应用PCR方法检测部分血液制品中HCV-RNA

应用市售丙型肝炎ＰＣＲ检测试剂盒,对１９９４ １９９６年用不同病毒灭活工艺生产的人凝血因子类制品、人静注丙球和人血白蛋白进行ＨＣＶ ＲＮＡ检测。结果表明,检测六种血液制品共２１批,１批阳性,占４８％。     

心律失常豚鼠、猕猴心脏心钠素和抗心律失常肽免疫组织化学研究

本文采用免疫组化方法观察高血钾诱发的心律失常豚鼠（２０例）和猕猴（３例）心脏心钠素（ＡＮＰ）和抗心律失常肽（ＡＭ）免疫反应。结果显示：心律失常组豚鼠和猕猴心脏的ＡＮＰ和ＡＡＰ免疫反应比正常组的明显（Ｐ＜０．０５）,心房肌的ＡＮＰ和ＡＡＰ免疫反应比传导系的明显,但心室肌未见棕色反应产物。ＡＮＰ和ＡＡＰ免疫反应在心脏不同部位反应强度不同可能与心脏传出神经和心肌钙通道的分布有关。     

HGV感染者的随访研究

采用ＲＴ－ｎＰＣＲ和合成肽包被的ＥＬＩＳＡ法对ＨＧＶ感染者进行随访研究和动态观察。２／１４ＨＧＶＲＮＡ和抗ＨＧＶ均阳性者３年后阴转;３／５单项抗－ＨＧＶ阳性者３年后阴转;２／７单项ＨＧＶＲＮＡ阳性者３年后阴转。２６例检出ＨＧＶ感染指标的献血员３年随访时仅１例ＡＬＴ为１４２。６例ＨＧＶ感染者１年动态观察显示,４例受血者１年内抗ＨＧＶ阳转,但仅１例受血者受血后２周时出现一过性ＡＬＴ升高。该研究证实ＨＧＶ可以经血传播并在体内有长期携带的趋势。６例ＨＧＶ感染者的动态观察未见ＨＧＶＲＮＡ或抗－ＨＧＶ与ＡＬＴ有相关。提示ＨＧＶ对肝脏的致病性较弱或致病需要辅助因子存在,应进一步加强ＨＧＶ的致病性研究和新型肝炎病毒的研究     

化学修饰对反义寡核苷酸稳定性及抗流感病毒活性的影响

为了探讨 Ａ Ｓ Ｏ Ｄ Ｎ 化学修饰形式与 Ａ Ｓ Ｏ Ｄ Ｎ 稳定性,体外细胞毒性以及抗流感病毒活性之间的关系,合成了 ７ 种不同化学修饰形式的 Ａ Ｓ Ｏ Ｄ Ｎ：硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰;３′与 ５′端硫代,中间为天然结构的混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰等．测定了 ７ 种修饰体在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液,含 ２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ培养液以及水中的稳定性,体外细胞毒性和在细胞水平抗流感病毒活性．结果表明,混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ,硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇的修饰形式在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液与含２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ 培养液中稳定性相对较高,作用 ２４～４８ ｈ 仅混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ 与硫代 Ａ Ｓ Ｏ Ｄ Ｎ 发生部分降解;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇修饰体在 ２４ ｈ 内大部分降解．所有 Ａ Ｓ Ｏ Ｄ Ｎ 修饰体在水中具有很高稳定性,４８ ｈ 内未见降解作用．７ 种 Ａ Ｓ Ｏ Ｄ Ｎ 修饰形式在 Ｍ Ｄ Ｃ Ｋ 细胞中未表现明显的细胞毒性．硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆     

用免疫单扩散法测定低pH静注丙球lgG含量及其影响因素初探

本文报道了低 pH静注丙球IgG含量测定免疫单扩散法的建立 ,并对其影响因素进行了初步试验。结果表明 ,该试验方法较为稳定 ,简单易行。在低pH静注丙球IgG含量检测时 ,进口Sigma琼脂糖效果优于国产琼脂糖(P <0 .0 1) ,国产 (东海 )琼脂糖不适于IgG含量的定量检测 ;应先将静注丙球pH调至中性后再进行IgG含量测定(P <0 .0 5 ) ;样品中含有的 10 %麦芽糖对试验结果无影响 (P >0 .0 5 )。     

伴刀豆蛋白A及其衍生物对培养软骨细胞蛋白多糖代谢的影响

观察了ＣｏｎＡ对培养软骨细胞ＰＧ合成代谢的影响,证实ＣｏｎＡ能够使培养的软骨细胞高分子硫酸化ＰＧ的合成增加３￣４倍,其分子量,硫酸化部位和硫酸化程度与对照组相比无明显差异,是具有正常结构的软骨型ＰＧ．ＣｏｎＡ对低分子型ＰＧ的合成未见明显的影响。     

不同贮藏温度下马铃薯的萌芽和内源激素含量变化

马铃薯品种晋薯２号休眠期间,ＡＢＡ含量下降,ＺＲ,ＧＡ３,ＧＡ３／ＡＢＡ增加,高温下种薯的ＡＢＡ含量下降时间,ＧＡ３和ＺＲ含量以及ＧＡ３／ＡＢＡ比值增加时间均提前,休眠期缩短,低温下种薯的ＡＢＡ含量下降,ＧＡ３含量和ＧＡ３／ＡＢＡ比值增加均缓慢,ＺＲ含量有升有降,休眠期延长。     

不同贮藏温度下马铃薯的萌芽和内源激素含量变化(简报)

马铃薯品种晋薯２号休眠期间，ＡＢＡ含量下降，ＺＲ、ＧＡ_３、ＣＡ_３／ＡＢＡ增加。高温（２５～２８℃）下种薯的ＡＢＡ含量下降时间、ＧＡ_３和ＺＲ含量以及ＧＡ_３／ＡＢＡ比值增加时间均提前，休眠期缩短。低温（７～９℃）下种薯的ＡＢＡ含量下降，ＧＡ_３含量和ＧＡ_３／ＭＢＡ比值增加均缓慢，ＺＲ含量有升有降，休眠期延长。     

蓝尾石龙子染色体组型与Ag-NORs分析

本文用骨髓细胞制片,分析了蓝尾石龙子的染色体组型和ＡｇＮＯＲｓ,其二倍体２ｎ＝２６,含６对大型的和７对小型的染色体,除３对小型的染色体呈点状外,其余均为中部着丝粒,ＮＦ＝４６。这个种的一对ＡｇＮＯＲｓ位于Ｎｏ．２长臂近着丝粒区。未见有异型的性染色体。     

臭氧胁迫下春小麦叶片胁迫乙烯产生与多胺含量的变化与调控

应用开顶式熏气装置,研究了０．７９６ ｍ ｇ／ｍ ３ Ｏ３ 浓度下,四叶期春小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍＬ．）叶片内胁迫乙烯产生和多胺含量的变化及其调控。结果表明：Ｏ３ 使胁迫乙烯的产生呈现先升后降的变化。ＣｏＣｌ２ 能强烈抑制胁迫乙烯产生。胁迫初期,精氨酸脱羧酶（ＡＤＣ）活性增强,当叶片伤害加重后,ＡＤＣ活性下降。对氯汞苯甲酸（ＰＣＭＢ）能抑制ＡＤＣ活性,并使腐胺（Ｐｕｔ）含量减少,而亚精胺（Ｓｐｄ）和精胺（Ｓｐｍ ）含量稍有增加。ＣｏＣｌ２ 对叶片ＡＤＣ活性影响不大,未见Ｐｕｔ的积累,Ｓｐｄ 和Ｓｐｍ 含量急剧增加,且一直保持较高水平,叶片所表现的伤害也较轻。较高浓度的Ｓｐｄ 和Ｓｐｍ 能抑制Ｏ３ 对植株的伤害,Ｓｐｄ 和Ｓｐｍ 的抑制作用大于Ｐｕｔ。由此认为,多胺含量变化是植物体对Ｏ３ 胁迫适应的调节机制之一     

l-Proline reduces IgG dimer content and enhances the stability of intravenous immunoglobulin (IVIG) solutions

Liquid intravenous immunoglobulin (IVIG) products offer improved convenience in preparation but often lack sufficient stability to allow room temperature storage. Furthermore, clinical tolerability may be affected due to formation of idiotype/anti-idiotype IgG dimers and/or aggregates. Here we report on the development of a 10% IVIG formulation with optimized stability achieved by the use of l-proline. The stability of concentrated liquid IVIG was strongly pH dependent. Aggregate formation, yellowish discoloration of the solution and loss of anti-hepatitis B surface antigen (HBs) antibody activity was minimal at intermediate pH (pH 4.8–5.3). Fragmentation of IgG was highest at low pH (pH 4.1). Idiotype/anti-idiotype IgG dimer formation was highest at neutral pH and was reduced with decreasing pH. The presence of l-proline further improved stability by inhibiting protein aggregation, reducing loss of anti-HBs antibody activity and decreasing coloring, particularly compared with glycine formulations. The IgG dimer content was up to 30% lower in solutions containing l-proline compared with those containing glycine or other stabilizers. In conclusion, a weakly acidic pH of approximately 5 and l-proline as stabilizer are optimal conditions for long-term stability of a liquid IVIG. l-proline, an amphiphilic, naturally occurring amino acid, is superior to glycine in restricting IgG dimer formation.     

A Comparative Structural and Bioanalytical Study of IVIG Clinical Lots

Intravenous immunoglobulin are important bio-therapeutics used in the replacement therapy for primary and secondary immunodeficiencies, chronic inflammatory disorders and several autoimmune haematologic disorders. Currently, a number of immunoglobulin intravenous (IVIG) products have been approved by the Food and Drug Administration (FDA) and are available commercially. It is known that small differences in the manufacturing processes as well as in the formulations may affect their clinical efficacy and tolerability. Therefore, given the complexity of the multi-step process required for the isolation of IVIG from human plasma, it is necessary to ensure a rigorous quality control of final products. We show here that a set of different bioanalytical techniques can be conveniently used to comparatively characterize, at a quantitative and qualitative level, different lots of IVIG preparations and to unveil randomly occurring impurities which can also affect the overall product stability. We have used circular dichroism, surface plasmon resonance and two-dimensional electrophoresis (2DE), and have demonstrated that this combination of bioanalytical approaches is very useful to improve the quality control of antibodies and to monitor the reliability of the IVIG manufacturing process.     

IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

High-dose intravenous immunoglobulin (IVIG) preparations are currently used for the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP). Although the mechanisms of IVIG efficacy remain enigmatic, some clinical and laboratory studies suggest that interaction of the Fc domain of IgG, especially the Fc domain of dimeric IgG, with its receptors (Fc gamma receptors; FcγRs) plays an essential role. In this study, IVIG was dimerized with chemical crosslinkers to augment its therapeutic efficacy. Dimerized IVIG was found to have a much higher affinity for FcγRs than monomeric IVIG. In a mouse ITP model, chemically dimerized IVIG abrogated the decrease in platelet numbers in the blood that was caused by an anti-platelet antibody at a dose that was one tenth of the required dose of IVIG. These results suggest that chemical dimerization of IVIG should greatly improve the efficacy of IVIG therapy of ITP.     

Concomitant Raman spectroscopy and dynamic light scattering for characterization of therapeutic proteins at high concentrations

A Raman spectrometer and dynamic light scattering system were combined in a single platform (Raman–DLS) to provide concomitant higher order structural and hydrodynamic size data for therapeutic proteins at high concentration. As model therapeutic proteins, we studied human serum albumin (HSA) and intravenous immunoglobulin (IVIG). HSA concentration and temperature interval during heating did not affect the onset temperatures for conformation perturbation or aggregation. The impact of pH on thermal stability of HSA was tested at pHs 3, 5, and 8. Stability was the greatest at pH 8, but distinct unfolding and aggregation behaviors were observed at the different pHs. HSA structural transitions and aggregation kinetics were also studied in real time during isothermal incubations at pH 7. In a forced oxidation study, it was found that hydrogen peroxide (H₂O₂) treatment reduced the thermal stability of HSA. Finally, the structure and thermal stability of IVIG were studied, and a comprehensive characterization of heating-induced structural perturbations and aggregation was obtained. In conclusion, by providing comprehensive data on protein tertiary and secondary structures and hydrodynamic size during real-time heating or isothermal incubation experiments, the Raman–DLS system offers unique physical insights into the properties of high-concentration protein samples.     

Passively administered pooled human immunoglobulins exert IL-10 dependent anti-inflammatory effects that protect against fatal HSV encephalitis

HSV-1 is the leading cause of sporadic encephalitis in humans. HSV infection of susceptible 129S6 mice results in fatal encephalitis (HSE) caused by massive inflammatory brainstem lesions comprising monocytes and neutrophils. During infection with pathogenic microorganisms or autoimmune disease, IgGs induce proinflammatory responses and recruit innate effector cells. In contrast, high dose intravenous immunoglobulins (IVIG) are an effective treatment for various autoimmune and inflammatory diseases because of potent anti-inflammatory effects stemming in part from sialylated IgGs (sIgG) present at 1-3% in IVIG. We investigated the ability of IVIG to prevent fatal HSE when given 24 h post infection. We discovered a novel anti-inflammatory pathway mediated by low-dose IVIG that protected 129S6 mice from fatal HSE by modulating CNS inflammation independently of HSV specific antibodies or sIgG. IVIG suppressed CNS infiltration by pathogenic CD11b(+) Ly6C(high) monocytes and inhibited their spontaneous degranulation in vitro. FcγRIIb expression was required for IVIG mediated suppression of CNS infiltration by CD45(+) Ly6C(low) monocytes but not for inhibiting development of Ly6C(high) monocytes. IVIG increased accumulation of T cells in the CNS, and the non-sIgG fraction induced a dramatic expansion of FoxP3(+) CD4(+) T regulatory cells (Tregs) and FoxP3(-) ICOS(+) CD4(+) T cells in peripheral lymphoid organs. Tregs purified from HSV infected IVIG treated, but not control, mice protected adoptively transferred mice from fatal HSE. IL-10, produced by the ICOS(+) CD4(+) T cells that accumulated in the CNS of IVIG treated, but not control mice, was essential for induction of protective anti-inflammatory responses. Our results significantly enhance understanding of IVIG's anti-inflammatory and immunomodulatory capabilities by revealing a novel sIgG independent anti-inflammatory pathway responsible for induction of regulatory T cells that secrete the immunosuppressive cytokine IL-10 and further reveal the therapeutic potential of IVIG for treating viral induced inflammatory diseases.     

Monitoring of the serum proteome in Kawasaki disease patients before and after immunoglobulin therapy

Kawasaki disease (KD) is a systemic vasculitis that mainly affects children younger than 5 years. The causal pathogen is unknown, therefore specific diagnostic biomarkers and therapy are unavailable. High-dose intravenous immunoglobulin (IVIG) is considered as the most effective therapy to reduce the prevalence of coronary artery lesion (CAL) in KD; however, it has side effects. This study aimed to (1) determine whether IVIG therapy is effective at the molecular level; (2) provide the first serum proteomic profile of KD under IVIG therapy; and (3) screen for monitoring biomarker candidates. We extracted serum proteins from samples of healthy individuals and from KD patients before and after IVIG therapy, and employed two-dimensional electrophoresis and MALDI-TOF/TOF mass spectrometry to identify differentially expressed proteins. The identifications were validated by Western blotting. We identified 29 differentially expressed proteins in KD patients and found that IVIG therapy restored most of these proteins to near-normal levels. Tracing the protein levels of single patients before and after IVIG therapy showed that the proteins, especially Transthyretin (TTR), are potential markers for therapeutic monitoring. Functional analyses of these proteins by PANTHER and String suggested that the key influence of KD lay in the immune system, which was targeted by IVIG.     

静注人免疫球蛋白中白喉抗体效价对其Fc段生物学活性检测结果影响的分析

目的分析静注人免疫球蛋白(IVIG)中白喉抗体效价对其Fc段生物学活性检测的影响。方法检测20批IVIG的白喉抗体效价水平和Fc段生物学活性结果,进一步探讨白喉抗体效价与Fc段生物学活性的关系。结果20批IVIG中白喉抗体效价水平均符合中国药典的要求,在3~60 HAU/g之间,其中有两批IVIG的白喉抗体效价水平相对较高,其他18批IVIG的白喉抗体效价水平变化趋势不明显。20批IVIG的Fc段生物学活性均较高,在60%~140%之间。白喉抗体效价水平高者,其Fc段生物学活性并非高,反之亦然。结论 IVIG的Fc段生物学活性与其白喉抗体效价水平无明显的相关性。     

层析法从血浆分离静注人免疫球蛋白及其初步检定

目的：实验尝试采用多步柱层析的工艺取代乙醇低温沉淀的方法,能高效的从血浆中获得IVIG产品。方法：通过亲和层析、离子交换一套工艺纯化出IVIG产品,进而按照枟中华人民共和国药典枠对静注人免疫球蛋白（pH4）及其关键指标进行了检测。在此基础上还采用免疫浊度法等方法,对其纯度和非目的蛋白构成与国际产品进行比较。结果：工艺获得IVIG产品的关键性指标检测均达到枟中华人民共和国药典枠的要求,其质量不低于现有国际同类产品。结论：本工艺能以较高回收率获得高质量的IVIG产品。     

Both the Fab and Fc domains of IgG are essential for ROS emission from TNF-α-primed neutrophils by IVIG

Intravenous immunoglobulin (IVIG) is currently a very important therapeutic used for not only infectious diseases, but also for autoimmune diseases such as idiopathic thrombocytopenic purpura (ITP). Untoward reactions of IVIG have been thought to result from complement activation by aggregated IgG in IVIG. In addition, the aggregates have been known to activate neutrophils, which may result in the untoward reactions. However, the effect and mechanism of IVIG on neutrophils remain unclear. In this study, we investigated the activation of neutrophils by IVIG in terms of their reactive oxygen species (ROS) emission to elucidate the mechanisms. IVIG-induced ROS emission from purified neutrophils was remarkably augmented by TNF-α priming of the cells. The ROS emission from TNF-α-primed neutrophils occurred by activation with whole gammaglobulin (GG) molecules, but not F(ab')(2), Fc, or a mixture of F(ab')(2) and Fc. ROS emission by GG was inhibited by the F(ab')(2) fragment and an inhibitory antibody against FcγRIII. These results suggest that binding of IVIG to not only surface antigen(s), but also FcγRIII on neutrophils, is involved in IVIG-induced ROS emission from TNF-α-primed neutrophils, and contribute to the untoward reactions of IVIG.     

Intravenous immunoglobulin G treatment increases live birth rate in women with recurrent miscarriage and modulates regulatory and exhausted regulatory T cells frequency and function

Exhausted T cells and regulatory T (Treg) cells have been recently proposed to be new risk factors for recurrent miscarriage (RM). Intravenous immunoglobulin G (IVIG) treatment reported to modulate various immune cells. In this study, the effects of IVIG on the frequency and function of exhausted T cells, exhausted Tregs, and Treg cells, as well as pregnancy outcome in women with unexplained RM (URM), were investigated. Ninety-four pregnant women with RM were enrolled. At the time of positive pregnancy, blood samples were drawn. Forty-four patients with URM were included as IVIG receiving treated group and received 400 mg/kg of IVIG and the rest fifty patients were considered as a control group and received no IVIG administration. IVIG was given intravenously every 4 weeks during 32 weeks of gestation. Blood samples of patients were collected after the latest administration. Exhausted T cells, exhausted Tregs, and Treg cells were evaluated pre- and posttreatment in both groups. IVIG induced a significant decrease in the frequency of exhausted Tregs population and function as well as a significant increase in Treg cells population, however, IVIG failed to affect population and the function of exhausted T cells. Pregnancy outcome was successful in IVIG treated women (86.3%) and were significantly different (P = 0.0006) in compared with the untreated URM subjects (42%). Therefore, employing of IVIG increases Treg cells and diminishes exhausted Tregs responses in RM patients with cellular immune anomalies throughout the pregnancy. Immunemodulatory effects of IVIG are probably associated with successful pregnancy outcome.