Production and preliminary characterization of monoclonal antibodies to human liver-specific lipoprotein (LSP)

ABSTRACT— Monoclonal antibodies (mc/anti-LSP) have been prepared by polyethylene glycol fusion of P3/NSI/Ag4–1 myeloma cells with spleen cells from Balb/c mice hyperimmunized with human liver-specific membrane lipoprotein (LSP). Ten hybridomas, cloned by limiting dilution, produced mc/anti-LSP reacting (by ELISA) with human LSP but not with normal human plasma proteins nor with a variety of other proteins likely to co-purify with LSP. Three of these (A15/7, A9/63 and B20), producing high-titre IgG1 mc/anti-LSP, were biosynthetically radiolabelled and used as index antibodies. By competitive inhibition of binding of the index antibodies to LSP in an immunoradiometric assay, the ten hybridoma products were classified into four distinct groups according to their specificities for different epitopes in LSP. None of the index antibodies reacted, on ELISA, with glutaraldehyde-fixed PLC/PRF/5, Chang, Daudi or HSB-2 cell lines nor with human peripheral blood leucocytes. However, A15/27 (but not A9/63 or B20) reacted with saponin-permeabilized PLC/PRF/5 and Chang cells and also with rabbit LSP. The results emphasize the polyantigenic nature of LSP and indicate that at least one of the mc/anti-LSP (A15/27) recognises a species crossreactive antigen that is present in liver-derived cell lines.     

Murine monoclonal antibodies against „liver specific lipoprotein” (LSP) defining three antigenic sites which differ in tissue- and species-distribution and subcellular location

ABSTRACT— The tissue and species cross-reactivity of three monoclonal antibodies against human liver-specific lipoprotein (LSP), as well as the subcellular locations of the respective target antigens, has been investigated using an enzyme-linked immunosorbent assay (ELISA). Antibody D6 was widely tissue cross-reactive and bound to human and rabbit but not rat, mouse or guinea pig tissues. This antibody bound to a particulate antigen localized in the microsomal fraction of rabbit liver, and distinct from enzyme markers for plasma membrane (5′-nucleotidase) and endoplasmic reticulum (glucose-6-phosphatase) in its sedimentation properties on sucrose density gradients. Antibody A9/63 bound to human liver and pancreas, but not kidney, spleen, adipose tissue, skeletal muscle, small intestine or heart, and also bound only to human and rabbit tissues. This antibody bound to a particulate antigen in pancreas, but a soluble antigen in liver. Antibody B20 bound to all tissues from all species tested, with the exception of guinea pig, and bound to particulate antigens in adipose tissue and pancreas but soluble antigens in other tissues (including liver). In addition to emphasizing the immunochemical complexity of LSP, these experiments demonstrate the suitability of monoclonal antibodies for analysis of its constituent antigens.     

Leucocyte-specific protein (LSP1) in malignant lymphoma and Hodgkin's disease

Biopsies from 319 haematopoietic neoplasms were immunostained for intracellular leucocyte-specific protein 1 (LSP1) to assess its distribution and to compare its diagnostic value with that of CD45 (leucocyte common antigen: LCA). Most small B-cell neoplasms expressed LSP1, but one third of diffuse large B-cell lymphomas (DLBCL) were LSP1 negative. Among the cases with DLBCL (76 samples) tested for both LSP1 and CD45, one fifth expressed only CD45, but five samples were LSP1-positive and negative for CD45. The latter pattern was also seen in four of nine myelomas. Five out of 14 T-lymphoblastic lymphomas co-expressed LSP1 and CD45, and three cases were LSP1 negative and CD45-positive. Most peripheral T-cell lymphomas co-expressed LSP1 and CD45. All anaplastic lymphoma kinase (ALK)-negative lymphomas of anaplastic large cell morphology (T and null phenotype) expressed LSP1 although the percentage of LSP1-positive tumour cells was variable, however, only seven out of 30 cases with ALK-positive lymphoma were LSP1 positive. LSP1 was expressed on Reed-Sternberg cells in 60 out of 66 cases with classic Hodgkin's disease but neoplastic cells were usually negative in lymphocyte predominant Hodgkin's. This study confirms the wide expression of LSP1 within haematopoietic neoplasms and its diagnostic value for a minority of lymphoid tumours that have lost CD45 expression. Furthermore, the strong expression of LSP1 in classic Hodgkin's disease, contrasting with its heterogeneous expression in ALK-negative anaplastic lymphomas, may help to distinguish the latter lymphomas from patients with tumour cell-rich Hodgkin's disease.     

电泳和免疫印迹分析副溶血弧菌和溶藻弧菌主要外膜蛋白和多糖抗原

目的　考察副溶血弧菌和溶藻弧菌的主要外膜蛋白 (MOMP)及其多糖抗原 (PSA)在不同菌株间的结构特征及其免疫学特性 ,从而进一步揭示其共同的保护性抗原。方法　一步超速离心法和蛋白酶K消化法分别制备 10株副溶血弧菌和7株溶藻弧菌的MOMP和PSA ,SDS -PAGE和免疫印迹分析其结构特点。结果　两种细菌的MOMP和PSA的结构和形态菌株间差别明显 ,但也有MOMP一致的菌株 ,免疫印迹显示 ,全部的 10株副溶血弧菌和 5株溶藻弧菌都具有分子量约为36kD的主要外膜蛋白 ,他们能够被副溶血弧菌全菌抗血清所识别 ,是两种细菌共同的具有强免疫原性的主要抗原。PSA分析未见经典LPS的O侧链结构。多糖成份在菌株间的交叉反应有限 ,可能受弧菌自身表面抗原复杂性的影响 ,凝集反应结果与免疫印迹试验结果不完全一致。此外 ,副溶血弧菌与溶藻弧菌的PSA也会产生免疫学上的交叉反应。结论　 36kD的MOMP是广泛存在于副溶血弧菌和溶藻弧菌的高丰度蛋白 ;与主要外膜蛋白相比 ,多糖抗原在菌株间具有更大的异质性 ;副溶血弧菌和溶藻弧菌的MOMP可能比PSA更具研制疫苗潜力。     

妊娠相关血浆蛋白A与急性冠脉综合征关系的研究

目的 探讨不同冠心病患者妊娠相关血浆蛋白A（PAPP-A）、高敏C反应蛋白（hs-CRP）的水平及临床意义。方法 采用ELISA方法分别检测35例急性冠脉综合征（ACS）组，17例稳定型心绞痛（SA）组和22例对照组的血清PAPP-A及hs-CRP水平，冠心病患者的冠状动脉病变支数，并比较各组间的差异。结果 ①血清PAPP-A和hs-CRP水平：ACS组明显高于对照组（P〈0．001）或SA组（P〈0．01）；②冠状动脉多支病变组的血清PAPP-A水平高于0支病变组。结论 PAPP-A是与ACS动脉粥样硬化斑块不稳定性相关的血清学指标。     

The plasma membrane redox system: a candidate source of aging-related oxidative stress

The plasma membrane redox system (PMRS) is an electron transport chain in the plasma membrane that transfers electrons from either intra- or extracellular donors to extracellular acceptors. Unlike the superoxide-generating NADPH oxidase of phagocytes and the homologous (but much less active) enzymes found in some other cells, the PMRS is still incompletely characterised at the molecular level. Much is known, however, concerning its function and affinity for both physiological and non-physiological substrates. A role for it in aging, the ‘reductive hotspot hypothesis’ (RHH), was proposed in 1998 as part of an explanation for the apparently indefinite survival in vivo of cells that have entirely lost mitochondrial respiratory capacity as a result of the accumulation of mitochondrial mutations. Stimulation of the PMRS might allow the cell to maintain redox homeostasis even while continuing to operate the Krebs cycle, which may be advantageous in many ways. However, the PMRS may, like the mitochondrial respiratory chain, be prone to generate superoxide when thus dysregulated – and in this case superoxide would be generated outside the cell, where antioxidant defences are more limited than inside the cell and where much highly oxidisable material is present. Cascades of peroxidation chain reactions initiated by this process may greatly amplify the oxidative stress on the organism that is caused by rare mitochondrially mutant cells. Since such cells increase in abundance with aging (though remaining rare), this is an economical hypothesis to explain the rise in oxidative stress seen in (and generally believed to contribute substantially to) mammalian aging. In an extension of previously published accounts of RHH, I propose here that the lysosomal toxicity of oxidised cholesterol derivatives (oxysterols) may contribute to the toxicity of mitochondrial mutations by affecting lysosomal function in many cell types in the same way as they have been proposed to do in arterial macrophages.     

Genetic basis and pathophysiological implications of high plasma Lp(a) levels.

Lipoprotein(a) or Lp(a) is a genetic variant of plasma low density lipoproteins (LDL) containing apoB100 covalently linked to apolipoprotein(a) or apo(a), the specific marker of Lp(a). Lp(a) is heterogeneous in size and density, accounting in part for the marked size polymorphism of apo(a), 300 to 800 kDa. The apo(a) size polymorphism is related to the different number of kringle repeats which are structurally similar although not identical to the kringle 4 of plasminogen. Recent studies on a genomic level have indicated that the apo(a) gene contains at least 19 different alleles varying in length between 48 and 190 kb, partially impacting on the plasma levels of Lp(a). High plasma levels of Lp(a) have been found to be associated with an increased prevalence of premature atherosclerotic cardiovascular disease by mechanism(s) yet to be established. Both atherogenic and thrombogenic potentials have been postulated and have been related to the LDL-like and plasminogen-like properties of Lp(a), respectively.     

ATP modulates sulfobromophthalein uptake in rat liver plasma membrane vesicles

The hepatic uptake of the bilirubin-bilirubin-sulfobromophthalein (BSP) group of organic anions is a carrier-mediated process and is accounted for by at least four distinct plasma membrane proteins (bilitranslocase, BSP/bilirubin-binding protein, organic anion-binding protein and the organic anion transport protein). In order to investigate the regulation of basolateral organic anion uptake, BSP transport was measured in rat basolateral liver plasma membrane vesicles in the presence of ATP. ATP significantly stimulated the electroneutral uptake of BSP with an increment in V_max compared with control (1.57±0.14 vs 0.73±0.06 nmol BSP/mg protein per 15 s, respectively; P< 0.001) while the apparent K_m was not changed significantly (12±1 vs 12±2 μmol/L). The stimulatory effect was dose-dependent for ATP (K_m 1.01±0.37 mmol/L). ATP had no detectable effect on the electrogenic component of BSP transport. Other nucleotides (ADP, AMP, GTP) and non-hydrolysable ATP did not enhance BSP uptake, suggesting that ATP hydrolysis was necessary for the effect. This was supported by the lack of effect on BSP uptake when ATP was added in the presence of vanadate. The addition of phorbol-12-myristate 13-acetate, an activator of protein kinase C (PKC), increased BSP uptake in a dose-dependent manner in the presence, but not in the absence, of ATP. Incubation of vesicles with staurosporine, an inhibitor of PKC activity, resulted in a dose-dependent inhibition of ATP-sensitive BSP transport. These data indicate that electroneutral BSP hepatic uptake is modulated by ATP. The effect is related to ATP hydrolysis and involves the activity of PKC.     

肺癌抑癌基因P53产物的免疫组织化学研究

用ＡＢＣ免疫组织化学方法，以抗Ｐ＿（５３）蛋白单克隆抗体ＰＡｂ＿（１８０１）为探针，研究了抑癌基因Ｐ＿（５３）产物Ｐ＿（５３）蛋白在７８例各型肺癌组织中的表达情况，发现Ｐ＿（５３）蛋白在肺癌组织中有较高的表达，总阳性率达８０．８％。在正常细胞未能发现突变Ｐ＿（５３），因而考虑Ｐ＿（５３）基因点突变是肺癌细胞的重要标志物。除文献报道的小细胞肺癌外，其他型肺癌也有很大一部分为细胞质阳性。提示在肺癌发生过程中Ｐ＿（５３）基因及产物的异常起着很重要的作用。     

脂蛋白(a)受体的研究

本文用蛋白免疫印迹方法发现了猴肝细胞膜上脂蛋白受体，为进一步研究这一导致动脉粥样硬化的脂蛋白的致病机理提供了新的研究内容，具体方法为：取新鲜猴肝匀浆，离心，超声波等处理制得可溶性的膜受体粗提液，经十二烷基磺酸钠－聚丙烯酰胺凝胶电泳纯化后转移至硝酸纤维膜，用蛋白质免疫印迹方法检测，出现特异的脂蛋白受体条带。     

心绞痛患者α-颗粒膜蛋白及D-二聚体的变化

目的 探讨血小板活化、凝血激活与不稳定性心绞痛（ＵＡ）的关系及其临床意义。方法 用酶联免疫双抗体夹以分别测定２２例ＵＡ患者急性期和缓解期血浆α－颗粒膜蛋白（ＧＭＰ－１４０）、Ｄ－二聚体浓度;并用相同方法测定１８例稳定劳累型心绞能（ＳＡ）患者和２０例健康对照组上述指标,对其结果进行统计学分析。结果 ＵＡ患者急性期血浆（ＧＭＰ－１４０、Ｄ－二聚体浓度显著高于ＳＡ组（Ｐ〈０．０１）和对照组（Ｐ〈０。．０     

Autoimmune reactivity of sera to hepatocyte plasma membrane in type 1 autoimmune hepatitis

Type 1 autoimmune hepatitis (AIH-1) is an organ-specific autoimmune liver disease for which no tissue-specific autoantigen has yet been identified. We examined the reactivity by sensitive immunoblotting with enhanced chemiluminescence (IB-ECL) of 43 sera from patients with AIH-1 and 182 sera from patients with other diseases on hepatocyte plasma membrane derived from rat or human liver (RHPM, HHPM) and separated by aqueous two-phase partition. The sera studied were from patients with AIH-1, primary biliary cirrhosis, chronic viral hepatitis, and systemic lupus erythematosus (SLE); and from normal subjects. Specificity of reactivity by IB-ECL was sought: (i) by testing sera on human or rat liver membrane; (ii) by testing sera on liver or kidney membrane; (iii) by serial titration of reactive sera; and (iv) by testing reactive sera from AIH-1 before and after successful treatment with prednisolone. The results were that in AIH-1 there were multiple reactive components which were not species-specific, since they were detected with both RHPM and HHPM, but were mostly tissue-specific for liver. There was no significant correlation between antinuclear antibodies (ANA) titer and the frequencies of sera reactivities against RHPM. Most of these reactive components were demonstrable at a lesser frequency in other liver diseases and in SLE. There was a striking decrease in reactivity by IB-ECL of AIH-1 sera with liver membrane after clinical remission, further suggesting that differences between AIH-1 and other inflammatory liver diseases and SLE are predominantly quantitative rather than qualitative. However, our study did point to candidate liver membrane antigens with molecular sizes of 136, 116, 81, and 49 kDa, additional to components previously described by others. The molecular identification of these prominent reactants with AIH-1 sera could prove informative for ascertaining pathogenesis. Received: July 23, 1999 / Accepted: September 24, 1999     

The intersection of bronchoscopy and extracorporeal membrane oxygenation

Central airway obstruction (CAO), which results from malignant, benign or iatrogenic etiologies, causes significant morbidity and mortality and can be seen in both the pediatric and adult patient population. Patients frequently present to the hospital with dyspnea, stridor, and respiratory distress, indicating impending respiratory failure. Heliox is used to help alleviate symptoms while procedural planning takes place. A multidisciplinary approach to airway management is often needed. Interventional pulmonologists treat CAO with rigid of flexible bronchoscopy in order to deliver therapeutic interventions under general anesthesia. In severe CAO where there is concern for total loss of the airway creating a life-threatening situation for the patient during procedural intervention, short term extracorporeal membrane oxygenation or ECMO has been successfully reported in the literature to provide ventilation and oxygenation support throughout the procedure. Venoarterial ECMO can be used to augment cardiac output in cases of central tumors with cardiac involvement. ECMO can also be used for the removal of tracheal stents when there is a concern that ventilation will be interrupted for a prolonged period of time. ECMO has also been reported as a salvage measure for patients with life threatening hemoptysis until more definitive interventions can be performed. Short term ECMO cannulation can be used with limited associated morbidity and a heparin-free approach can be pursued when there is a concern for bleeding. We will briefly review the anesthetic considerations in CAO as well as review cases of CAO where ECMO was employed to safely alleviate the airway compromise.     

低分子肝素在人工肝血浆置换治疗重型肝炎中的应用观察

观察低分子肝素在人工肝(ALSS)血浆置换(PE)治疗重型肝炎中的临床效果。将30例重型肝炎患者随机分为两组,应用低分子肝素抗凝组(LMWH组)15例,应用普通肝素抗凝组(SH组)15例,两组行ALSS-PE治疗前后均记录出血、凝血情况及有关凝血功能指标变化。30例患者行60例次PE治疗后,LMWH组在出血倾向、血浆分离器及管路凝血发生率方面均优于SH组(P<0．05),且其检测有关凝血功能指标的改善方面均明显优于SH组(P<0．05)。低分子肝素在重型肝炎PE治疗中的有效性和安全性均优于普通肝素。     

心脑血管病患者血浆Lb(a)水平及其与血脂相关性的临床研究

本文报告185例心脑血管病患者的血浆Lp(a)与139例正常中老年健康人的Lp(a)和其与血脂的相关性临床研究,结果表明185例患者血浆Lp(a)水平显著高于正常健康人(P<0.01).Lp(a)水平与其心脑血管病各项血脂指标相关性研究发现,脑溢血组中HD-C/TC和HD-C与Lp(a)具有相关性(r=0.554和r=0.484,P<0.05);而与其他心脑血管病的各项血脂指标无明显相关性,提示Lp(a)是独立于其他血脂指标的心脑血管病的危险因子.     

不同空腹血糖水平与非酒精性脂肪性肝病的相关性研究

目的 探讨不同FPG水平与非酒精性脂肪性肝病(NAFLD)的相关性. 方法 选取7896名研究对象进行统一问卷调查、血液生化和肝脏超声检查,根据FPG水平将研究对象分为NGT、IFG和T2DM组,分析NAFLD影响因素. 结果 NGT、IFG、T2DM组NAFLD检出率分别为43.0％、63.0％和67.6％,IFG、T2DM组发生NAFLD的OR值分别是NGT组的1.31、1.77倍(95％CI:1.07～1.60、1.42～2.20,P＜0.01). 结论 IFG和T2DM是发生NAFLD的独立危险因素.     

Protein-Restricted diet alters concentration of plasma membrane glycoproteins in rat liver

Malnutrition is known to have adverse effects on the physiology and morphology of the liver. The aim of this investigation was to examine the effect of protein restriction on the content of plasma membrane proteins residing in the sinusoidal and bile canalicular domains of rat liver. Post-weanling rats maintained on low protein isocaloric diets showed marked growth retardation concomitant with reduced liver protein concentration compared to control animals. The content of leucine aminopeptidase, a bile canalicular enzyme, and asialoglycoprotein receptor, a sinusoidal receptor, in livers of protein-restricted rats was 66% and 50%, respectively, of control livers. In contrast, the relative concentrations of dipeptidyl peptidase IV and a cell adhesion molecule (GP 110), both canalicular proteins, were 160% and 121%, respectively, in rat livers upon protein restriction. After a 4-week rehabilitation period, the concentrations of all canalicular membrane proteins were similar to those in control livers, while the sinusoidal receptor was only 68% of control values. Protein restriction was found to adversely affect the concentrations of protein constituents, but not their localization in the hepatocyte plasma membrane. In general, altered concentrations of hepatocyte membrane proteins were reversed on the administration of a normal protein diet.     

The transition state for folding of an outer membrane protein

Inspired by the seminal work of Anfinsen, investigations of the folding of small water-soluble proteins have culminated in detailed insights into how these molecules attain and stabilize their native folds. In contrast, despite their overwhelming importance in biology, progress in understanding the folding and stability of membrane proteins remains relatively limited. Here we use mutational analysis to describe the transition state involved in the reversible folding of the β-barrel membrane protein PhoPQ-activated gene P (PagP) from a highly disordered state in 10 M urea to a native protein embedded in a lipid bilayer. Analysis of the equilibrium stability and unfolding kinetics of 19 variants that span all eight β-strands of this 163-residue protein revealed that the transition-state structure is a highly polarized, partly formed β-barrel. The results provide unique and detailed insights into the transition-state structure for β-barrel membrane protein folding into a lipid bilayer and are consistent with a model for outer membrane protein folding via a tilted insertion mechanism.     

Rapid regulation of plasma membrane insulin receptors

Summary Injection IP of insulin at a dosage of 1 g/g body weight into normal rats produced a rapid rise in serum insulin levels from < 1 to 298 ng/ml, and a rapid decrease in specific ¹²⁵I-insulin binding to its receptors in purified liver plasma membranes. A fall in binding was seen as early as 10 minutes after injection and binding remained decreased for up to 60 min. At 10 min, ¹²⁵I-insulin binding had fallen to 59% of controls; in contrast, ¹²⁵I-glucagon binding remained unchanged. Extraction of these plasma membranes followed by radioimmunoassay for insulin did not reveal appreciable amounts of exogenous insulin. The ¹²⁵I-insulin dissociation rate from plasma membranes of control and insulin treated rats was the same, also indicating a lack of exogenous insulin. Scatchard analyses indicated that the decreased binding seen after insulin injection was due primarily to a change in the number of insulin receptors and not their affinity. These studies suggest, therefore, that high doses of insulin in vivo can rapidly regulate the number of plasma membrane insulin receptors in liver.