Normal serum cytotoxicity for P32-labeled smooth Enterobacteriaceae. 3. Isolation of a gammag normal antibody and characterization of other serum factors causing P32 loss

Spitznagel, John K. (University of North Carolina School of Medicine, Chapel Hill). Normal serum cytotoxicity for P(32)-labeled smooth Enterobacteriaceae. III. Isolation of a gammaG normal antibody and characterization of other serum factors causing P(32) loss. J. Bacteriol. 91:401-408. 1966.-Gram-negative bacteria lost metabolically incorporated P(32) when suspended in serum only if the serum contained heat-labile in addition to heat-stable factors. Gram-positive bacteria labeled with P(32) and included for comparison lost P(32) in heat-inactivated as well as in fresh normal serum. Further investigation of gram-negative bacteria showed that a smooth Escherichia coli (O117:H27) lost P(32) only if suspended in serum containing complement fractions C'1, C'2, C'3, and C'4 "normal" antibody and lysozyme. The normal antibody was recovered by absorption on and subsequent elution from E. coli O117:H27 cell walls. Immunoelectrophoresis showed that it was a gammaG-globulin. Its P(32)-releasing activity was destroyed by 2-mercaptoethanol. Lysozyme was found to potentiate the P(32)-releasing action of normal antibody plus complement. Evidence was obtained suggesting that beta(1C) globulin was the component absorbed to zymosan during serum absorption at 15 C. Reduction of the beta(1C) level evidently upsets an important balance that exists in normal serum among complement, antibody, and lysozyme. This balance is essential for maximal P(32) release from labeled bacteria, or possibly for a maximal antibacterial effect from normal serum. The possible relationships of bactericidal, bacteriolytic, and opsonic action of normal serum are discussed.     

The significance of regression line slope changes in the quantitative titration of complement components

The hypothesis is discussed that dissociable complexes are formed between elements of a test complement (C') and the reagent used to titrate it for a particular component. Calculations are presented which demonstrate that such complex formation may give considerable changes in slopes of percentage hemolysis-log dose regression lines obtained with mixtures of C' and reagent. It is shown that marked change of slope may occur with relatively little change in the value of the 50 per cent intercept.     

Partial characterization of a Trypanosoma cruzi-released decomplementing factor

Trypanosoma cruzi releases a factor (SCAF) when grown in vitro which decomplements normal mouse, human, and guinea pig sera. The production and potency of SCAF was dependent on the density of cultured parasites, parasite viability and proliferative capacity, and duration of culture. The in vitro interaction between SCAF and serum complement (C') occurred rapidly and was complete within 30 min of mixing. The administration of SCAF to normal mice resulted in up to 50% reduction in hemolytic C' activity, whereas SCAF had no effect on the C' levels in mice infected wit T. cruzi for more than 10 days. The active moiety of SCAF was shown to be a nonproteinaceous substance(s) with a molecular weight of approximately 23,000 daltons.     

Analysis of sequential stages in serum bactericidal reactions

Michael, J. Gabriel (House of the Good Samaritan, Children's Hospital Medical Center, Boston, Mass.), and Werner Braun. Analysis of sequential stages in serum bactericidal reaction. J. Bacteriol. 87:1067-1072. 1964.-The bactericidal reaction of "normal" human serum against Escherichia coli was found to be separable into two distinctive stages. The early (first) stage of the reaction lasts for a relatively short period of time, and involves factors that are present in sufficient amounts only in slightly diluted serum. The later (second) stage needs more time and requires factors present in highly diluted serum. The first stage depends on the presence of Ca(++) and Mg(++) and on the activity of all components of complement; the second stage does not require divalent cations and C'1, C'2, and C'4, but requires factors that can be removed by zymosan. Under our conditions, removal of lysozyme did not influence either stage of the reaction. Bacteria exposed to concentrated serum for a short time, during the first stage, are essentially unaffected as far as their potential for subsequent multiplication is concerned; the actual damage to cellular integrity occurs only during the second stage of the reaction. In the absence of cell division, the "sensitization" produced during the first stage can be preserved for prolonged periods, and the bactericidal reaction can be completed later by exposure to antibody-free, highly diluted serum (second stage). Cell multiplication abolishes the sensitizing effects of the first stage.     

Influence of Complement on the Neutralization of Murine Cytomegalovirus by Rabbit Antibody

Antiserum against murine cytomegalovirus produced in the rabbit contained complement (C')-requiring neutralizing (CRN) antibody. The proportion of CRN was extremely high (up to 98%) during the early portion of an immunization procedure, whereas the antisera produced late had a much lower proportion that required C'. The antiserum produced was specific for MCMV with or without C'.     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.     

IgG antibodies to asialoGM1 are more sensitive than IgM antibodies to kill in vivo natural killer cells and prematured cytotoxic T lymphocytes of mouse spleen

IgG and IgM antibodies, which were isolated from the anti-asialoGM1 (GA1) serum, had different effects against natural killer (NK) and prematured cytotoxic T cells (CTLs) in in vivo administration and in in vitro treatment. In in vitro treatments, the IgM antibody killed NK cells of nude mouse spleen in the presence of complement (C') 12 times more potently than the IgG antibody did, and either antibody with C' killed pre-CTL. In in vivo administrations, only the IgG antibody was effective in diminishing NK activity of the nude mouse spleen cells and in suppressing antigen-specific CTL induction from primed spleen cells by in vitro stimulation with X-irradiated tumor cells. The IgM antibody was not effective at all in either system. The in vivo effect of the IgG on NK activity was blocked by preadministration with silica or carrageenan but not by that with cobra venom factor (CVF). These results indicate that in vivo administration of anti-GA1 antiserum leads to macrophage-mediated depletion of CTL precursors as well as NK cells.     

Complement reversal of immunosuppression induced with plasma of rats infected with Trypanosoma brucei rhodesiense

Suppression of antibody production by splenic lymphocytes from rats immunized with sheep red blood cells (SRBC) after incubation with plasma from rats infected with Trypanosoma brucei rhodesiense was confirmed. Suppressive activity became evident in plasma after the sixth day of infection and was manifested by reduction in the number of hemolytic Jerne plaques produced by the treated cells. The activity was temporally associated with increased amounts of soluble immune complex (SIC) reduced titers of lytic complement, elevated titers of immunoconglutinin (IK) and anemia. Treatment of suppressive plasma with hemolysin sensitized SRBC alexinated with horse complement to reduce IK did not reduce suppressive activity, and the activity appeared to have been enhanced when the plasma was heated to inactivate the remaining complement (C'). When fresh rat C' was added to the treated cells, the suppression was largely, though not completely, reversed. Treatment of spleen cells with SIC prepared in vitro from bovine serum albumin (BSA) and rabbit antiBSA also suppressed the plaque forming capacity of the cells. Complexes of BSA-antiBSA-C' and complexes of BSA-antiBSA-C'-IK were equally suppressive. Again, addition of fresh C' to cells treated with these complexes largely, though not completely, reversed the suppressive effect on the cells. From the results it is suggested that immunosuppression associated with experimental T. b. rhodesiense infection may be in part a suppression of the capacity of induced lymphocytes to produce antibody. It is possible that the suppression was mediated by SIC present in the plasma of the infected rats and this effect was probably enhanced by reduced levels of complement in the suppressive plasma.     

Diagnosis of bovine respiratory syncytial virus infection by complement fixation test.

The complement fixation test by the microtiter method was applied to the serological diagnosis of bovine respiratory syncytial (RS) virus infection. When used as complement fixing antigens, untreated infected cell culture fluid, fluorocarbon-treated, and ether-treated materials showed no differences in antigenicity among them. The complement fixing antigenicity of bovine RS virus appeared in bovine kidney and Vero cell cultures for the first time 4 days after inoculation. Both the infectivity and complement fixing antigenicity reached a maximum 6 days after inoculation. In detecting complement fixing antibody from infected cattle, the most outstanding specific reaction was obtained when 5% fresh normal calf serum had been added to the diluent of complement. Neutralizing and complement fixing antibodies were examined in serum samples from two cattle in the course of experimental infection. It was found that both antibodies turned to be positive 2 weeks after inoculation. There was a linear correlation between neutralizing and complement fixing antibody titers, when serum samples from 40 natural cases were tested in the acute and convalescent stages. In addition, common antigenicity was demonstrated between the virus of bovine origin and the Long strain of human RS virus by complement fixation test.     

C'3 polymorphism of human complement in North-East England.

The C'3 polymorphism of human complement was investigated in a sample of 268 unrelated individuals in North-East England. The two common genes were comparable in frequency with other European populations investigated so far. Two individuals were found to have rare variant C'3 types.     

Determination of the genetic polymorphism of the 3d complement component (C'3) by vertical polyacrylamide gel electrophoresis

A variant of the vertical polyacrylamide gel electrophoresis method, hitherto not applied in determination of C'3, is used. The method allows simultaneous determination of the C'3 phenotypes and other serum proteins in human blood. In this paper, the influence of aging of the serum samples and of the Tf polymorphism on the C'3 electrophoregram is discussed. The gene frequencies in Mongolian population (MPR) are: C'3S = 0.9605, C'3F = 0.0375, and the rare variants C'3Srare = 0.0013, C'3Frare = 0.0007; in Chukchi these are 1.0, 0.0, 0.0, respectively; in Eskimos - 0.9556, 0.0222, 0.0111; Evenki - 0.9359, 0.0641, 0.0; Yakuts - 0.9091, 0.0909, 0.0; Komi-Zyrians - 0.8507, 0.1493, 0.0; Komi-Permiyak - 0.8176, 0.1698, 0.0068.     

Evaluation of the usefulness for the latex agglutination test for serodiagnosis of Mycoplasma pneumoniae infections

The usefulness of latex agglutination test prepared in our laboratory for the diagnosis of M. pneumoniae infections was assessed. A total of 628 serum samples obtained from patients with respiratory tract infections were tested by complement fixation test and by latex test, from among them 274 serum samples were additionally tested by ELISA--Ig A/--IgG/--IgM and by immunoelectroprecipitation test. The highest sensitivity and specificity was displayed by the latex test in relation to ELISA when determining mycoplasmal antibodies of IgM class (respectively 82.1% and 89.6%) and to the complement fixation test (81.0% and 89.0%). Positive latex test results in our investigations were associated only with the presence of IgM antibodies and were not dependent on the IgA and IgG antibody classes. The latex agglutination test may be used in routine serodiagnosis of mycoplasmosis under condition that the results obtained in this test will be confirmed by the complement fixation test or ELISA.     

Leishmania amastigotes: resistance to complement-mediated lysis is not due to a failure to fix C3

Amastigote forms of Leishmania major are sensitive to lysis by fresh serum, whereas those of L. donovani are resistant. To understand the basis for this resistance we have examined the interaction of complement with amastigotes of seven strains of leishmania. Complement activation was determined by measuring the ability of amastigotes to consume complement from normal serum and by identifying parasite surface-bound C3. All of the strains that were tested activated complement, including both those that are resistant and those that are susceptible to inactivation by fresh serum. Complement consumption by amastigotes was measured as a decrease in the ACH 50 titers of serum exposed to parasites. L. major, L. donovani, and L. mexicana mexicana (strain 1VLM) amastigotes decrease titers by 35.7, 33.5, and 40.3%, respectively. The binding of C3 to amastigotes was judged qualitatively by immunofluorescence and quantitatively by a C3 radiobinding assay. L. major amastigotes bind an average of 6.6 X 10(4) molecules of C3 per parasite. L. mexicana amazonensis, L. mexicana mexicana, and L. donovani bind an average of 3.9 X 10(4), 5.9 X 10(4), and 3.7 X 10(4) molecules, respectively. In all cases, C3 binding is the result of alternative pathway activation requiring Mg++ but not Ca++. Amastigotes of the disseminating strains of leishmania represent the first example of a group of protozoa that activate early complement components leading to fixation of C3, but that are resistant to inactivation by complement.     

Antibodies elicited by defined oligodeoxyribonucleotide sequences.

Antibodies to the oligodeoxyribonucleotides d(pT)3, d(pT)4, d(pT)6 and d(pA-A-T-T) were elicited in rabbits by immunization with electrostatic complexes of the respective haptens with methylated bovine serum albumin (MBSA). The antisera were assayed by complement fixation using denatured DNA's of various sources as antigens. The specificities of the antibodies were determined by estimating the inhibition of the complement fixation reaction by defined oligodeoxyribonucleotides. The antibodies were shown to be specific for the sequence of the oligode-oxyribonucleotides or parts of it.     

Estimation and Identification of Streptococcus dysgalactiae Protective Antibody in Sera of Rabbits and Cattle

Rabbit and cow anti-Streptococcus dysgalactiae sera were tested by bacterial agglutination, complement fixation, hemagglutination, and immunodiffusion for the presence of antibody. The results of these tests were compared with mouse-protection studies on the same serum to estimate which in vitro test would best reflect the in vivo protective capacity of serum. Identification of the antibody constituents responsible for the mouse protection, hemagglutination, and complement fixation titers were established by reacting whole and diluted antisera with mercaptoethanol before and after testing. Results indicate that the complement fixation test may be a more accurate indicator of IgG protective bovine and rabbit antibody, whereas the hemagglutination test may more readily reflect a wider range of protective antibody levels and IgM. The complement fixation test showed some shared responses to IgG and IgM in both the rabbit and cow, whereas the IgM components seemed to be the predominant factor influencing hemagglutination titers in the rabbit and more so in the bovine. Mouse protection tests with mercaptoethanol-treated cow and rabbit sera indicate that the protective capacity of these antisera is shared between IgM and IgG components.     

Actomyosin content of Physarum plasmodia and detection of immunological cross-reactions with myosins from related species

The content of myosin in plasmodia of the myxomycete Physarum polycephalum was measured by an immunological technique, quantitative microcomplement (C') fixation. Migrating plasmodia (starved after growth on rolled oats) contained 0.60 +/- 0.08 (SD) mg myosin per g fresh plasmodia. Myosin comprised 0.77% +/- 0.05 (SD) of the total plasmodial protein. When total plasmodial proteins were separated by electrophoresis on SDS-polyacrylamide gels, a large amount of protein appeared in a band comigrating with muscle actin. Densitometry performed after Coomassie blue staining indicated that as much as 15- 25% of the total protein in the plasmodium could be actin. This gives an actin/myosin ratio by weight in the myxomycete plasmodium as high as 19-33, a very "actin-rich" actomyosin compared with rabbit skeletal muscle actomyosin with an actin/myosin ratio of 0.6. Starvation stimulates rapid migration and is correlated with a higher percent of both myosin and actin in the total protein of the plasmodium compared with normally growing cultures. Immunological cross-reaction of myosins from a variety of species was measured by C' fixation using an antiserum produced against purified native myosin from P. polycephalum. Although myxomycete and vertebrate striated muscle myosins have very similar morphological and biochemical properties, and apparently possess similar binding properties to F-actin, only myosins from myxomycetes in the order Physarales, rather closely related to P. polycephalum, gave detectable cross-reactions. This finding suggests that many amino acid sequences in myosin have been variable during evolution.     

MECHANISM OF HEMOLYSIS BY COMPLEMENT : I. COMPLEMENT FIXATION AS AN ESSENTIAL PRELIMINARY TO HEMOLYSIS.

1. Sensitization confers upon the red cell the property of adsorbing complement from solution. The submicroscopic film of immune serum protein deposited upon the cell surface during sensitization, and completely analogous to the precipitate formed in a soluble antigen-antibody reaction (e.g., sheep serum vs. rabbit anti-sheep serum) acts as absorbent, the degree of sensitization (size of the film) determining the amount of complement "fixed" (adsorbed). 2. This adsorption of complement by the sensitized cell is an essential preliminary to hemolysis, and when inhibited, even large quantities of demonstrably active complement have no hemolytic action. The marked influence of electrolytes and of the hydrogen ion concentration upon hemolysis is due primarily to corresponding effects upon the fixation of complement by the sensitized cell. In the case of salts with monovalent cations, complement fixation (and hemolysis) is completely inhibited at any concentration < 0.02 M or > 0.35 M. Electrolytes with bivalent cations are much more inhibitory, and in low as concentration 0.07 M completely prevent fixation (and hemolysis). The optimal reaction for complement fixation (and hemolysis) is pH 6.5 to 8.0. In slightly more acid range both are inhibited. But at a reaction pH 5.3, and in the alkaline range, there is an irreversible inactivation of complement, complete at pH 4.8 and 8.8 respectively. It is perhaps more than a coincidence that complement fixation, and therefore, hemolysis, are prevented by just those factors which suppress the ionization of serum proteins, and lead to an increased aggregation state. Between a suspension of macroscopically visible particles of euglobulin in distilled water, and a solution is physiological saline, there is certainly a gradual transition, manifested at low electrolyte concentrations by the opacity of the solution. At pH 7.4, globulin would ionize as a Na-salt, an ionization inhibited as the isoelectric point (5.3) is approached, with a coincident greater tendency of the globulin to separate from solution. And the cataphoretic velocity of particles of globulin, as well as all the other properties which are a function of its ionization (viscosity, osmotic pressure, etc.), are suppressed by electrolytes, the degree of suppression being determined by the concentration and valence of the cation (on the alkaline side of the isoelectric point). The analogy with complement fixation is too complete to be dismissed as fortuitous. 3. The fact that the degree of complement "fixation" increases with the degree of sensitization explains one of the most puzzling phenomena in hemolysis,—that immune serum and complement are, to a certain extent, interchangeable, a decrease in either factor being compensated by an increase in the other (8), (20), (22). The explanation is evident from Figs. 1,2, and 3. The exact quantitative relationships involved will be developed in a later paper. With increasing sensitization there is an enormously more complete and more rapid fixation of complement, and correspondingly more rapid hemolysis, exactly the effect produced by increasing the quantity of complement instead of amboceptor (Fig. 3). All other variables being constant, the velocity of hemolysis is determined by the amount of complement adsorbed. With more amboceptor, a greater proportion is "fixed" by the cell; with more complement, a smaller proportion, but a larger absolute amount. The result is the same: more complement adsorbed, and a corresponding acceleration of hemolysis. If this mobilization of complement is the sole function of immuneserum (and there is as yet no reason to assume any other), then the accepted terminology, in which amboceptor, immune body, and hemolysin are used synonymously, is erroneous. The immune body would function only as an "amboceptor," mobilizing the effective hemolysin, complement, upon the surface of the cell. Nothing has been said of the multiple components into which complement may be split. A priori, it would be expected that the adsorption demonstrated is of the so called midpiece fraction.     

Antibody Response of Animals to Mycoplasma pneumoniae Measured by Latex Agglutination

The standardization, application, and usefulness of latex agglutination for Mycoplasma pneumoniae antibody titration were investigated and compared with tetrazolium reduction inhibition and complement fixation. The sera of guinea pigs and monkeys reacted in a specific fashion, whereas rabbit serum required pretreatment to eliminate its nonspecific agglutinin. Human serum given such pretreatment still contained a nonspecific agglutinin. The latex agglutination procedure compared favorably with complement fixation and metabolic inhibition tests for evaluating vaccine antigenicity.     

A comparative study of detection methods for Aleutian disease viral antibody.

Four methods of detecting and quantitating mink antibody against Aleutian disease (AD) virus were compared. Counterelectrophoresis, modified, counterelectrophoresis, immunofluorescence, and complement fixation were performed blindly on 274 serum samples. All four methods were reliably specific for AD antibody. Immunofluorescence was less reproducible than the other systems. Immunofluorescence complement fixation were 4- to 8-fold more sensitive than regular or modified counterelectrophoresis, but were limited by background staining and anti-complementary activity, respectively, when used to detect small amounts of antibody in undiluted sera.     

Monoclonal antibody against the carboxy terminal peptide of pp60src of Rous sarcoma virus reacts with native pp60src.

A monoclonal mouse antibody has been prepared against a synthetic peptide corresponding to the six carboxy-terminal amino acids (C' peptide) of the src gene product pp60v -src of Rous sarcoma virus (RSV). The antibody was able to precipitate pp60v -src and to bind pp60v -src kinase activity in a competition test, indicating that this peptide can serve as an antibody-binding site (epitope). Furthermore, the finding that three out of 28 pp60src-specific tumor-bearing rabbit (TBR) sera contained antibody against the C' peptide argues for an in vivo role for the carboxy terminus of pp60src. C' peptide-specific IgG was purified from one TBR serum using affinity chromatography, and was shown to precipitate significant amounts of pp60src, and bind most of the pp60src kinase activity from SRA, PrA, and B77-C strains of avian sarcoma virus (ASV), but not endogenous pp60c -src, a cellular homologue to the viral pp60v -src. Similar results were obtained with IgG isolated from a C' peptide immune rabbit serum. None of the three C' peptide-specific IgGs could serve as a phosphate acceptor in an immune complex protein kinase reaction.