胰岛素自身免疫性抗体对血清胰岛素测定的影响

目的 :了解胰岛素自身免疫性抗体(IAA)对血清胰岛素测定的影响。方法 :对29例IAA阳性的1型糖尿病患者去除IAA前后血清胰岛素浓度(用RIA测定)进行比较 ,另选29例IAA阴性的1型糖尿病患者作为对照。结果 :IAA阳性的糖尿病患者去除IAA前 ,胰岛素浓度高于IAA阴性组(分别为12.08±2.15、8.31±2.27 ,P<0.01) ;去除IAA后 ,IAA阳性组胰岛素明显下降(8.19±2.80,P<0.01) ,而IAA阴性组则无明显变化(8.24±2.50) ;血清IAA的存在可使血清胰岛素测定值升高4.44 %～72 % ;IAA阳性组去除IAA前后总体数差值的点估计为3.89μIu/ml,其95 %可信区间为3.03～4.75μIu/ml。IAA阳性组去除IAA前后胰岛素浓度的变化大小与IAA强弱呈直线相关。结论 :IAA存在对血清胰岛素的放射免疫分析有明显干扰 ,使结果假性升高     

Binding of Helix Pomatia A Hemagglutinin to Human Erythrocytes and their Cells. Influence of Multivalent Interaction on Affinity

The binding of ¹²⁵I-labelled intact (hexavalent) and partially reduced (divalent) Helix pomatia A hemagglutinin to human A.₁, A.₂, A.₃, A₁B and A₂B erythrocytes, human lymphocytes, human lymphoblastoid and other tumor cell lines was investigated. The essential finding was that the association constants calculated for the interaction between hemagglutinin and A-erythrocytes were many orders of magnitude higher than the intrinsic association constant for the interaction between the hemagglutinin and the blood group A determinant. The latter value was 5–10³1/mole. The K-values for intact hemagglutinin and A and AB erythrocytes were in the order of 10¹⁰1/mole at 18–22 °C and pH 7.3. For partially reduced hemagglutinin the K-values were in the order of 5–10⁷ 1/mole. Multivalent interaction would seem to be the essential factor responsible for the high K-values in the cell binding experiments. Intact hemagglutinin reacted against A₁ and A₂ erythrocytes or against a human osteogenic sarcoma cell tine (2T) gave homogeneous binding curves in Scatchard's plot. Human lymphocytes and most lymphoblastoid cell lines lacked hemagglutinin receptors.     

胸腺肽α1对老年脓毒血症患者免疫功能的影响

目的探讨老年脓毒血症患者细胞免疫功能的变化,观察胸腺肽α1的治疗效果。方法120例老年脓毒症患者随机分为两组：干预组和对照组,每组60例。对照组给予常规治疗,干预组在常规治疗基础上加用胸腺肽α1 1．6mg,／d,皮下注射,疗程1周。比较两组患者治疗前后细胞免疫功能、APACHEⅡ评分和住院死亡率。结果两组患者治疗前CD4^＋、CD8^＋、CD4^＋／CD8^＋、APACHEⅡ评分差异无统计学意义（P〉0．05）。治疗后干预组CD4^＋、CD4^＋／CD8^＋较常规治疗组明显增高（P〈0．001）,CD8^＋明显降低（P〈0．001）,APACHEⅡ评分和住院死亡率亦明显降低（P〈0．05）。结论老年脓毒血症患者细胞免疫功能明显降低,胸腺肽α1能改善此类患者的细胞免疫功能,提高临床治疗效果。     

A Simple Assay for Evaluating Inhibitors of Proteoglycan-Ligand Binding

Proteoglycans, once thought to primarily serve as structural components of extracellular matrix, are now being focused on for their role in tissue and cell regulation, particularly angiogenesis. Many growth factors, notably the fibroblast growth family (FGF) which now numbers 19 members, bind to heparin and heparan sulfate proteoglycans and this binding has been shown to have a significant impact on the availability and activity of these growth factors. Proteoglycans can serve as both temporal and spatial regulators and effective inhibitor design may depend on disruption of these interactions. We have developed a simple assay for evaluating small inhibitors of proteoglycan-ligand binding. The assay is based on cell-free incubation of the reactants and filtration across a cationic membrane. Conditions were established that allow one to semiquantitatively determine binding constants for both direct proteoglycan as well as soluble inhibitor affinity. The assay has been demonstrated using a model heparan sulfate proteoglycan preparation (perlecan from cultured bovine endothelial cells) and FGF-2. Protamine sulfate, sucrose octasulfate, and heparin were analyzed as model inhibitor molecules. This type of assay may have wide application as a fast and easy screening tool for small potential agonists and antagonists of proteoglycan-protein interactions. © 2000 Biomedical Engineering Society. PAC00: 8714Ee, 8715Kg, 8715Rn, 8780-y     

Use of Lymphocyte Platelet Binding Assay for Detecting a Preimplantation Factor: A Quantitative Assay

PROBLEM: To evaluate the ability of the lymphocyte/platelet binding assay to identify a preimplantation factor (PIF). METHOD: Percentages of binding of lymphocytes by platelets in the presence of sera from 30 known pregnant and 30 nonpregnant individuals were compared using a novel lymphocyte/platelet binding assay. The assay is performed using a combination of a heat inactivated sera with donor O+ lymphocytes, activated complement and an antibody against CD₂(T11, Ortho Pharmaceuticals). RESULTS: In nonpregnant females (23.6 ± 6.5%) and males (17.7 ± 4.7%) the percentage of lymphocytes bound by platelets was significantly different from pregnant women (56.1 ± 15.9%) (P < 0.0001). Serial sampling of blood in five women undergoing IVF/ET who had normal pregnancies showed the detection of PIF by 4 days after transfer. The lymphocyte/platelet binding assay was not influenced by hCG, progesterone and estradiol. The interassay and intraassay variabilities were <3%. CONCLUSIONS: The lymphocyte/platelet binding assay is a simple, reproducible, specific and cost efficient assay for measurement of PIF. Application of this assay will provide investigative and diagnostic tools for identifying and monitoring early pregnancy events.     

The Production of High Affinity Monoclonal Antibodies to Human Chorionic Gonadotropin and Their Application to Immunoradiometric Assay

Abstract

Production of monoclonal antibodies against hCG has been studied using hCG as the antigen. This study reports the successful isolation of hybrid clones secreting monoclonal antibodies specific for hCG with an affinity constant higher than 10¹⁰M^?l. Of 23 fusions, only 17 fusions have produced positive clones which secrete antibodies giving high levels of binding with ^l25I-labelled hCG in the supernatant. Finally, 6 different monoclonal antibodies have been isolated; 4 of them, specific for the β-subunit, with a Ka approximately 1.1–4.0 ± 10¹¹M^?1 and 2 others, specific for the α-subunit, presenting an affinity of 2.5 ± 10¹⁰M^?l. When the antibodies specific for the β-subunit are used, specific and highly sensitive radioim-munoassays are obtained after only 3 hrs of incubation. Using iodinated monoclonal antibodies specific for the α-subunit and tubes coated with antibodies against the β-subunit, we have developped sensitive immunoradiometric assays.     

In Vitro Effects of Tp5 Analogs on E-Rosette Formation and Cell Division

The effects of seventeen synthetic analogs of thymopentin (TP-5) have been studied in the active and azathioprine-inhibited E-rosette tests.

Thymopentin was gradually shortened from the C terminus to peptides and single amino acids. Thymopoietin 32-34 (Arg-Lys-Asp-RGH-0205-TP-3) (II) and thymopoietin 32-35 (Arg-Lys-Asp-Val-RGH-0206-TP-4) (I) were the most active peptides.

Dipeptide Arg-Lys produced significant stimulatory effect on azathioprine (ED₇₅) inhibited E-receptor. Treatment of azathioprine (ED₇₅-inhibited E-rosette forming cells (ERFC) with arginine or especially lysine increased the number of ERFC.

Some of TP-4 analogs decreased further the number of ERFC decreased by azathioprine ED₃₀. These “suppressive” peptides as well as TP-3 caused a partial arrest of K 562 cell proliferation up to 96 hours.

Results suggest that TP-5 is not the smallest active fragment of thymopoietins, since peptides (TP-3 and TP-4) exhibit similar or higher T-cell membrane activation on E-receptor. Arginine, lysine, and acidic aspartyl residue seem to be a necessary basic structure to produce a cumulative chemical signal on the activity of T-lymphocytes.     

Action of Levamisole on E-Rosette Forming Cells and Serum Adenosine Deaminase in Hodgkin's Disease

The interaction of levamisole and/or adenosine in vitro an E-rosette forming cells (RFC) of healthy subjects and on low RFC of patients affected with various diseases was investigated. Levamisole did not enhance normal RFC. The drug enhanced low RFC in some patients (responders) but not in others (non-responders). Adenosine inhibited normal and low RFC and this inhibition could be reversed by levamisole.

Further, levamisole 150 mg 2 days a week was given to 12 Hodgkin's disease (HD) responders for 2 weeks, then 150 mg 1 day weekly for 2 1/2 months After one week of treatment RFC increased and the enhancement could be maintained during the whole treatment period.

Lastly levamisole in vitro and in vivo diminished serum adenosine deaminase activity in normal subjects and HD patients.     

The Influence of Antibody Functional Affinity on the Effector Functions Involved in the Clearance of Circulating Immune Complexes Anti-BSA IgG/BSA

A systematic study was carried out to investigate the role of antibody functional affinity in the capacity of immune complexes (IC) to activate the complement system and to trigger subsequently the molecular events involved in the handling of IC by providing a clearance mechanism. For this purpose, two populations of polyclonal anti-BSA IgG antibodies of different affinities were prepared, with values of 1.89 × 10⁸ M^?1 and 4.94 × 10⁸ M^?1. First we studied the capacity of IC formed at equivalence with both antibodies to activate the classical and the alternative pathways of human complement and the ability of the complexes to bind to erythrocyte C3b-C4b receptors (CR1; CD35). The data showed that the highest affinity antibodies were more efficient in activating complement by both pathways. However, their binding to erythrocyte CR1 was significantly lower compared to the binding of the lowest affinity IgG. Second we compared these IC in terms of their ability to stimulate the respiratory burst of neutrophils (PMN) and to induce the release of PMN lysosomal enzymes. In general, both of these PMN functions were better stimulated by the IC prepared with the IgG antibodies having a highest affinity, although the effects were variable for different IC concentrations. The suggestion to be drawn from the data is that the antibody affinity has an influence on the formation of the immune complex lattice, modulating its three-dimensional structure and the arrangement of the antibody Fc fragments, interfering with complement activation and access to the neutrophil IgG receptors. The significance of these observations for the understanding of how affinity influences the precise biological mechanism that participates in the fate of IC is discussed.     

Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

Background  

Immunoglobulin A nephritis (IgAN) is the most common primary glomerulonephritis worldwide. It is caused by accumulation of IgA1-containing immune complexes in the kidney resulting in renal failure, which is thought to be due to altered glycosylation of IgA with a decrease of 2–3-sialylated galactosides (NeuAcα2-3Gal).     

Stability of Peripheral Blood Lymphocyte Count and E-Rosette Forming Lymphocytes in Healthy Adults

Highly reproducible peripheral blood lymphocyte (PBL) count and percent E-rosette forming lymphocyte (%E-RFL) assays were developed by modifying existing procedures. PBL count assay variation was reduced by using replicate electronic white blood cell (WBC) counting and 2,000 WBC differentials. The %E-RFL assay modifications reduced variation and include the use of: (a) a capillary buffy coat isolation procedure that recovered more than 85% of the PBL without the use of chemical separation media, (b) centrifugation temperatures of 20–22°C, and (c) toluidine blue staining that allowed enumeration of %E-RFL and not E-rosette forming cells, by exclusion of nonlymphoid cells. Day-to-day variation of PBL count and %E-RFL was defined by making serial determinations over a period of months. The data indicate that healthy adults maintain PBL counts and %E-RFL within narrow ranges that are specific for each individual.     

人体25羟维生素D的竞争蛋白结合测定及冬夏季正常值

本文采用二氯甲烷/乙醇粗提血浆,高压液相色谱精分,竞争蛋白结合测定法求得25羟维生素D值,夏季北京居民正常值为13.2±5.9ng/ml(33.0±14.8nmol/L)(n=68),其中男性为13.0±5.7ng/ml(32.5±14.3nmol/L)(n=39),女性为13.5±6.2ng/ml(33.8±15.5nmol/L)(n=29);冬季正常值为10.4±3.8ng/ml(26.1±9.6nmol/L)(n=45),其中男性为10.2±4.2ng/ml(25.4±10.4nmol/L)(n=22),女性为10.7±3.6ng/ml(26.6±8.9nmol/L)(n=23),性别间无显著性差异,夏冬季P值均大于0.5;夏季值之间差异显著(P<0.01)。 文中还对响影测定的有关问题作了讨论。     

用分离胶真空采血管保存血清对ALT测定结果的影响

观察用分离胶真空采血管保存血清对谷丙转氨酶(ALT)测定结果的影响.采集静脉血,分别置于两管分离胶真空采血管中,分离血清,立即测定ALT后,用原管保存血清,一管放室温,一管放4℃冰箱保存,连续4天测定两管血清ALT,对两组下降率进行比较.表明第2、3、4、5天测定结果表明,分离真空胶采血管室温保存血清ALT明显下降,下降率平均为16.2%、32.2%、47.0%、57.6%;将第1天和第2、3、4、5天的结果进行配对t检验,结果有显著性差异(P＜0.05);同时测定4℃冰箱保存血清AST,平均每天下降率为6.9%、7.7%、10.2%、12.2%,下降率无显著性差异.这表明这种分离胶真空采血管室温保存血清,不能用于第2天以后检查及复查ALT.     

Theoretical Analysis of the Parr Antibody Assay with a Computer Model: Importance of Antigen Concentration and Antibody Affinity

The Farr assay for antibody was analyzed theoretically in order to determine the maximum sensitivity of the assay, the quantitative relationship between actual antibody concentration and antibody estimates by the Farr technique, and the relationship between antibody affinity and the Farr avidity index. Analysis shows that sensitivity is limited by the antigen concentration when large amounts of antigen are used in the assay. Sensitivity at low antigen concentrations is maximal and varies with the affinity of the antibody studied. Antibody titers obtained by the Farr technique using low antigen concentrations vary with antibody affinity as well as antibody concentration. Titers measured at high antigen concentration are less affected by affinity and correlate better with antibody concentration. Antibody measurements expressed as Antigen Binding Capacity reflect antibody concentration only when the antigen concentration used in the assay exceeds a value equal to ten times the reciprocal of the antibody affinity constant. The Farr avidity index correlates with antibody affinity over a narrow affinity range. Different ranges of affinity can be examined by changing the antigen concentrations.     

Stability of Peripheral Blood Lymphocyte Count and E-Rosette Forming Lymphocytes in Healthy Adults

Highly reproducible peripheral blood lymphocyte (PBL) count and percent E-rosette forming lymphocyte (%E-RFL) assays were developed by modifying existing procedures. PBL count assay variation was reduced by using replicate electronic white blood cell (WBC) counting and 2,000 WBC differentials. The %E-RFL assay modifications reduced variation and include the use of: (a) a capillary buffy coat isolation procedure that recovered more than 85% of the PBL without the use of chemical separation media, (b) centrifugation temperatures of 20-22°C, and (c) toluidine blue staining that allowed enumeration of %E-RFL and not E-rosette forming cells, by exclusion of nonlymphoid cells. Day-to-day variation of PBL count and %E-RFL was defined by making serial determinations over a period of months. The data indicate that healthy adults maintain PBL counts and %E-RFL within narrow ranges that are specific for each individual.     

Studies on the Influence of a Mutation of MASP-2 on the Binding to MBL and Ficolins

Chronic hepatitis B virus (HBV) infection affects about 200–400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. Control over the HBV infection is achieved mainly by vaccination with Hepatitis B surface antigen (HBsAg). HBsAg contains N -linked glycosylation side and is recognized by both MBL-A and MBL-C in a Ca-dependent manner. HbsAg–MBL complexes activate complement and may thus affect humoural immunity. To investigate the role of MBL in humoural responses to HBsAg, we immununized mice that lack both MBL-A and MBL-C proteins with soluble HBsAg. It has been shown that deficiencies in other complement components like C1q, C4 and C3 result in decreased antibody responses. However, MBL double KO animals mounted dramatically increased humoural responses. After priming, MBL double KOs mounted HbsAg-specific IgM responses, which were threefold higher than WT controls. After boosting the HBsAg, total IgG was 10-fold higher in MBL KO than in WT control animals. Similar to the response to HbsAg, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in MBL double KO animals, suggesting that MBL plays an important role in a negative feedback regulation of adaptive immunity. Reconstitution experiments with rMBL partially rescued the KO phenotype. We propose that the clearance of glycoprotein antigens in MBL KO is handled differently from the WT, resulting in better stimulation of humoural responses. Alternatively, glycoprotein-Ag-MBL-rich complexes inhibit B-cell responsiveness via putative MBL receptors.     

抗人白细胞间素-2 McAb亲和力排列及亲和指数的测定

利用蛋白浓度校正的ELISA稀释曲线快速确定了抗人白细胞间素-2(IL-2)单克隆抗体(McAb)的亲和力排列。该排列与用竞争ELISA测定的亲和常数(K_D值)相一致。为了更实际地确定McAb的用途,又用十二烷基磺酸钠(SDS)等解离因素,建立了反映 McAb在实际应用时亲和力变化强度的吸附-解离ELISA,测定McAb的亲和指数(ID)。根据亲和力排列和亲和指数可更准确地取舍McAb,供检测或亲和色谱使用。     

Binding Affinity of Human Autoantibodies: Studies of Cryoglobulin IgM Rheumatoid Factors and IgG Autoantibodies to Albumin

The binding affinity of cryoglobulin IgM rheumatoid factors (RF) for human IgG and of human IgG anti-albumin autoantibodies for HSA was measured by the molecular sieving technique. The binding affinities of the two autoantibodies were consistently low (10⁴-10⁵ I/M) as compared to the affinities of corresponding hyperimmune animal antibodies (10⁶-10⁸ I/M). The findings were discussed in relation to theories on human autoimmunity. The existence of strict autotolerance at the T cell level and of autoreactivity at the low affinity B cell level was considered to be best compatible with the findings of this study and with the major known facts of autoimmunity.     

Flow and High Affinity Binding Affect the Elastic Modulus of the Nucleus,Cell Body and the Stress Fibers of Endothelial Cells

Cell mechanical properties are important in the adhesion of endothelial cells to synthetic vascular grafts exposed to shear flow. We hypothesized that the local apparent elastic modulus of the nucleus and the cell body would increase to a greater extent for cells adherent via the dual ligand (integrin-fibronectin/avidin-biotin) and exposed to flow, than for cells treated with either ligand alone. High affinity avidin-biotin bonds and in vitro flow exposure were used to improve adhesion to grafts thereby altering the mechanical properties of endothelial cells. Introduction of the dual ligand chemistry at the cell-substrate interface increased the apparent elastic modulus of the cells as compared to cells adherent with the fibronectin-integrin bonds only. Cells cultured on the dual ligand surface exhibited higher elastic moduli of the nucleus and cell body relative to cells cultured on fibronectin alone. Exposure of cells to flow increased the apparent elastic modulus of the cell body, nucleus, and stress fibers of cells adherent to the fibronectin surface. A similar effect was seen for cells adherent to the dual ligand surface, although there was little effect on the elastic modulus of the nucleus. While the dual ligand surface produces an increase in adhesion strength, focal contact area and elastic modulus, the change in elastic modulus after exposure to flow is due only to an increase in stress fibers and not an increase in contact area.