Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography

The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart.     

Suppression of cell-mediated tumor cell lysis and complement-induced cytotoxicity by trypan blue.

Different forms of cell-mediated cytotoxicity were suppressed in the presence of trypan blue. The systems affected included lysis of antibody-coated tumor cells by normal and C. parvum-stimulated mouse peritoneal cells and lysis of allogeneic targets by immune effector cells. The inhibition, measured in a 4-hr 51Cr release assay, was reversible and did not occur in the presence of 30% fetal calf serum or albumin. Binding between effector and target cells through Fc receptors was not affected, and lysis of allogeneic cells was inhibited at the lytic step rather than at the binding step. In contrast, lysis of sensitized erythrocytes was not inhibited by trypan blue, suggesting that lysis of these targets may not involve the steps required in tumor cell lysis. Trypan blue blocked the function of antibody before binding to target cells and also suppressed complement-induced cytolysis. Most individual complement components were susceptible to the inhibitory action of trypan blue. These results reveal an affinity of trypan blue for proteins in general that may be responsible for many of its biologic actions.     

Membrane factors responsible for homologous species restriction of complement-mediated lysis: evidence for a factor other than DAF operating at the stage of C8 and C9

Species-restricted lysis of complement refers to the relative inefficiency of complement to lyse cells from the homologous species. Restriction occurs at least at the steps involving C3/C5 convertase formation and the C9 insertion phase of the complement cascade, and is presumed to be mediated by inhibitory factors in the target cell membrane. In this study, we have examined whether decay accelerating factor (DAF), a membrane protein known to modulate C3/C5 convertase activities on cell surfaces, acts as a regulatory protein in species-restricted lysis of human erythrocyte (E). The role of DAF was assessed in homologous lysis by the classic pathway, in reactive lysis, and in lytic steps requiring C8 and C9. The results indicated that DAF participated in regulating C3/C5 deposition on the surface of homologous E, but had no effect on homologous restriction in reactive lysis and in the reaction of C8 and C9 with antibody-sensitized E C1-7. Treatment of E with pronase or with dithiothreitol (DTT) abolished the restricting effect of homologous C8/C9, indicating that species-restricted lysis by C5b-9 involves membrane factor(s) sensitive to pronase and DTT.     

Inhibition of cytolytic lymphocytes by homologous restriction factor. Lack of species restriction

Homologous restriction factor (HRF) has been shown to inhibit complement-mediated lysis in a species-restrictive manner. Human HRF is able to block lysis by human complement but not by complement from other species. HRF has also been found in the membrane of lymphokine-activated killer (LAK) cells. When this HRF is inserted into sheep erythrocyte membranes, it is able to protect the erythrocyte from LAK cell lysis. In this report, we show that while HRF can inhibit human complement but not rat complement-mediated hemolysis, it is able to inhibit LAK cell lysis by both human and rat LAK cells. HRF is therefore a more general protective protein than has been previously thought.     

Neuraminidase reversal of resistance to lysis of herpes simplex virus-infected cells by antibody and complement.

The susceptibility to lysis by antibody and complement was examined in four human cell lines. The cells were infected with herpes simplex virus type 1 and lysis was assessed by the 51Cr release test by using antibodies to herpes simplex virus and guinea pig serum as a source of complement. The four cell lines were found to differ in their susceptibility to lysis, although virus replication was readily demonstrated in the different cell lines. By indirect immunofluorescence, no differences in the expression of virus antigens at the surface of the cells could be found between the different cell lines. Treatment of cells with neuraminidase markedly enhanced the sensitivity of the cells which were relatively insensitive to lysis. The enhancement of susceptibiltiy to lysis by neuraminidase occurred if cells were treated before reaction of the cells with antibody and if the cells were reacted with antibody before treatment with the enzyme. No enhancement was observed when cells were reacted with antibody and complement before neuraminidase treatment. Neuraminidase treatment did not seem to enhance appreciably the quantity of antibody which reacted at the cell surface. The observations suggest that surface properties of certain cells render the cells resistant to lysis by antibody and complement and that the resistance to lysis can be abrogated by treating the cells with neuraminidase.     

A reexamination of the role of clusterin as a complement regulator.

Clusterin is a highly conserved glycoprotein which has been proposed to protect host cells against complement-mediated cytolysis. We tested the hypothesis that clusterin is a complement regulator using erythrocytes and cells which had been stably transfected with a membrane-anchored form of clusterin as targets for complement-mediated cytolysis. Clusterin gave dose-dependent protection of antibody-coated sheep erythrocytes against complement-mediated lysis by diluted normal human serum. There was a linear relationship between the concentration of clusterin giving 50% protection and the concentration of serum; extrapolation of this to the case of undiluted human serum showed that a clusterin concentration at least two orders of magnitude greater than its physiological plasma concentration would be needed to confer protection against complement-mediated cytolysis under physiological conditions. Physiological concentrations of clusterin did not protect rabbit erythrocytes against alternative complement pathway-mediated lysis using dilute human serum. Exogenous clusterin had no effect on lysis of human erythrocytes triggered by the addition of inulin to autologous human serum. Induction of cell-surface clusterin expression by L929 (murine fibroblast) cells which had been stably transfected with cDNA for human clusterin linked to DNA coding for the 44 C-terminal amino acid residues of CD55 did not protect the cells against complement-mediated lysis by either normal or clusterin-depleted human serum. These data suggest that clusterin may not be a physiologically relevant regulator of complement activation.     

A kinetic model for the quantitative analysis of complement

A simple model has been developed which accurately predicts the time course of complement mediated lysis of sensitized red cells. The model assumes that the one hit theory of immune hemolysis is applicable and that the rate of lysis is directly proportional to the concentration of a complement component present in rate limiting amounts. It also assumes that the rate of lysis is dependent on the fraction of cells lysed. The model can be related to the classical von Krogh equation for end point complement analyses and can be used to estimate the rate constant for the critical step in hemolysis, as well as the efficiency of the critical complement component in the rate limiting step. Parameters derived from the model can be quantitatively related to complement concentration and can be used as the basis for a quantitative assay of complement activity. The model can also be used to calculate, for a particular sample, the concentration at which complement activity becomes undectable, the complement activity of the pure, undiluted sample, and the time required for the sample to produce complete lysis of the available cells.     

Lysis of leukemia cells with a monoclonal antibody-cocktail (e.g. VIP-pool) and human complement

During the lysis of leukemic cells with a monoclonal antibody cocktail (the so-called VIB pool) and complement the attempt was made to replace rabbit serum as a complement source by human serum. For identifying the lysis of leukemic cells the complement-dependent in vitro cytotoxicity test was used and for excluding stem cell toxicity the CFU-c test according to PIKE and ROBINSON. In combination with the applied monoclonal antibody pool against B and c-ALL the human complement could be shown to be suitable to produce a lysis in the same manner as rabbit complement. Similarly to the pretested rabbit serum the treatment with the human complement had no impact on stem cell recovery. An optimal cytotoxic activity (95% against ALL blasts of patients, 100% against NALM) could be identified up to an antibody dilution of 1:32 with a volume percentage of 50% of human complement, an incubation temperature of at least 37 degrees C and an incubation time of 30 mins. With proved high reactivity against leukemic cells and lacking impairment of the haemopoietic power of the bone-marrow, this method can be recommended for "purging" protocol with the possibility of using human serum as a source of complement having advantages as far as clinical application is concerned.     

Effects of a Quaternary Ammonium Compound on Escherichia coli

Increasing amounts of tetradecyldimethylbenzyl ammonium chloride (TAC) were lethal to an increasing proportion of an actively growing culture of Escherichia coli. The loss of nucleic acid material by actively growing E. coli did not appear to play a major role in the lethal effect. It was found that lag-phase cells were more sensitive than logarithmic-phase cells to the lethal effect of TAC. The effect of TAC on the lysozyme sensitivity of the test organism was compared with that obtained using disodium dihydrogen ethylenediaminetetraacetate (EDTA). Although TAC was found to render the test organism susceptible to lysozyme, the degree of lysis never reached that attained with EDTA.     

Avidin-induced lysis of biotinylated erythrocytes by homologous complement via the alternative pathway depends on avidin's ability of multipoint binding with biotinylated membrane.

It was reported that avidin and streptavidin induce lysis of prebiotinylated red blood cells via the alternative pathway of both homologous and heterologous complement. Both of these proteins have four biotin-binding sites, providing a polyvalent interaction with biotinylated components of the erythrocyte membrane. We have compared the effects of mono- and multipoint avidin attachment on the sensitivity of biotinylated erythrocytes to lysis by the complement system. In the presence of anti-avidin antibody, avidin-bearing biotinylated erythrocytes were rapidly lysed by heterologous serum. This lysis was independent from the mode of avidin attachment, implying that complement activation by the classical pathway triggered by interaction between C1 and avidin-bound antibody on the erythrocyte surface is independent from the avidin's ability of polyvalent (multipoint) binding with biotinylated membrane components. In the absence of anti-avidin antibody, biotinylated erythrocytes bearing polyvalently attached avidin were lysed by homologous complement better than cells bearing avidin, which possesses reduced ability for multipoint binding with biotinylated erythrocyte. Two independent approaches to reduce avidin's ability of multipoint binding were used: decrease in surface density of biotin on the erythrocyte membrane and blockage of biotin-binding sites of avidin. Both methods result in reduced lysis of avidin-bearing erythrocytes as compared with erythrocytes bearing an equal amount of polyvalent-bound avidin. Thus the activation of homologous complement via the alternative pathway depends on avidin's ability to 'cross-link' to the biotinylated components of the erythrocyte membrane.     

The modifications of the final stages of the complement reaction by alkali metal cations.

We studied the effects of alkali metal cations on the terminal stages of complement lysis of human and sheep HK erythrocytes. Sensitized erythrocytes (EA) were reacted with limited amounts of complement for 1 hr at 37 degrees C in buffer containing 147 mM NaCl (Na buffer), which resulted in 10-40% lysis. The unlysed cells were washed with Na buffer at 0-2 degrees C and incubated for 1 hr at 37 degrees C in buffers containing 147 mM of the various alkali metal cations. Although additional lysis (25 to 65%) occurred with K, Rb, or Cs buffer, only minor degrees developed with Na or Li buffer, only minor degrees developed with Na or Li buffer. Intermediate levels occurred with 100 mM of the divalent alkali cations. Halogen ions and SCN-(147 MM), Ca++ (0.15mM), and Mg++ (0.5 mM) did not alter the effect of the alkali metal cations. Lysis occurring in K+, Rb+ or Cs+ proceeded without lag, was temperature dependent with an optimum of 43 degrees C, and had a pH optimum of 6.5. Lysis in K and Na buffers was unaffected by 10(-3) to 10(-5) M ouabain. Experiments with mixtures of cations indicated that Na+ had a mild inhibitory effect that could be totally overcome by K+, partially by Rb+, and not at all by Cs+. Li+ had a strong inhibitory effect, 6 X 10(-5) M causing 50% inhibition in buffers containing 147 mM K+, Rb+, or Cs+. By using intermediate complexes of EA and purified complement components we demonstrated that K+ enhances the lytic action of C8 on EAC1-7 as well as that of C9 on EAC1-8. It was known that Li+ facilitates lysis when acting on the entire complement reaction. We found that Li+ enhanced the lytic action of C8 on EAC1-7, with a kinetic that differed from that of the K+ effect. In addition, Li+ inhibited the enhancing effect of K+ upon lysis of EAC1-8 by C9. This occurred at concentration of Li+ similar to those which inhibited the additional lysis by K+, Rb+, and Cs+ of cells that were pretreated in Na buffer with the entire complement sequence. We propose that the major effects of alkali metal cations on complement lysis are due to their interaction with C8 and/or membrane constitutes.     

Chemiluminescent determination of total antioxidant capacity during winemaking.

The total antioxidant capacity (TAC) of five different wines (four red and one white) was determined in five different steps of winemaking carried out in a commercial wine cellar by a chemiluminescence (CL) assay. The CL method is suitable to determine the antioxidant capacity of beverages, and preliminary trials showed that the TAC immediately after the bottle was opened was greater than the day after (about 25% decrease). Immediate analysis or a correct sample storage is therefore necessary. The wines were characterized by different levels of total phenolics and TAC: these differences were related to grape composition and winemaking technologies. The TAC values were the highest immediately after devatting. The TAC suffered the highest decrease (30-50%) after the clarification procedure, which may be due to the fining agents used and to oxygen contact, then remained more or less constant in the subsequent steps.     

Murine trophoblast resists cell-mediated lysis. II. Resistance to natural cell-mediated cytotoxicity

The susceptibility of murine trophoblast cells to natural cell-mediated cytotoxicity has been assessed. Primary short-term cultures of murine trophoblast cells isolated from 14-day placentas were found to be resistant to endogenous and interferon-activated natural killer (NK) cells and natural cytotoxic cells. That the relevant target structures are expressed on the surface of trophoblast cells and accessible to the effectors was demonstrated by their ability to inhibit the lysis of NK-sensitive target cells (YAC-1) in a dose-dependent manner. The lytic resistance of trophoblast cells was unaffected by neuraminidase treatment, inhibition of protein synthesis, or extending the assay time to 12 hr. Moreover, trophoblast cells were resistant to antibody-dependent cell-mediated cytotoxicity when coated with an alloantibody capable of mediating their lysis in the presence of heterologous complement. Neither the preincubation of effector cells in concentrated trophoblast culture supernatants nor the direct exposure of effectors to monolayers of trophoblast cells inhibited their NK lytic activity, indicating that the secretion of a suppressive factor or the direct inactivation of the NK cells was not responsible for the observed resistance to lysis. These observations, together with previous results showing the resistance of trophoblast to cytotoxic T cell-mediated lysis, reveal that murine trophoblast cells possess a resistance mechanism against several forms of cell-mediated lysis. This feature of trophoblast cells at the maternal-fetal interface is likely to play an important role in protecting the fetoplacental allograft from immune rejection.     

Identification of RNA as a complement inhibitory component in an extract of Ehrlich ascites tumor cells.

A factor capable of inhibiting complement was obtained from intact Ehrlich ascites tumor cells by mild extraction with phosphate-buffered saline (PBS). The inhibitor caused a decrease in extent of lysis of EAC14 with a concomitant extension of Tmax. EA, EAC1, EAC4 and EAC142 were all less susceptible to complement-mediated lysis after treatment with the tumor cell extract. Partial purification of a complement inhibitor was accomplished. The inhibitor was rich in RNA and its activity was totally destroyed by RNAase but not DNAase. RNA from mouse tissues, yeast, and Escherichia coli also inhibited complement hemolytic activity. The partially purified material only inhibited lysis of EAC1 and EAC14. Slow inhibition of fluid phase C1 was also demonstrated. In addition, RNA-rich partially purified tumor cell extract was capable of precipitating with purified human C1q.     

Suppression of measles virus expression by noncytolytic antibody in an immortalized macrophage cell line.

Immune regulation of measles virus (MV) expression was studied in a persistently infected mouse macrophage cell line. Synthesis of both membrane-associated and internal MV antigens was suppressed when infected macrophages were treated with polyclonal rabbit anti-MV antibody that was specific for MV proteins. Persistently infected macrophages were treated for 3, 5, or 7 days with increasing doses of anti-MV antibody. All MV proteins were down-regulated 2 days after treatment was terminated. One week after treatment was terminated, down-regulation was still evident but to a lesser degree. MV protein synthesis was suppressed whether or not complement components were inactivated by heating all serum supplements and antibodies. However, when complement was active, cell lysis accounted for some of the reduced MV protein synthesis. When lytic destruction of infected cells by antibody and complement was prevented by inactivation of complement, antibody alone reduced the cellular synthesis of viral proteins by noncytolytic mechanisms. The absence of cell death in the absence of complement was confirmed by the lack of 51Cr release from labeled cells, the lack of reduction in cell number, and the lack of a decrease in total protein synthesis when radiolabeled infected cells were treated with antibody. It is noteworthy that low doses of antibody were optimal for suppression in the longer-term experiments and did not cause lysis, even in the presence of active complement. Since infected macrophages disseminate virus in measles infection, noncytolytic regulation of these cells by antibody may supplement viral clearance by cytolytic T cells and other immune mechanisms.     

Targeted receptor trafficking affects the efficiency of retrovirus transduction

We describe the development of an experimental system to test the hypothesis that the efficiency of retrovirus transduction is dependent on the pathway of virus entry into the host cell and the intracellular trafficking itinerary of the cellular receptor with which it interacts. The experimental system consists of three model target cell lines, derived from HeLa cells, that stably express one of three interleukin-2 receptor alpha chain (CD25) chimeras, TAC, TAC-CD16, and TAC-DKQTLL, which have identical extracellular domains but different intracellular trafficking itineraries, and a targeted amphotropic murine leukemia retrovirus whose envelope proteins were modified to include a binding site for TAC at their N-termini. We found that the efficiency of retrovirus transduction was affected by the distribution and trafficking itinerary of the TAC receptors. Transduction of cells that expressed TAC-DKQTLL was nearly 4-fold lower than transduction of control cells that did not express any of the TAC receptors. In contrast, transduction of cells that expressed TAC was 1.6-fold higher than transduction of control cells, whereas transduction was not significantly affected by the expression of TAC-CD16. Our results suggest that in the course of designing a targeted retrovirus it may be prudent to target only those receptors that internalize retroviruses via pathways that most efficiently support post-binding steps of infection.     

Relationship between trinitrophenyl and H-2 antigens on trinitrophenyl-modified spleen cells. I. H-2 antigens on cells treated with trinitrobenzene sulfonic acid are derivatized.

Spleen cells were treated with TNBS in order to determine if cell surface H-2 antigens are derivatized with TNP. By labeling the cell membrane of the TNP-modified cells with 125I, followed by detergent lysis and immune precipitation with anti-TNP, it was determined that no H-2 antigenic activity remained in the supernatant. Further, by the use of an antibody-induced antigen redistribution assay it was found that previous exposure to TNP-modified cells to anti-TNP in the absence of complement rendered these cells resistant to lysis by anti-H-2 in the presence of complement. Together these data indicate that at the concentration of TNBS used for modification, H-2 antigens are derivatized with TNP. However, in addition to H-2, other proteins including immunoglobulin were also derivatized with TNP. Anti-TNP cytotoxic effector cells were blocked from their cytotoxic activity by anti-TNP antiserum. These data indicate that TNP directly couples to H-2 antigens on the cell surface of TNP-modified cells and that TNP is associated with the antigenic determinant that the cytotoxic T cell recognizes.     

Complement receptor type three-dependent degradation of opsonized erythrocytes by mouse macrophages

The role of the complement receptor type 3 (CR3) on thioglycollate-elicited peritoneal macrophages (TG-PM) in the destruction of opsonized particles was studied. We found that sheep red blood cells (E) that were opsonized with an IgM monoclonal anti-Forssman antibody and complement (E-IgM-C) were lysed by TG-PM, whereas there was little lysis of E pretreated with either the antibody or the complement source alone. Furthermore, this lysis could be inhibited by anti-CR3 monoclonal antibodies that had previously been shown to inhibit binding of E-IgM-C to the CR3. Kinetic studies of phagocytosis and lysis indicated that lysis of E-IgM-C occurs after phagocytosis, suggesting that lysis is an intracellular event. Further findings suggested that intra-cellular lysis was promoted by CR3 bound to the phagocytosed target, because a monoclonal anti-CR3 antibody decreased the rate of phagocytosis of E-IgM-C but not its magnitude, whereas the rate and extent of lysis were strikingly inhibited. Furthermore, TG-PM that had already internalized unopsonized E selectively lysed E-IgM-C that were added later. These data confirm that the interaction of the CR3 with its ligand on E-IgM-C promotes rapid phagocytosis, and further suggest that the CR3 facilitates degradation of the target particle once internalization has occurred.     

Use of some snake venoms in the diagnostics of paroxysmal nocturnal hemoglobinuria (PNH)

The crude toxin of Agkistrodon piscivorus was found to produce hemolysis of PNH erythrocytes by way of complement activation, however the degree of PNH blood cell lysis was lower than in other techniques used. The crude toxins of Naja naja and Naja oxiana cause much higher hemolysis though lower hemolysis takes place in normal blood cells. In some normal persons and different blood diseases it is responsible for high hemolysis even when the complement is absent. From Naja naja toxin a fraction can be isolated which has a "specific" complement effect only on PNH blood cells. This fraction can thus be utilized in a specific test for this disease.