Activation of hemolytic complement by mouse monoclonal IgG1 antibody: comparison with rabbit IgG

We investigated the ability of a mouse anti-hapten monoclonal IgG1 antibody (Ab) to bind to cell-bound specific hapten and to fix and activate C1 and thus the lytic sequence of complement (C). In a comparative study with polyclonal rabbit anti-hapten IgG Ab, we found that about 6 times more monoclonal Ab molecules than polyclonal were necessary for the generation of 1 hemolytic site/cell: the data were interpreted to mean that a cluster of four cell-bound monoclonal Ab molecules was necessary to bind C1 and activate C-mediated hemolysis. Experiments performed under conditions of low density of cell-bound hapten and excess of antibody showed that both monoclonal and polyclonal IgG Abs were able to react only with 20-30% of the cell-bound hapten and that both Abs recognized the same hapten specificity. We also found that even though monoclonal IgG1 Ab was able to bind strongly to a protein A-Sepharose column and could be eluted only by a low-pH buffer, the purified Ab, when bound to cell surface hapten, showed a weak ability to react with free protein A.     

Effect of EDTA and citrate on the functional activity of the first component of complement, C1, and the C1q subcomponent

The first component of complement, C1, is a calcium-dependent complex of the three distinct subcomponents, C1q, C1r, and C1s. Earlier observations revealed that treatment of C1 with EDTA led to a loss of hemolytic C1 activity even after recalcification. Therefore, it was of interest to study whether EDTA has an additional effect on C1 and its subcomponents, beside its chelating capacity. The chelating effect of EDTA was compared to that of citrate. It was found that treatment of C1 or C1 with EDTA followed by addition of Ca++ led to a loss of hemolytic activity up to 90%, depending on EDTA concentration. Even pretreatment of EDTA with varying amounts of Ca++ did not prevent the inactivation of C1 or C1. In contrast, after dissociation of C1 or C1 by citrate, 100% of the original C1q activity is recoverable on addition of C1q deficient serum as source of C1r and C1s. EDTA-treated serum, however, showed a concentration-dependent loss of hemolytic C1q activity, indicating an inhibitory effect of EDTA on C1q. EDTA-treated C1q, fluid phase or bound to EA, was no longer able to form an hemolytically active C1 complex by interaction with C1r and C1s.     

Physicochemical characterization of fluid phase (SC5b-9) and membrane derived (MC5b-9) attack complexes of human complement purified by immunoadsorbent affinity chromatography or selective detergent extraction

Immunoadsorbent affinity chromatography, employing a monospecific caprine anti-human C5 (IgG)-agarose bead matrix, was applied to the purification of complement attack complexes from insulin activated normal human serum (SC5b-9). Gel filtration chromatography (Biogel A-15M) of the SC5b-9 complexes eluted from the anti-C5 column by guanidine-HCl was utilized as a second step to obtain a highly purified SC5b-9 preparation. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoretic (SDS-PAGE) analysis of SC5b-9 isolated by immunoadsorbent chromatography revealed the appropriate subunit composition. Complement attack complexes extracted from complement lysed erythrocyte membranes (MC5b-9) upon solubilization with either Triton X-100 (TX-100) or deoxycholate (DOC) were also purified by anti-C5 immunoadsorbent and gel filtration chromatography, but with somewhat less than satisfactory results. Thus, an alternative purification procedure was developed based upon the ability of zwitterionic detergents to selectively extract MC5b-9 from erythrocyte membranes. Zwitterionic detergent solubilized MC5b-9 complexes were isolated in high yield in a one-step purification procedure by gel filtration column chromatography (Biogel A-15M). SDS-PAGE analysis of MC5b-9 isolated in this manner revealed a subunit composition which was similar to SC5b-9 except for the complete absence of S protein.Sucrose density gradient ultracentrifugal analysis of MC5b-9 complexes solubilized by TX-100, DOC, or zwitterionic detergents revealed similar sedimentation profiles with the major portion of MC5b-9 expressing a sedimentation coefficient of29S with minor peaks of 23S and 34S or greater. Thus MC5b-9 complexes solubilized from biological membranes by nonionic, anionic, or zwitterionic detergents form a heterogeneous population of high molecular weight, ordered, oligomeric structures with the 23S form representing MC5b-9 monomer, 29S the MC5b-9 dimer and 34S the MC5b-9 trimer or possibly tetrameric structure. These results clearly suggest a possible relationship between the observed MC5b-9 complex physical heterogeneity and the heterogeneity in the effective size of functional complement lesions.     

Prognostic differences in 8th edition TNM staging of esophagogastric adenocarcinoma after neoadjuvant treatment

Background

Prognostic differences between pTN- and ypTN-categories and the prognostic accuracy of the 8th edition UICC-pTNM- and AJCC-ypTNM-staging-system for esophageal and gastric adenocarcinoma are unclear.

Isolation of the fourth component of guinea pig complement and its single polypeptide chain precursor from plasma

Guinea pig C4 (C4^gp)4 was isolated from plasma by polyethyleneglycol-4000 (PEG-4000) precipitation, lysine-Sepharose affinity chromatography removal of plasminogen and plasmin, DEAE-Sephacel, SP-Sephadex chromatography, and Sepharose CL-6B gel filtration. Both phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetraacetate (EDTA) were present in all Chromatographie buffers, and each C4 pooled concentrate was made 0.5 mM in DFP. The overall yield for three consecutive preparations averaged 3.1% of the initial plasma hemolytic C4 activity and the average specific activity rose to 139,223 U/mg, representing an average purification factor of 157-fold. The preparation of C4 gave a single band on Ouchterlony analysis with antiserum to guinea pig serum or to purified C4. Analytical alkaline discontinuous gel electrophoresis revealed a dominant protein band and functional C4 eluted from replicate gels in this region. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of unreduced C4 gave a mol. wt of 200,000, whereas SDS-PAGE of reduced and alkylated C4 demonstrated subunits of 93,000, 75,000 and 33,000 mol. wt as well as a small amount of non-reducible 200,000 mol. wt material. Elution of the 200,000 mol. wt material recognized by SDS-PAGE under reducing conditions revealed antigenic relatedness to either the α- or β-chain of C4, suggesting that it represents a pro-C4 single chain polypeptide.     

离心性环状红斑相关潜在恶性疾病及系统性疾病的研究现状

离心性环状红斑(Erythema annulare centrifugum，EAC)是一组原因不明，以四肢、躯干、臀部出现环状红斑，缓慢离心性扩大，可以自然消退，但反复发作为特征的疾病。EAC可能的病因包括感染(真菌、细菌、病毒、寄生虫)、药物(青霉素、抗疟药等)、食物(发霉乳酪)、激素改变(月经周期)、胆石病、特别是以一些系统性疾病(系统性红斑狼疮、Graves病、干燥综合征、某些肝脏疾患)及一些全身潜在的恶性疾病有关。推测其可能的发病机制是机体对感染病原体、食物、药物等各种不同的变应原的一种超敏反应，或一些自身免疫性疾病及潜在恶性疾病引起的免疫紊乱导致的皮肤反应。但其确切的痛因和临床病例之间的因果联系尚未得到临床研究的证实，其具体的发病机制仍不十分清楚，本文对近年来所报道的离心性环状红斑与潜在恶性疾病及全身系统性疾病进行综述，描述其在这些疾病中的表现及与原发病之间的关系，揭示EAC可能为上述疾病发生、发展和疾病活动的一个先兆表现或伴随症状，为进一步明确EAC的发病机理，探讨与这些疾病之间可能的内在联系提供参考，对伴有潜在恶性疾病及全身系统性疾病的EAC患者提供早发现、早诊断、早治疗的依据。