the glucose transporter (GLUT1) proteins each contain a single site of N-glycosylation that is heterogeneously glycosylated. Lectin binding and enzymatic deglycosylation assays showed that the polylactosaminyl oligosaccharide structure of these glycoproteins was altered to a high mannose or hybrid glycan form in three patients with hereditary erythroblastic multinuclearity, with a positive acidified-serum lysis test (HEMPAS). Offspring from one of the HEMPAS patients had intermediate levels of polylactosaminyl oligosaccharide associated with AE1 and GLUT1, suggesting they may have been heterozygous for the genetic defect. The array of polylactosaminyl-containing glycoproteins present in EBV-transformed lymphoblasts derived from fresh blood of HEMPAS patients was similar to control lymphoblasts. HEMPAS lymphoblasts do not therefore express the defect in polylactosamine synthesis found in erythroid cells, indicating that lymphoid cells are not deficient in the processing enzymes or contain an alternative oligosaccharide processing pathway. Purified HEMPAS band 3 had an unaltered oligomeric structure but dimers aggregated more rapidly in detergent solution than normal band 3. The altered oligosaccharide structure did not affect the sensitivity of band 3 to proteolytic digestion in intact red cells but a greater amount of HEMPAS band 3 was associated with the cytoskeleton. The transport activities of AE1 and GLUT1 in HEMPAS erythrocytes were similar to those in normal controls. This shows that the HEMPAS glycosylation defect does not impair the functional accumulation of these two important erythrocyte membrane transporters even though it produces subtle structural changes in band 3 that result in its increased cytoskeletal interaction and self association in detergent solution.     

采用多种抗原测定静注人免疫球蛋白(pH4)中抗体Fc段生物学活性的初探

目的初步探讨采用多种抗原测定静注人免疫球蛋白(IVIG)(pH4)中抗体的Fc段生物学活性,了解IVIG中抗体的Fc段生物学活性。方法采用补体活化的经典途径中免疫复合物激活补体的方法,将不同浓度的麻疹病毒、风疹病毒、乙肝表面抗原(HBsAg)、破伤风类毒素、脑膜炎球菌P64k外膜蛋白和白喉类毒素6种抗原分别致敏人O型血红细胞形成红细胞-抗原结合物;然后,6种致敏红细胞分别与IVIG孵育,与特异性抗体形成红细胞-抗原-抗体复合物;最后,此复合物与补体反应,在541 nm波长处读取吸光值,并绘制溶血反应动力学曲线,分别计算IVIG中针对上述6种抗原IgG的Fc段生物学活性。采用此方法,用6种抗原致敏的红细胞测定IVIG的Fc段生物学活性10次,验证此方法的重复性。结果麻疹病毒、风疹病毒、HBsAg、破伤风类毒素和脑膜炎球菌P64k外膜蛋白致敏的红细胞分别与供试品和补体反应后,测定的溶血反应动力学曲线较平缓,而白喉类毒素致敏的红细胞与供试品和补体反应后,测定的溶血反应动力学曲线下降明显,呈典型的"S"型曲线。计算结果显示,IVIG中针对此六种抗原的抗体Fc段生物学活性相对于参考品均大于80%。Fc段生物学检测方法重复性较好。结论采用多种抗原分别致敏红细胞,可以用来检测IVIG中多种抗体的Fc段生物学活性,为深入了解IVIG制品中的多种抗体的Fc段生物学活性奠定了基础。     

Dimerization of chemokine receptors and its functional consequences

It became clear over the recent years that most, if not all, G protein-coupled receptors (GPCR) are able to form dimers or higher order oligomers. Chemokine receptors make no exception to this new rule and both homo- and heterodimerization were demonstrated for CC and CXC receptors. Functional analyses demonstrated negative binding cooperativity between the two subunits of a dimer. The consequence is that only one chemokine can bind with high affinity onto a receptor dimer. In the context of receptor activation, this implies that the motions of helical domains triggered by the binding of agonists induce correlated changes in the other protomer. The impact of the chemokine dimerization process in terms of co-receptor function and drug development is discussed.     

Purification and characterization of Tora-bean (Phaseolus vulgaris) lectin

A lectin purified from the Tora-bean (Phaseolus vulgaris) by affinity chromatography with Con-A Sepharose was shown to be a glycoprotein containing 7.8% neutral sugars (D-mannose, N-acetyl-D-glucosamine, L-fucose, and D-xylose, in a molar ratio of 9.6 : 2.0 : 0.6 : 0.7). Its molecular weight was 130,000, as estimated by exclusion gel chromatography, and SDS gel electrophoresis showed that it consists of four subunits of molecular weight 32,000. The lectin reacts with various glycoproteins, i.e., blood group substances, human parotid salivary glycoprotein, fetuin, and bovine submaxillary mucin. Divalent cations, such as Ca2+, Mn2+, and Mg2+, appear to stimulate its reactivity. Inhibition tests using the glycopeptide fragment from fetuin and some oligosaccharides, as well as the binding test with 14C-N-acetyl-lactosamine suggest that the sequence of D-galactose, N-acetyl-D-glucosamine, and D-mannose residues in the carbohydrate chain of fetuin is essential for binding.     

Analysis of serum protein glycosylation by a differential lectin immunosorbant assay (dLISA)

Background

Lectin immunosorbant assays (LISAs) have been widely used for analyzing protein glycosylation. However, the analysis of serum samples by LISAs could suffer from high sample-dependent background noise. The aim of this study is to develop a differential lectin immunosorbant assay (dLISA) with reduced background interferences.

Methods

For the analysis of protein glycosylation, dLISA establishes a dose–response curve for every serum sample. The sample is split into five aliquots. Four aliquots undergo differential removal of the glycoprotein of interest by immunoprecipitation. Then, all five aliquots are subject to two measurements: protein by immunoassay and protein glycans by LISA. A dose–response curve is established by plotting glycans signals on the y-axis and protein levels on the x-axis for all the aliquots. Slope of the curve, calculated by linear progression analysis and expressed as fluorescence per concentration of protein, is used for the measurement of protein glycosylation in the serum sample.

Results/conclusions

To demonstrate the feasibility of the dLISA approach, we used recombinant, fucosylated tissue inhibitor of metallopeptidase 1 (TIMP-1) as the target glycoprotein. Magnetic beads based TIMP1 immunoassay and TIMP-1 UEA LISA were developed for the measurement of TIMP1 protein and terminal α1, 2 fucosylated glycans on TIMP1, respectively. Serum samples supplemented with differentially fucosylated recombinant TIMP-1 were used to demonstrate that the slopes measured the TIMP-1 fucosylation, and were less prone to background interference.