The effect of some epsilon-aminocaproic acid derivatives on platelet responses

epsilon-Aminocaproic acid (EACA) is a synthetic low molecular drug with antifibrinolytic activity. However, treatment with this drug can be incidentally associated with an increased thrombotic tendency. The aim of the present work was to test synthetic EACA derivatives for their antiplatelet activities. We investigated the effect of three EACA derivatives with antifibrinolytic activity: I. epsilon-aminocaproyl-L-leucine hydrochloride (HClH-EACA-L-Leu-OH), II. epsilon-aminocaproyl-L-(S-benzyl)-cysteine hydrochloride (HClH-EACA-L-Cys(S-Bzl)-OH) and III. epsilon-aminocaproyl-L-norleucine (H-EACA-L-Nle-OH) on platelet responses (aggregation and adhesion) and on their integrity. It was found that: 1. as judged by LDH release test, none of the tested compounds, up to 20 mM, was toxic to platelets, 2. in comparison with EACA, all the synthetic derivatives inhibited much stronger the ADP- and collagen-induced aggregation of platelets suspended in plasma (platelet rich plasma) and aggregation of these cells in whole blood, 3. EACA and its derivatives exerted a similar inhibitory effect on the thrombin-induced adhesion of platelets to fibrinogen-coated surfaces. Since platelet activation and blood coagulation are tightly associated processes, the antiplatelet properties of EACA derivatives are expected to indicate reduced thrombotic properties of these derivatives compared to EACA.     

Inhibition of the development of the cellular slime mould Dictyostelium discoideum by omega-aminocarboxylic acids.

Four omega-aminocarboxylic acids - epsilon-aminocaproic acid (EACA), trans-4-aminomethylcyclohexane-1-carboxylic acid (t-AMCHA), p-aminomethylbenzoic acid (PAMBA) and omega-aminocaprylic acid (OACA) -- prevented fruiting body formation of the cellular slime mould Dictyostelium discoideum. At concentrations of 40 mM, 75 mM, 10 mM and 5 mM, respectively, they allowed aggregation but prevented all further development at 24 degrees C. At lower concentrations, EACA allowed fruiting body formation but with a reduced number of spores per fruiting body. Only t-AMCHA had a significant inhibitory effect on the growth of myxamoebae. EACA affected development only if it was present between 8 and 16 h after the cells were deposited on the filters. Its effect was enhanced by high salt concentrations and by higher temperature, and was also dependent on the manner in which the cells were grown. Only strains capable of axenic growth displayed this sensitivity to EACA, although strains carrying only one of the genetic markers for axenic growth (axe A) were partially sensitive.     

Effectors of the activation of human [Glu1]plasminogen by human tissue plasminogen activator

The activation of human [Glu1]plasminogen [( Glu1]Pg) by human recombinant (rec) two-chain tissue plasminogen activator (t-PA) is inhibited by Cl-, at physiological concentrations, and stimulated by epsilon-aminocaproic acid (EACA), as well as fibrin(ogen). Chloride functions as a result of its binding to [Glu1]Pg, with a Ki of approximately 9.0 mM, thereby rendering [Glu1]Pg a less effective substrate for two-chain rec-t-PA. EACA stimulates the activation in Cl-(-)containing solutions, with a Ka of approximately 4.0 mM, primarily by reversal of the Cl-(-)inhibitory effect. Fibrinogen appears to exert its stimulatory properties mainly through effects on the enzyme, two-chain rec-t-PA, with a Ka of approximately 3.7 microM in activation systems containing physiological levels of Cl-. Analysis of the results of this paper reveals that normal plasma components, Cl- and fibrinogen, exert major regulatory roles on the ability of [Glu1]Pg to be activated by two-chain rec-t-PA, in in vitro systems. The presence of Cl- inhibits the stimulation of [Glu1]Pg activation that would normally occur in the presence of fibrinogen, a result of possible importance to the observation that some degree of systemic fibrinogenolysis accompanies therapeutic use of tissue plasminogen activator.     

A monoclonal antibody to the epsilon-aminocaproic acid binding site on the kringle 4 region of human plasminogen that accelerates the activation of Glu1-plasminogen by urokinase

A monoclonal antibody, 10-F-1, previously shown [V. A. Ploplis, H. S. Cummings, and F. J. Castellino (1982) Biochemistry 21, 5891-5897] to interact with a particular epsilon-aminocaproic acid (EACA)3 binding site on the kringle 4 (K4) region of human Glu1-plasminogen (Glu1-Pg), has been employed to assess the contribution of this particular EACA site toward the enhancement, by EACA and its analogs, of the urokinase (UK)-catalyzed activation of Glu1-Pg. As is the case with EACA-like compounds, the presence of antibody 10-F-1 accelerates the activation of Glu1-Pg by UK, but does not enhance the similar activation of Lys77-plasminogen. In the presence of concentrations of antibody 10-F-1 which saturate its binding site on Glu1-Pg, the Km of Glu1-Pg activation by UK is raised from 1.4 +/- 0.2 microM, a value obtained in the absence of antibody, to 17.0 +/- 2.0 microM. On the other hand, the kcat for this activation, 0.038 +/- 0.005 s-1, is elevated to 2.45 +/- 0.2 s-1 at saturating concentrations of antibody 10-F-1. The kcat/Km for activation under these conditions is 0.027 s-1 microM-1 in the absence of antibody, and 0.144 s-1 microM-1 in the presence of saturating levels of antibody 10-F-1. This demonstrates that the interaction of this antibody with its epitope results in a fivefold stimulation of the activation rate of Glu1-Pg by UK. The availability of antibody 10-F-1 allows for a specific means of probing the function of one of the four to five thermodynamically equivalent weak EACA sites on human plasminogen. From this particular study, it is concluded that the weak binding site for EACA on the K4 domain of Glu1-Pg is either in-part or in-whole responsible for the enhancing effect of EACA on human Glu1-Pg activation by UK.     

Isolation of three separate anaphylatoxins from complement-activated human serum

Summary Recent methodologies used in preparing anaphylatoxins from complement-activated serum are described. Activation of the alternative pathway generates C3a and C5a; however, activation of the classical pathway is required to generate the anaphylatoxin from C4. This article describes an activation scheme that simultaneously generates all three of the anaphylatoxins (e.g., C3a, C4a and C5a) in human serum and outlines a procedure for isolating each as homogeneous products. Purification of intact anaphylatoxins directly from complement-activated serum takes place only if an exopeptidase in serum, known as carboxypeptidase N (SCPN), is properly inhibited. A new series of mercapto derivatives of arginine analogs are introduced as potent and effective inhibitors of SCPN. These inhibitors permit normal complement activation but prevent degradation of the released activation fragments C3a, C4a or C5a.The SCPN inhibitor previously used was 6-aminohexanoic acid (EACA), but it required a 1 M concentration for effective inhibition, the substituted mercapto-guanido compounds prove to be effective in the mM range.     

Human IgA1 initiates complement-mediated killing of Neisseria meningitidis

We studied the effect of human IgA1, the predominant IgA subclass in serum, on C-mediated killing of Neisseria meningitidis. We purified monomeric IgA1 from normal human serum and tetravalent meningococcal polysaccharide vaccinate serum by using the following successive chromatographic steps: jacalin lectin affinity, Superose 12 FPLC gel filtration, Mono Q FPLC anion exchange, and anti-IgG affinity. SDS-PAGE, ELISA, and Western immunoblot analyses of the IgA1 detected no trace of contaminating IgG or IgM. IgA1 initiated partial or complete lysis (62 to 100%) of nine group C strains by using either normal, hypogammaglobulinemic, factor B-depleted, or properdin-deficient human serum as a C source, but IgA1 was unable to effect killing in serum chelated with 10 mM MgCl2 and 10 mM EGTA. Lytic activity was dependent on the group C strain and the source of the IgA1; neither IgA1 preparation was bactericidal for all nine strains. Removal of the Fc portion of IgA1 with pepsin completely abolished bactericidal activity. We purified and radiolabeled C component C3, and found that IgA1 did not increase C3 deposition. With the use of a group C polysaccharide ELISA, we found that the vaccinate IgA1 had a high titer of group C polysaccharide antibody, whereas the IgA1 purified from normal human serum had no detectable group C polysaccharide specificity. Absorption of the vaccinate IgA1 with alum-bound group C polysaccharide did not affect the killing of a sensitive strain, but it did potentiate the killing of a previously resistant strain. Western immunoblots of whole cell lysates, outer membrane complex, and purified lipooligosaccharide showed that the bactericidal IgA1 was specific for several outer membrane proteins. Four of the proteins recognized by both IgA1 preparations had apparent Mr of 29, 42, 66, and 74 kDa. We conclude that IgA1, when bound to specific outer membrane proteins, can initiate lysis of group C meningococci via the classical C pathway, and that initiation of lysis is an Fc-dependent event which occurs without an increase in C3 deposition.     

Construction, expression, and purification of recombinant kringle 1 of human plasminogen and analysis of its interaction with omega-amino acids

An Escherichia coli expression vector, containing the alkaline phosphatase promoter and the stII heat-stable enterotoxin signal sequence, along with the cDNA of the kringle 1 (K1) region of human plasminogen (HPg), has been employed to express into the periplasmic space amino acid residues 82-163 (E163----D) of HPg. This region of the molecule contains the entire K1 domain (residues C84-C162) of HPg, as well as two non-kringle amino-terminal amino acids (S82-E83) that are present in their normal locations in HPg and a carboxyl-terminal amino acid, D163, that results from mutation of the E163, normally present at this location in the HPg amino acid sequence. After purification of r-K1 by chromatographic techniques, we have investigated its omega-amino acid binding properties by titration calorimetry, intrinsic fluorescence, and differential scanning microcalorimetry (DSC). The antifibrinolytic agent, epsilon-aminocaproic acid (EACA), possesses a single binding site for r-K1. The thermodynamic properties of this interaction, studied by calorimetric titrations of the heats of binding with this ligand, reveal a Kd of 12 +/- 2 microM at 25 degrees C and pH 7.4, a corresponding delta G of -6.7 +/- 0.1 kcal/mol, a delta H of -3.6 +/- 0.1 kcal/mol, and a delta S of 10.5 +/- 0.8 eu. The intrinsic fluorescence of r-K1 decreases by approximately 44% when its binding site is saturated with EACA, and titrations of this perturbation with EACA lead to calculation of a Kd of approximately 13 microM, a value in good agreement with that obtained from titration calorimetric analysis. EACA represents the strongest binding ligand of a variety of simple aliphatic omega-amino acids examined. A cyclic analogue of EACA, trans-4-(aminomethyl)cyclohexanecarboxylic acid, interacts with r-K1 with an approximate 12-fold tighter Kd (1.0 +/- 0.2 microM). Investigations by DSC, at pH 7.4, demonstrate that a significant stabilization of the r-K1 structure occurs when EACA binds to this domain. The temperature of maximum heat capacity change (Tm) in the thermal denaturation of r-K1 increases from approximately 340.8 to 359.1 K as a consequence of EACA binding. These studies demonstrate that a fully functional EACA-binding kringle from HPg can be expressed and secreted in E. coli, purified by techniques that do not require refolding, and investigated as an independent structural unit.     

Functional and biochemical properties of the early classical complement system of mice

Mouse serum and EDTA plasma were subjected to low ionicity precipitation, gel filtration, and ion exchange chromatography in an attempt to purify C1, C4, and C2 to functional and chemical homogeneity. In marked contrast to human and guinea pig components, those of the mouse could not be separated by these techniques. Except for partial separation of C1 from C4 and C2 on DE-52 cellulose columuns with EDTA in the eluting buffers, there was no separation of those three components on ion exchange chromatographic columns. Sephadex G-200 gel filtration columns, or with precipitation of euglobulins from serum or plasma. Generation of EAC142 by incubation of EA in whole serum followed first order kinetics when mouse serum was used and second (or greater) order kinetics when human or guinea pig sera were used. Generation of EAC142 by incubation of EA in whole mouse serum followed by incubation in EDTA containing buffers resulted in rapid loss of all three activities from the cell. These experiments indicated that there were significant differences between the early classical C system of mice and those of human and guinea pig. In addition, they indicated that under a variety of in vitro conditions, murine C1, C4, and C2 behaved biochemically and functionally as a unit. The reasons for the major differences in behavior of the murine C components with not become clear until methods to stabilize their function are found so that they can survive multiple purification steps.     

Requirement of serum pretreatment for induction of alkaline phosphatase activity with prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in human liver cells continuously cultured in serum-free medium

A cell line (HuL-1) derived from normal fetal human liver was adapted to grow continuously in a modified Eagle's minimum essential medium without serum or hormones. The population doubling time of this adapted cell line (HuL-1-317) was about 72 h and the modal number of chromosomes was 54. The morphology of HuL-1-317 cells was round in the absence of serum, but at 37 degrees C with the addition of serum (1-10%), the cells flattened. HuL-1-317 cells had a low level of alkaline phosphatase activity. However the enzyme activity was slightly enhanced by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and a hypertonic concentration of NaCl after 3 days of incubation at 37 degrees C. The increase in alkaline phosphatase activity with the four agents was further amplified dose-dependently by the pretreatment of the cells with serum. The stimulatory effect of the serum was evident at concentrations as low as 1%, and was maximal at 20%. The half life of the effect of serum on alkaline phosphatase induction was 48 h at 37 degrees C. Serum alone could not enhance the enzyme activity without the four agents. The present results indicate that serum contributes to the regulation of alkaline phosphatase induction by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in fetal human liver cells (HuL-1-317).     

Growth characteristics of human esophageal epithelial cells in primary explant and serial culture

Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm²) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to platic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium. Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared in Clin. Res. 30: 93A; 1982.     

The relative contribution of somatomedin to the serum-stimulated growth of human fibroblasts

Culture conditions were defined allowing to demonstrate a stimulatory effect of both serum-contained and purified Somatomedin activity on incorporation of [3H]thymidine and replication of cultured normal human fibroblasts. The use of dialyzed human serum in MEM medium supplemented by 0.2 mM serine offered the necessary and sufficient culture conditions. A significant difference between normal and hypopituitary patients sera was found in their effect on the rate of [3H]thymidine incorporation (p < 0.0001) and on cell replication (p < 0.01). Purified Somatomedin-C, in MEM without serum, is a poor mitogen. Its activity was strongly enhanced by the addition of 0.1 % dialyzed serum and 0.2 mM serine without, however, exceeding the stimulatory level of 1 % whole normal serum. The requirement of concomitant presence, for optimal $\text{in}\text{vitro}$ cell growth, of different low and high MW serum components is discussed.     

Complement-dependent induction of DNA synthesis and proliferation of human diploid fibroblasts

The effects of fresh human serum (FHS) and heat-inactivated human serum (HHS) on the DNA synthesis and proliferation of human diploid fibroblasts were assessed. FHS activated significantly more quiescent fibroblasts to undergo DNA synthesis and proliferation than did HHS. The stimulatory effect occurred consistently over a serum concentration range of 0.1–10%. Using bromodeoxyuridine selective killing techniques, it was shown that this FHS stimulatory effect was on a specific subpopulation of fibroblasts unresponsive to HHS. The involvement of the complement system, and specifically of C1, was shown by the inability of Clq-depleted FHS to support enhanced DNA synthesis whereas Clq-depleted serum reconstituted with purified Clq was effective. Purified Clq did not restore activity when added to heated serum, nor was it mitogenic when tested in basal medium without serum. The addition of purified Clq to fresh serum inhibited the enhancement of DNA synthesis, and at Clq concentrations of 4γ/ml and greater, the fresh serum effects were abrogated. Thus, it appears that binding of the assembled C1 complex to the fibroblast surface was required for FHS-mediated enhancement of fibroblast proliferation, with Clq subcomponent serving as the recognition site. The results from several experiments indicated that antibody was not required for the complement-dependent fibroblast activation. FHS was not cytotoxic, and autologous serum was as effective as allogeneic sera. A 20-fold molar excess of Fab' from pooled human IgG did not alter the FHS effects. FHS from which IgG was more than 99% depleted was still effective. These results suggested an antibody-independent role for complement in the activation of a subpopulation of human diploid fibroblasts.     

Human erythrocytes inhibit complement-mediated solubilization of immune complexes

Incubation of precipitable immune complexes (IC) with fresh human serum or guinea pig serum resulted in solubilization of IC. When packed human E were added to human serum or guinea pig serum, binding of IC to the E occurred and IC solubilization was significantly inhibited. By contrast, SRBC did not bind IC nor inhibit IC solubilization. Because IC binding to human E is mediated by CR type 1 (CR1) we evaluated whether CR1 was responsible for the inhibition of IC solubilization. Human E were treated with trypsin or anti-CR1 mAb. Both treatments abrogated IC binding to human E but did not affect the ability of the human E to inhibit IC solubilization. Human E inhibited C activation by IC. Thus, incubation of IC in human serum caused significant activation of C3 and C5, but not C4. However, when IC were incubated in whole blood or with isolated human E and serum, C3 activation by IC was inhibited significantly. In addition, we demonstrated that the C3b generated during C activation by IC deposited on both IC and human E. Thus, human E may compete for nascent C3 generated during C activation by IC. In conclusion, human E inhibit both complement-mediated solubilization of IC and C activation by IC.     

The effect of proteases on endothelial cell migration in vitro

This communication reports on the role of proteases in the migration of endothelial cells in vitro. Endothelial cell (EC) migration was assayed by wounding confluent monolayers of bovine aortic endothelial cells with a razor blade and counting the number of cells crossing the wound per unit time. Treatment with mitomycin C inhibited wound-induced proliferation of endothelial cells without affecting migration, indicating that in this assay migration could be measured independent of proliferation. Migration of endothelial cells in vitro in 10% serum was not affected by depletion of plasminogen, which inhibited plasmin production, or by various protease inhibitors: soybean trypsin inhibitor, Trasylol, E-amino caproic acid (EACA), ovalbumin, p-tpsyl-1-arginine-methyl ester (TAME), and benzamidine. However, migration and proliferation of endothelial cells in vitro was inhibited by acid-treated serum, a procedure commonly used to inactivate protease inhibitors. Migration of bovine smooth muscle cells, 3T3 cells and SV40-3T3 cells was inhibited by plasminogen-depleted serum; reconstitution with purified plasminogen reversed the depressed migration of only SV40-3T3. These results indicated that endothelial cell migration in vitro is not dependent on plasminogen, which may be another unique property of endothelial cells.     

The blockage of the high-affinity lysine binding sites of plasminogen by EACA significantly inhibits prourokinase-induced plasminogen activation

Prourokinase-induced plasminogen activation is complex and involves three distinct reactions: (1) plasminogen activation by the intrinsic activity of prourokinase; (2) prourokinase activation by plasmin; (3) plasminogen activation by urokinase. To further understand some of the mechanisms involved, the effects of epsilon-aminocaproic acid (EACA), a lysine analogue, on these reactions were studied. At a low range of concentrations (10-50 microM), EACA significantly inhibited prourokinase-induced (Glu-/Lys-) plasminogen activation, prourokinase activation by Lys-plasmin, and (Glu-/Lys-) plasminogen activation by urokinase. However, no inhibition of plasminogen activation by Ala158-prourokinase (a plasmin-resistant mutant) occurred. Therefore, the overall inhibition of EACA on prourokinase-induced plasminogen activation was mainly due to inhibition of reactions 2 and 3, by blocking the high-affinity lysine binding interaction between plasmin and prourokinase, as well as between plasminogen and urokinase. These findings were consistent with kinetic studies which suggested that binding of kringle 1-4 of plasmin to the N-terminal region of prourokinase significantly promotes prourokinase activation, and that binding of kringle 1-4 of plasminogen to the C-terminal lysine158 of urokinase significantly promotes plasminogen activation. In conclusion, EACA was found to inhibit, rather than promote, prourokinase-induced plasminogen activation due to its blocking of the high-affinity lysine binding sites on plasmin(ogen).     

Trypsin-activated complex of human factor B with cobra venom factor (CVF), cleaving C3 and C5 and generating a lytic factor for unsensitized guinea pig erythrocytes. I. Generation of the activated complex.

A complex formed between cobra venom factor (CVF) and isolated human factor B (B) was found to be converted by trypsin to a stable enzyme, CVF-B which cleaved the third component (C3) and the fifth component (C5) of human complement. The formation of CVF-B by trypsin required divalent cations, whereas the formation of the lytic factor from human serum occurred even in the presence of EDTA. CVF-B purified by gel filtration could initiate the hemolysis of unsensitized guinea pig erythrocytes when incubated with human complement components C5 to C9 in 0.01 M EDTA buffer. C3 was not required for the lysis of guinea pig erythrocytes initiated by CVF-B because of the beta1C precipitation line formed between human serum and anti-beta1C antibody did not inhibit the hemolysis by CVF-B in agarose gel. Treatment of beta1C and beta1F globulins in whole human serum with CVF-B in the presence of 0.01 M EDTA converted them to components with higher mobilities on immunoelectrophoresis.     

Development of an improved medium for germination of Cajanus cajan (L.) Millsp. pollen in vitro.

A simple, reliable medium for pollen germination of Cajanus cajan was developed by modifying Brewbaker and Kwack (BK) medium. Past attempts of C. cajan pollen germination in artificial media were not successful. A medium containing polyethylene glycol 4000 (PEG) showed more than 90% germination for C. cajan var. Pusa 33 only when the young buds (36 h before anthesis) were kept in pollen germination medium (PGM) for 36 h before pollen extraction. Supplementation of PGM with epsilon-amino caproic acid (EACA), an amino acid, showed improved pollen germination in Pusa 33 and also helped to avoid preconditioning of young buds before pollen extraction. It was also observed that there is a genotypic difference in the level of EACA required for in vitro pollen germination. Thus a complete medium for C. cajan genotypes consists of 37.5% sucrose+ 15% PEG 4000+250 mg l(-1) boric acid+300 mg l(-1) calcium nitrate+100 mg l(-1) potassium nitrate+ 200 mg l(-1) magnesium sulphate+1% agar+EACA (0, 100, 250, 500, 750 or 1000 mg l(-1)).     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Role of complement component C1q in the IgG-independent opsonophagocytosis of group B streptococcus.

We investigated the role of complement component C1q in the IgG-independent opsonophagocytosis of type III group B Streptococcus (GBS) by peripheral blood leukocytes. We report that C1q binds to type III GBS both in normal human serum deficient in IgG specific for type III capsular polysaccharide and in a low-ionic strength buffer. The dissociation constant Kd ranged from 2.0 to 5.5 nM, and the number of binding sites Bmax ranged from 630 to 1360 molecules of C1q per bacterium (CFU). An acapsular mutant strain of GBS bound C1q even better than the wild type, indicating that the polysaccharide capsule is not the receptor for C1q. In serum, binding of C1q to GBS was associated with activation of the classical complement pathway. However, normal human serum retained significant opsonic activity after complete depletion of C1q, suggesting that the serum contains a molecule that is able to replace C1q in opsonization and/or complement activation. Mannan-binding lectin, known to share some functions with C1q, appeared not to be involved, since its depletion from serum had little effect on opsonic activity. Excess soluble C1q or its collagen-like fragment inhibited phagocytosis mediated by normal human serum, suggesting that C1q may compete with other opsonins for binding to receptor(s) on phagocytes. We conclude that, although C1q binds directly to GBS, C1q binding is neither necessary nor sufficient for IgG-independent opsonophagocytosis. The results raise the possibility that additional unknown serum factor(s) may contribute to opsonization of GBS directly or via a novel mechanism of complement activation.     

The role of protease inhibitors and albumin on the membrane shedding of human erythrocytes.

Human erythrocytes incubated at 37° C in a physiological solution containing divalent cations but without glucose release up to 55% of the total sialoglycopeptides of the membrane within 60 hrs. The inhibition of the release by soybean trypsin inhibitor (SbTI), trasylol and epsilon amino-n-caproic acid (EACA), suggests that proteases bound to the membrane are probably responsible for the membrane shedding. Human albumin reduces significantly the membrane proteolysis.