bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement and Feline Leukemia
Authors: O'Neill G. J. , Hardy W. D. , Kassel R. , Old L. J. , Day N. K.
Published: January 2023

Our interest in complement and leukemia has emerged from studies by Kassel et al. (J. Exp. Med., 138:925, 1973) and Hardy et al. (unpublished results) that infusion of normal plasma in mice, cats, and dogs causes massive dstruction of leukemic cells. Evidence in the mouse indicates that the anti-leukemic factor is a component of the complement system, C5. We have therefore begun to assay C and C component levels in cat serum. With functionally pure C1 and C2 and intermediate cells (EAC1, EAC4, EAC1,4) prepared from cat serum, the measurement of C1, C4 and C2 can be accomplished when the source of terminal C components, C3 to C9 is guinea pig serum in EDTA. With cat serum in EDTA, lysis does not occur. The blocking effect appears to be due to a potent inhibitor present in cat serum. This inhibitor is anti-complementary to guinea pig and human serum.
Complement Dependent Adherence, Phagocytosis and Hemolysis Initiated by C-Reactive Protein (CRP)
Authors: Osmand A. P. , Mortensen R. F. , Gewurz H.
Published: January 2023

Interactions of CRP with several substances have been shown to result in activation of the C system at the level of C1. By using sensitized C-polysaccharide-coated erythrocytes (E-CPS), we have investigated both the qualitative and quantitative differences between IgG antibody and CRP in their ability to promote a variety of C-dependent reactions. Sensitization with CRP or antibody to CPS (1 to 10 µg/ml) rendered E-CPS susceptible to C-dependent lysis during incubation with suitably absorbed human or guinea pig sera. Extensive hemolysis (60 to 85%) in C4- and C2-deficient sera and potentiation by human C1q of hemolysis by guinea pig serum were suggestive both of the ability of CRP and antibody to activate the C1-bypass and of the sensitivity of the cells to this C pathway. The ability of CxRP, the analogous protein in the rabbit, to promote these reactions indicated that these properties are a general function of C-reactive proteins.
C1q Binding Substances in Cancer and Complement Deficient Sera
Authors: Teshima H. , Kunkel H. G. , Day K. Noorbibi
Published: January 2023

We recently reported the presence of C1q-binding substances in serum of a patient with CLL and circulating antigen-antibody complexes (Day et al., Clin. Res. 23:410a, 1975). We have extended this observation to study of patients' sera with various forms of malignancies and with genetically inherited C deficiencies (c1r, C2, and C3). Two methods are used: one by a modified agar diffusion (Agnello et al., Immunology 19:909, 1970) and two, by the C1q deviation test (Bokisch et al., Clin. Res. 23:409a, 1975). The modified agar diffusion test correlated with C1q levels determined by the Mancini technique (normal: 17 to 23.5 µg/ml). Fifteen of 50 malignant sera (2/3 breast, 2/2 melanoma, 5/12 acute myelogenous leukemia, 1/1 chronic lymphatic leukemia, 1/1 oat cell, 2/3 esophagus, 1/5 rectal, 1/5 ovary, 1/1 bone) wath low levels of C1q (<10 µg/ml) were strongly positive by the precipitation test. These low sera also showed high C1q-binding activity in the C1q deviation test.
Activation of the C1r Subunit of C1 by Proteolytic Cleavage
Authors: Ziccardi R. J. , Cooper N. R.
Published: January 2023

C1r, isolated by an improved method, was found to be a 190,000-dalton glycoprotein consisting of two non-covalently linked 95,000 dalton polypeptide chains. Antisera to C1r detected a protein with a sedimentation coefficient of 19S on ultracentrifugation of serum in the presence of calcium and a 7S protein on analysis in EDTA. No reaction with C1r-deficient sera was observed. Isolated C1r formed C1 in the presence of C1q and C1s. Binding of 125I-C1r to sensitized sheep erythrocytes required the presence of C1q and calcium but not C1s, whereas the binding of 125I-C1s required C1q and C1r and calcium. Thus C1r serves as the physical link between C1q and C1s is macromolecular C1.

Acquisition of ability to activate C1s was associated with cleavage of each of the noncavalently bonded 95,000 dalton chains into disulfide linked subunits of 60,000 and 35,000 daltons, respectively. Thus, C1r is converted from a precursor form by proteolytic cleavage into an enzyme, C, able to cleave and activate C1s.
Opinion
Authors: Kilbourne Edwin D.
Published: January 2023

Influenza persists in this last quarter of the 20th century as an important cause of wholesale human incapacity and death. Paradoxically, it does so in the face of recent improvements in inactivated vaccines developed 30 years ago and striking advances in the understanding of the structure, replication, and genetics of the influenza viruses during the past decade. Neither prior infection nor artificial immunization provides lasting immunity against the constantly changing virus of influenza. In part, the transient protection induced by presently available inactivated viral vaccines is related to rapid decline of vaccine-induced homologous antibody; in part, vaccine obsolescence is the result of viral mutation to antigens unlike or less like those contained in the vaccine.

The threat of pandemic influenza is so awesome that major efforts are required for its curtailment upon even the hint of its inception, as was the case with swine influenza during 1976 (1).
Nonspecific Activation of Murine Lymphocytes
Authors: Goodman Michael G. , Weigle William O.
Published: January 2023

Recent evidence has indicated that addition of 2-mercaptoethanol (2-ME) to culture medium is able to activate murine lymphocytes to undergo blastogenesis, to synthesize polyclonal antibody, and to develop cytotoxicity to both autologous and heterologous target cells. In order to explore the basis for these phenomena, a study of the physical interaction between the cell and 2-ME was undertaken by using a radiolabeled preparation of 2-ME. Uptake of labeled 2-ME increased over the initial 24 hr of culture, after which a steady state was achieved. Cells were found to have maximal susceptibility to activation by 2-ME after incubation for 24 hr in the absence of the thiol compound. This observation was not explicable in terms of any alteration in the kinetics of 2-ME uptake. The amount of labeled 2-ME taken up was a function of the 2-ME concentration with which the cell was incubated, with the exception of the concentration range that is optimal for mitogenesis. At this range, the curve was suggestive of a saturation effect. Uptake by B cell cultures was found to exceed that by T cell cultures. Uptake was shown to result from interaction with protein, to be independent of metabolic energy, to be governed by temperature-dependent kinetics, and to be highly specific.
Genetic Control of Immunologic Unresponsiveness to Adjuvant-Free Solutions of β-D-Galactosidase
Authors: Rotman Boris
Published: January 2023

Two genetic loci, Ir-Z1 and ir-Z2, controlling the immune response to adjuvant-free bacterial β-D-galactosidase (Z) are present in inbred mouse strains SJL/J and CE/J, respectively. Each locus segregates as a single, autosomal gene: Ir-Z1 as dominant and ir-Z2 as recessive. The response is characterized by production of activating and precipitating IgG. Maximal levels of circulating IgG occur between 16 and 20 days after immunization with a single, 50-µg dose of enzyme. Failure of proteins other than Z to elicit an immune response indicates that the Ir-Z control is specific for determinant(s) of this enzyme. The immunogenicity of β-D-galactosidase preparations cannot be attributed to either the catalytic activity of the enzyme or adjuvant contamination. Non-responder mice acquire immunologic memory without detectable increase in circulating specific IgG under the same conditions that elicit antibody production in responder strains.
Patterns of Expression of Human “Ia-Like” Antigens during the Terminal Stages of B Cell Development
Authors: Halper J. , Fu S. M. , Wang C. Y. , Winchester R. , Kunkel H. G.
Published: January 2023

The “Ia-like” antigens characteristically present on the membrane of B lymphocytes were shown to be absent in most terminally differentiated Ig-producing plasma cells. This was most evident in analyses of myeloma plasma cells that uniformly lacked the Ia-antigens. Also, in B lymphoid cell lines, the lymphoblasts were uniformly Ia-positive but the plasma cells were negative. In contrast, however, the majority of plasma cells produced after pokeweed mitogen stimulation remained positive. Plasma cells in tonsil and in the tissue of peripheral blood of patients with Waldenström's macroglobulinemia were also sometimes Ia-positive although the majority were negative. Studies of membrane IgM and IgD indicated a similar loss in some instances. However, the SIg and Ia antigens were not invariably associated. These results lead to the conclusion that Ia-antigens are differentiation antigens for B cells and are usually lost by the end stage cells in this series.
Characterization of Lymphocyte-Activating Factor (LAF) Produced by a Macrophage Cell Line, P388D1
Authors: Mizel Steven B. , Oppenheim Joost J. , Rosenstreich David L.
Published: January 2023

Unstimulated P388D1 cells, as well as P388D1 cells stimulated with PHA-activated guinea pig T lymphocytes or LPS, produced a lymphocyte activating factor (LAF). In order to have a chemical basis for comparing this LAF with the LAF produced by normal macrophages, we have analyzed several biochemical characteristics of the P388D1-derived LAF. Sephadex G-75 chromatography of concentrated LAF-containing supernatants from cultures of unstimulated and T cell stimulated P388D1 cells demonstrated that the cell line LAF had a m.w. of approximately 16,000. On DEAE cellulose, the T cell-induced LAF fractionated into at least three major peaks and one minor peak. By using hydroxylapatite chromatography, two of the major peaks of LAF activity were separated from residual contaminating Lowry positive material.

LPS-stimulated P388D1 also produced LAF with a m.w. of 16,000. However, the LPS-induced LAF appeared to lack one of the DEAE peaks of LAF activity observed with the T cell-derived LAF. In contrast to LPS, T cells may induce the synthesis and/or release of an additional LAF component or enzymatically modify one or more of the LAF species that are produced in response to both stimulants.

Based on the results of chemical characterization studies, the P388D1-derived LAF appears to be similar in size and charge to the lymphocyte activating factor produced by normal macrophages.
The Activation of Lymphocytes by the Ionophore A 23 187
Authors: Resch Klaus , Bouillon Dorothea , Gemsa Diethard
Published: January 2023

The possible mechanism by which a divalent cation ionophore, A 23 187, initiated the activation of lymphocytes was investigated. Rabbit lymphocytes were cultured in the presence of the ionophore, the T cell mitogen Con A, or the B cell mitogen GARIG serum. The proliferative response, assessed by incorporation of 3H-thymidine into DNS, as well as the increase in incorporation of 3H-uridine induced by the ionophore was comparable to that caused by the receptor-binding B mitogen in T-B mixtures. Thymocytes responded weakly and inconsistently to the ionophore, whereas purified B lymphocytes proved to be highly reactive, suggesting that A 23 187 is primarily a B mitogen in rabbits. Removal of macrophages did not impair the activating capacity of the ionophore. To initiate cell activation the ionophore had to be present for at least some hours.

At optimal concentrations of Ca++ (1 mM), increasing doses of Mg++, which has the same binding affinity to the ionophore but does not support activation, did not suppress the response to the ionophore. Similarly, Mn++ which binds to the ionophore with a 100-fold higher affinity than Ca++ was not able to interfere with the activation of lymphocytes. Both ions, however, inhibited the ionophore-dependent Ca++ influx at concentrations not affecting activation. Within 1 hr of contact the ionophore increased the turnover of the phospholipid fatty acids. The dose response curve for the activation of the membrane phospholipid metabolism and that for the stimulation of RNA or DNA synthesis were identical.

These data suggest that the ionophore A 23 187 interacts directly with rabbit (B) lymphocytes to induce cell activation. The activating capacity does not depend on the translocation of Ca++. We propose that the ionophore intercalates into the lipid phase of the plasma membrane, initiating the same changes in the membrane itself that are induced by mitogens that bind to surface receptors, i.e., increasing the turnover of membrane phospholipids.
Progressive Changes in Cytotoxic Potential during Mixed Lymphocyte Culture
Authors: Touton Mary H. , Clark William R.
Published: January 2023

In a previous study it was shown that at least one round of DNA synthesis is required for initial expression of cytotoxic function in mouse lymphocytes responding to alloantigen in vitro. In the experiments reported here we ask whether subsequent rounds of cell division are required simply for clonal expansion of this initial level of cytotoxic function within the population, or whether the amount of cytotoxicity per cytotoxic cell is altered during subsequent rounds of cell division. The amount of cytotoxicity per unit number of cells at various stages of culture was compared with the frequency of cytotoxic cells as estimated principally by effector-target cell conjugates. Our results strongly suggest that the amount of cytotoxicity per cell (cytotoxic potential) is not a static property of cytotoxic cells, but can be modulated up or down during the course of a reaction.
Melanoma-Associated Immunosuppression through B Cell Activation of Suppressor T Cells
Authors: Ninnemann John L.
Published: January 2023

Using normal human lymphocytes isolated by sedimentation and cotton column adherence, we have developed a reliable assay of immunosuppression of PHA-induced blastogenesis by serum from selected melanoma patients. These lymphocyte cultures contained both responder cells (subpopulation x) and regulator cells (subpopulation y). Lymphocytes isolated by gradient centrifugation on sodium metrizoate-Ficoll contained responder cells (x) but no regulator cells (y). Cultures of lymphocytes isolated by this method were stimulated by PHA but were not suppressed by the addition of melanoma serum. When lymphocytes were isolated by a cotton column adherence/Lymphoprep centrifugation-double separation, subpopulations (x) and (y) were isolated. We have established that both subpopulations are necessary for suppression to occur, and that (y) operates as the regulator of (x). Finally, by manipulating B cell and T cell populations isolated by nylon column adherence or AET rosette separation, we have determined that the regulator ability of subpopulation (y) is the result of B cell activation of suppressor T cells.
Trypan Blue Inhibits Complement-Mediated Phagocytosis by Human Polymorphonuclear Leukocytes
Authors: Guckian James C. , Christensen Wayne D. , Fine Douglas P.
Published: January 2023

Trypan blue completely inhibited attachment of human polymorphonuclear leukocytes (PMN) to Sepharose beads coated with C3 and to sheep erythrocytes coated with IgM plus C3, but it did not inhibit attachment to erythrocytes coated with IgG. These results suggested that trypan blue inhibited C-mediated attachment to PMN membranes. Corroborative studies were performed with a strain of Staphylococcus aureus that requires C but not antibody, for opsonization and that activates the alternative pathway. Trypan blue was not toxic to PMN or bacteria, did not interfere with immunoglobulin or C interactions, and did not affect attachment of opsonins to bacteria. However, the dye impaired PMN killing of S. aureus in normal nonimmune serum by inhibiting bacterial attachment to and ingestion by PMN. Further evidence that the inhibition was at the C3 receptor level came from the observations that, 1) once staphylococci were attached to PMN at either 37°C or 0°C, addition of trypan blue did not inhibit killing; and 2) trypan blue inhibited killing of bacteria opsonized with serum sufficient in C but previously absorbed at 0°C with the same strain of organism to deplete specific antibody. Further studies with this agent may elucidate the roles of opsonic receptors on human phagocytes.
Mechanisms of Regulation of Cell-Mediated Immune Responses
Authors: Greene Mark I. , Sugimoto Masanobu , Benacerraf Baruj
Published: January 2023

A new method for inducing contact sensitivity (CS) responses has been developed. The subcutaneous injection of trinitrophenyl (Tnp)-derivatized syngeneic spleen cells into BALB/c mice, without adjuvant, could lead to significant ear swelling CS responses upon challenge of the ear with the same hapten, i.e., picryl chloride (PCI). This response was transferred by lymphoid cells obtained from immunized animals. Such cells were judged to be thymus-derived (T) cells as they could be obtained by nylon wool passage and were sensitive to treatment with AKR anti-Thy 1.2 serum and C. The response displayed the characteristic time course of cell-mediated immune reactions and was maximal at 24 hr after challenge. Histologic investigation revealed a marked mononuclear cell infiltration into the lesion site.

The i.v. administration of either 2,4,6-trinitrobenzene-sulfonic acid (TNBS), or the same moieties capable of eliciting CS, viz, Tnp cells or Tnp cell membranes, could suppress the generation of Tnp cell-induced CS reactions. It was also possible to suppress the CS response by the i.v. transfer of suppressor T cells or factor(s) obtained from suppressed mice.

Furthermore, it was found that Tnp-derivatized syngeneic macrophage (Mø) were more capable of inducing CS responses than similarly derivatized spleen cells or thymocytes, suggesting that Mø surface was a better presentation template for the induction of CS. The notion that the route of presentation of hapten-modified syngeneic membranes dictates the response evoked, i.e., immunity when presented subcutaneously but suppression when the encounter is via the i.v. route, is discussed.
Role of Interferon in the Pathogenesis of Viral Diseases of Mice As Demonstrated by the Use of Anti-Interferon Serum
Authors: Virelizier Jean-Louis , Gresser Ion
Published: January 2023

Potent sheep anti-mouse interferon globulin has been used to determine the role of virus-induced interferon in mouse hepatitis virus type 3-infected susceptible (C57BL/6), semiresistant (C3H/He), and resistant (A/J) strains of mice. Injection of anti-interferon globulin accelerated the onset of death in C57BL/6 mice, induced almost 100% mortality in C3H/He mice that usually do not die of acute disease, and caused death in 4- and 6-week-old A/J mice, but not in older mice. We conclude that interferon is an important host defense factor in the initial response of different strains of mice to MHV-3 infection. Other factors, however, such as the capacity of macrophages to restrict viral multiplication probably underlie the genetically determined susceptibility or resistance of mice to MHV-3 infection.
Inhibition of the Alternative Pathway of Complement by Gold Sodium Thiomalate in Vitro
Authors: Burge Joseph J. , Fearon Douglas T. , Austen K. Frank
Published: January 2023

Gold sodium thiomalate (AuTM) in molar concentrations comparable to those observed in plasma of patients undergoing treatment of rheumatoid arthritis inhibits the interaction of C3b, B, and D̄ to form the amplification C3 convertase (C3b,Bb). The inhibitory action of AuTM is dose related and occurs whether or not P is present to stabilize the C3b,Bb convertase. When the convertase is formed in the absence of D̄ so as to be C3b,B, a higher concentration of AuTM is required for inhibition, suggesting an action not only to prevent the association of B with C3b but also on the presentation of the bimolecular complex as a substrate for D̄. Thiomalate alone was not inhibitory and AuTM had no irreversible effects on cell-bound C3b or fluid phase B, D̄, or P. AuTM did not enhance the decay or inhibit the active site of the preformed C3 convertase generated with cleaved or uncleaved B, with or without P stabilization. AuTM is most active in inhibiting convertase formation on cellular intermediates bearing the lowest number of 125I-C3b per cell and requiring development with the highest B concentration, suggesting the binding site on C3b for B as the locus of AuTM action. This interpretation is compatible with the ability of AuTM to prevent B proteolysis during the fluid phase interaction of C3b, B, and D̄. AuTM is a reversible inhibitor of the formation of C3b,Bb, the amplification C3 convertase of the complement system and thus modulates a critical step in the entire reaction sequence.
Murine Ia Antigens: Identification and Mapping of Ia.23 and Further Definition of the I-E Subregion
Authors: David Chella S. , Cullen Susan E.
Published: January 2023

The Ia.23 specificity is a private specificity of the H-2d haplotype which we have assigned to the I-E subregion. It is found on the same Ia molecule that bears Ia.7, a public specificity previously assigned to the I-C subregion of haplotypes H-2d, H-2k, H-2p and H-2r. We suggest a reassignment of specificity Ia.7 to the I-E subregion to simplify the interpretation of the data presented in this paper and elsewhere, and to permit an orderly description of that part of the I region to the right of I-J.
Inhibition of the Induction of Cytolytic T Lymphocytes with Alloantisera Directed against H-2K and H-2D Gene Products
Authors: Lemonnier Francois , Burakoff Steven J. , Mescher Matthew , Dorf Martin E. , Benacerraf Baruj
Published: January 2023

Alloantisera directed at gene products of the H-2Kd or H-2Dd loci on the stimulator cell were shown to inhibit specifically the generation of cytolytic T lymphocytes to those antigens. Thus, masking the antigens of one H-2 locus on the stimulator cell inhibits the induction of CTL to products of that locus but does not inhibit the induction of CTL to antigens of another H-2 locus. Alloantisera inhibition of the induction of cytolytic T lymphocytes occurred with both normal and primed responder cells and also occurred when the stimulating antigens were on whole cells or purified plasma membrane. Absorption on the appropriate spleen cells removed the inhibitory capacity of these alloantisera.
Antibody-Dependent Cell-Mediated Cytotoxicity by Human Lymphocytes
Authors: Fuson E. W. , Whitten H. D. , Ayers R. D. , Lamon E. W.
Published: January 2023

A study has been made of the ability of IgM and IgG to initiate ADCC by normal human peripheral blood lymphocytes (PBL) in a 51Cr release assay with sheep erythrocytes as target cells. The immunochemical purity of the IgM and IgG preparations was confirmed by ion-exchange and affinity chromatography. Passage of the IgM over DEAE-cellulose removed its C-dependent hemolytic as well as ADCC activity, whereas no activity was removed from IgM by passage over a Sepharose 4B-Protein A column. The opposite was observed with IgG; DEAE-cellulose removed no activity and the protein A column effectively depleted IgG activity in both C-dependent hemolysis and ADCC. The IgM-induced ADCC was compared to IgG-induced ADCC on the basis of kinetics, dose-response, and effector-target cell ratios required. In addition, it was shown that preincubation of lymphocytes facilitated IgM-induced ADCC but had little effect on IgG-induced ADCC. When preincubated PBL were used as effector cells the following was found: The time requirements for induction of cellular cytotoxicity by IgM and IgG were similar when the antibodies were used at concentrations near their hemolytic end point titers. Dose-response curves of IgM and IgG indicated that lymphocytes were induced to be cytotoxic more efficiently by IgM when the antibody classes were compared on the basis of molecules/target cell required to produce 50% lysis. The effector-target cell ratio producing optimal lysis in the assay was the same for both classes of antibody. In contrast, the cytotoxicity produced by freshly isolated PBL against IgM-sensitized target cells was significantly lower than against IgG-sensitized target cells in all comparisons.
Primary Structural Analysis of Human C4
Authors: Andrews David W. , Capra J. Donald
Published: January 2023

C4 has been isolated from human plasma by a three-step procedure and its component chains have been separated. Partial amino acid sequence analysis of these chains reveals considerable polymorphic variability. Chains isolated from individual donors have been examined to define this polymorphism at the primary structural level. The structure of the γ-chain has been more intensively investigated using fragmentation with CNBr, and the resulting peptides have been partially sequenced. The longer CNBr fragments of the γ-chain have been further fragmented with trypsin after citraconylation. Sequence data concerning these peptides will be presented and discussed.
In Vivo and in Vitro Inhibition of Complement by Chlorazol Fast Pink
Authors: Bauman N. , Brockman J. A.
Published: January 2023

Chlorazol Fast Pink (CFP) is a sulfonic acid azo dye, first described as an in vitro complement inhibitor in 1952 (Lambert & Richley, B. J. Exp. Path. 33, 327, 1952). We have examined CFP in vivo and in vitro and found that it inhibits complement dependent lesions in laboratory rodents, and that these effects are correlated with depression of serum complement. In some cases, CFP has been compared with pentosan polysulfate (PPS), another anionic complement inhibitor.

CFP and PPS, injected intraperitoneally, block Forssman shock and Forssman cutaneous vasculitis in guinea pigs and the reverse passive Arthus reaction in guinea pigs and rats. Inhibition of these reactions is accompanied by an inhibition of serum complement, demonstrable by an assay of “undiluted” serum. In this assay, serum is laid over EA embedded in an agarose matrix in a capillary tube, and the zone of lysis measured with an optical micrometer.
Identification of Properdin, B, D, and C3 As Biosynthetic Products of Guinea Pig Peritoneal Macrophages and Influence of Culture Conditions on Their Secretion
Authors: Brade Volker , Fries Wolfgang , Bentley Carolyn
Published: January 2023

Monolayers of adherent cells (a.c.) were prepared from peritoneal cells obtained from guinea pigs injected three days previously with starch gel. In serum-free medium, a.c. were cultured for approximately 40 hr under the following conditions: 1) On a plastic surface in MEM. 2) On a plastic surface in MEM plus 0.2% lactalbuminhydrolysate (LH). 3) On a plastic surface coated with glutaraldehyde-linked BSA (BSA-surface) in MEM. 4) On a BSA-surface in MEM plus LH. The culture conditions were found to be slightly improved by LH and the BSA-surface alone, or in combination, as judged by lactatdehygrogenase and lysosomal enzyme determinations, trypan blue exclusion tests, and cell number. When culture supernatants (c.s.) from monolayers cultured these four different conditions were tested in the same order for functional activity of C3 and D, the respective values were found to be 5.7 × 107 site forming units (SFU) C3 and 70 U D; 4.5 × 107 SFU C3 and 27 U D; 1.1 × 107 SFU C3 and 2 U D; 0.3 × 107 SFU C3 and 6.5 U D.
Granulocyte (GR) Aggregometry: A New Assay for the Detection of C5a and Complement (C) Activation
Authors: Craddock Philip R. , Lammi Carol J. , Bowers Timothy K.
Published: January 2023

Products of C-activation (particularly C5a) induce chemotaxis, phagocytosis, and lysosomal exocytosis by GR, and thus profoundly influence the role of GR in inflammation. We have recently reported that C5a will induce GR aggregation in vitro. This aggregation is specifically dependent on C5a in that aggregating activity can be generated in normal but not C5-deficient plasma by incubation with zymosan (ZYM); the aggregating activity in normal ZYM-treated plasma is limited to a single peak of 17,000 daltons, is heat stable (56° × 30 min), and is inhibited by incubation with anti-C5 but not anti-C3 antisera. The amplitude of the aggregator response is proportional to log2 [C5a]. GR aggregation might therefore be used as an assay for C5a and thus for C activation. The aggregation response of GR to C5a is amplified tenfold by the addition of cytochalasin B (CB).
Complement Expression on Lymphoid Cells
Authors: Ferrone S. F. , Pellegrino M. A. , Gorski J. P. , Cooper N. R.
Published: January 2023

Earlier studies showed that peripheral human lymphoid cells (PBL) and cultured lymphoblastoid cells (CL) express, but do not secrete, C4. Membrane expression of C4 is detectable by immunochemical, radiochemical and immunofluorescence techniques. CL absorbed C4 α- and β-chain antibodies, as assayed by radioimmunoassay, thus CL express C4 α- and β-chain determinants. CL also produce other C components since several rabbit antisera to human CL of B and T cell origin contain antibodies to human C1q, C1r, C3, C4, C5, C6, C7, C8 and P.

Our earlier studies showed that some, but not all, antisera to human C4 inhibited the mixed lymphocyte reaction (MLR) between allogeneic human PBL, suggesting a role for C4 in immune recognition phenomena. Because of variable MLR inhibition with different antisera by us and by other workers, we have further investigated the effect of anti-C4 on the MLR.
Purification of the Third Component of Rabbit Complement
Authors: Giclas Patricia C. , Henson Peter M.
Published: January 2023

Rabbit C3 has been isolated from normal rabbit plasma by a method based on that developed for human C3 by Tack and Prahl (Biochem. 15:4513, 1976). Fractionation of the rabbit plasma with polyethylene glycol was followed by DEAE-sepharose chromatography, gel filtration and final separation on hydroxyl apatite. The C3 obtained by this technique is hemolytically active and represents 7–15% of the plasma C3.

Rabbit C3 migrates as a single band on SDS gels with an estimated molecular weight of 186,000, and can be separated on SDS gels into α and β chains of 132,000 and 70,000, respectively, under reducing conditions.

Preliminary studies of the biologic activities of the rabbit C3 preparations indicate that it binds to rabbit peripheral blood neutrophils. The C3 remaining in the supernatant after incubation with the neutrophils is no longer hemolytically active, and SDS gels of this material show that it has been cleaved into two major fragments. Studies are in progress to determine the nature of the interaction between rabbit C3 and the neutrophils.
The Effect of Danazol in Acquired C1̄ Inhibitor Deficiency: Changes in C1̄ Inhibitor and Complement Levels
Authors: Hauptmann G. , Mayer S. , Lang J. M. , Oberling F. , Mayer G.
Published: January 2023

Several cases of acquired C1̄ inhibitor (C1̄ INH) deficiencies and angioedema have been reported in association with lymphoproliferative diseases. An acquired C1̄ INH deficiency can be distinguished from the hereditary angioedema by the adsence of C1̄ INH deficiency in other family members, by the profound reduction of the C1 level in addition to the reduction of C4 and C2 levels, and by the presence, in most cases, of serum immunoglobulin abnormalities and of a high C1 depleting activity in the serum of a patient suffering from a lymphoproliferative disease. In addition to the three cases previously described (Blut 32: 195, 1976) we have now observed a fourth case of acquired C1̄ INH deficiency and we have studied the effect of the treatment with Danazol on the C1̄ INH and complement levels.

A 57-year-old man presented with a lymphoproliferative disease, IgM dysglobulinemia and the characteristic complement profile of an acquired C1̄ INH deficiency.
Deviated Lysis: Formation of a Properdin Stabilized C3/C5 Convertase As the Active Principle Underlying the Phenomenon
Authors: Kolb W. P. , Podack E. R. , Müller-Eberhard H. J.
Published: January 2023

Deviated lysis (d.l.), as described by Rother et al. (Z. Immun.-Forsch. Bd. 148:172, 1974), is the ability of activated complement, either on the surface of the activating particle or in the fluid phase, to lyse nonsensitized target cells in the presence of EDTA. We wish to report experiments conducted to characterize the active principle responsible for this activity. D.1. activity was generated by incubating NHS with inulin for 10 to 20 min at 37°C. The reaction was terminated by the addition of EDTA (10 mM final concentration) and centrifugation to separate pellet-associated and fluid phase activities, which were assayed for their ability to lyse chicken erythrocytes (ChE) in GVB-10 mM EDTA (GVBE).

Characterization of the sucrose-stabilized fluid phase d.l. activity revealed a major peak possessing a m.w. of 250–350,000 daltons and an S-rate of 14 to 16 S as determined by chromatography with Bio-Gel A-0.5 M and sucrose density gradient ultracentrifugation. D.1. activity present in column and gradient fractions was completely inhibited by monospecific antisera to properdin, factor B or C3 but not by anti-C4.
A Quantitative Comparison of the Biologic Activity of Membrane-Bound C3 and C4 In Vitro
Authors: Kurlander R. J. , Rosse W. F.
Published: January 2023

Human red cells (E) were specifically and singly coated with either C4, C3b, or C3d by using human serum as the source of complement, and the amount of each component fixed was measured quantitatively by antiglobulin consumption tests. The immune adherence (IA) of E-C4, E-C3b and E-C3d to peripheral blood monocytes and polymorphonuclear leukocytes (PMN) was dependent upon the amount of the component on the red cell surface. The amount of adherence per molecule of component fixed was slightly greater with E-C4 than with E-C3b and much less than either with E-C3d. However, E-C3d adherence was more stable to washing or mechanical disruption than E-C3b adherence; E-C4 adherence was less stable than either. Unlike E-C3b when incubated with heated serum, resulting in E-C3d with continued IA activity, E-C4 incubated with serum completely lost IA activity.

51Cr-labeled E-C4, E-C3b and E-C3d were not lysed during incubation with monocytes even though they readily adhere to monocytes.
C6–C7: A Further “Complement Supergene”
Authors: Lachmann P. J. , Hobart M. J.
Published: January 2023

The genetic polymorphism of C6 is well established. The two common alleles have frequencies of 0.6 and 0.35 which makes C6 a very useful genetic marker. Genetic polymorphism of C7 has now also been demonstrated. Three alleles have been found, of which only the commonest occurs at a frequency above 0.05. Family studies have so far shown no cross-overs between C6 and C7 in 21 informative meioses, indicating close linkage between the loci.

There is no significant evidence for linkage between the C6, C7 locus and any other marker tested. These include C3, HLA, Gm, and many red cell antigenic and enzyme markers. From rather limited family data, close linkage to C1̄-inhibitor and C3b inactivator deficiencies can also be excluded. Further evidence for C6,C7 association is provided by an individual with a remarkable genetic complement deficiency affecting both C6 and C7. The propositus, who is in good general health, has low, but detectable, levels of both C6 (c1%) and C7 (c3%); other components being within the normal limits.
The Role of C1q in the Internal Activation of the First Component of Complement (C1): Studies in Serum of a Selective C1q-Deficient Patient
Authors: Loos Michael , Thesen Rolf , Berkel A. Izzet
Published: January 2023

The serum of a 10-year-old boy was found to be totally deficient in hemolytic C1 activity. The lack of C1 activity was due to a selective deficiency of C1q whereas the titres of the other components including C1r and C1s were found within the normal range. We looked for C1q functionally as well as immunochemically. There was no decrease of serum immunoglobulins IgG, IgM, or IgA. Addition of highly purified human C1q to the serum restored the whole complement activity as well as the C1 activity. In ultracentrifugation studies the sedimentation rates of the restored C1 molecules were found mostly in the 18.6S region when the experiment was performed with 0.06 µ VBS containing gradients; however, C1 activity was found only in the 12.6S region when a gradient with 0.15 µ VBS was used. The dose-response curve of the restored C1 molecule showed the same nonlinear shape as that of C1 in normal human serum.
Responses of Human Platelets to Immunologic Stimuli: Independent Roles for Complement (C), Fibrinogen and IgG in Zymosan Activation
Authors: Martin S. E. , Breckenridge R. T. , Rosenfeld S. I. , Leddy J. P.
Published: January 2023

We have employed zymosan (Z), 1 particle/15 platelets, as an in vitro probe for the study of the effects of immunologic reactions on human platelets. Studies by others had shown a) that human platelets respond with aggregation and release reactions when exposed to Z previously treated with normal plasma; b) that an intact alternative C pathway as well as fibrinogen are required; and c) that the critical plasma reactants are Z bound. Using genetically C-deficient human plasmas, we have previously reported requirements for C5, C6, and C7 but not C8 in the formation of the active Z-complex.

Utilizing agammaglobulinemic (agamma) plasma and washed human platelets, we have demonstrated a requirement for IgG in this reaction. Agamma plasma did not support Z-induced platelet activation, whereas Z previously incubated in normal plasma activated agamma platelets. This agamma plasma defect was corrected by purified normal IgG, but not by F(ab′)2 or Fc fragments.
Multiple Forms of Properdin
Authors: Medicus R. G. , Müller-Eberhard H. J.
Published: January 2023

Properdin exists in at least three functionally different forms. The form which is isolated by the most rapid (two-step) procedure from fresh serum we designate native properdin (nP). Purified nP is characterized by its inability to bind to solid phase C3b, to stabilize the labile C3/C5 convertase, to form a soluble C3 convertase in serum, and by its capacity to reconstitute P-depleted serum with respect to C3 consumption by zymosan or inulin. The second form of P, designated pre-P, is characterized by its ability to bind reversibly to solid phase C3b, to stabilize the labile C3/C5 convertase formed with multiple C3b molecules, and to reconstitute P-depleted serum in a manner analogous to nP. The third form is active P, referred to as P̄. It is characterized by its ability to bind irreversibly to solid phase C3b, to stablize the labile C3/C5 convertase as well as the single C3b C3 convertase and to assemble the soluble C3 convertase when added to serum independent of zymosan or inulin.
Mechanism of Action of β1H Globulin on C3b
Authors: Ruddy Shaun , Carlo Jaime , Conrad Daniel
Published: January 2023

The activity of the “C3b Inactivator Accelerator,” described at the Sixth Complement Workshop, has since been found to reside in β1H globulin, a 150,000 m.w. glycoprotein (K. Whaley and S. Ruddy, Science 193:1011–1013, 1976; and J. Exp. Med. 144:1147–1163, 1976). This protein accelerates the rate of inactivation of the hemolytic activity of the C3b by C3b Inactivator and also increases the rate of proteolysis of C3b by C3b Inactivator. In addition, β1H has direct effects on C3b, preventing its participation in either the classical or alternative pathway C5 convertases, and increasing the rate of decay of factor B from the alternative pathway convertase, .

Some of these apparent diverse effects of β1H may be explained by its binding to C3b. This is evidenced by 1) agglutination of cellular intermediates exposed to β1H by monospecific antibody to β1H, 2) immunofluorescent staining of such intermediates with fluorescein-labeled anti-β1H, 3) depletion of β1H activity from the fluid phase of reactions containing C3b.
Complement Activation in Human Serum by Urate Crystals, Evidence for a Role for IgG
Authors: Russell I. J. , Papaloannou C. , McDuffie F. C.
Published: January 2023

Previous studies of complement activation in serum by urate crystals have not supported a role for immunoglobulins (Naff and Byers, J. Lab. Clin. Med. 81:797, 1973). The recent demonstration by Kozin and McCarty (J. Lab. Clin. Med. 89:1314, 1977) of adsorption of IgG to urate crystals prompted us to reexamine this question. Addition of urate crystals (10 mg per ml.) to hypogamma-globulinemic serum reduced hemolytic activity only slightly but caused virtually complete depletion from normal serum. Addition of 7S IgG (10 mg per ml) but not IgA (10 mg/ml) nor IgM (10 mg/ml) restored the ability of such serum to demonstrate depletion of C activity by urate crystals. Urate crystals were able to inactivate C3 in normal human serum but not in C2 deficient human serum nor in C4 deficient guinea pig serum. This effect is mediated primarily through the classical pathway.
Translation of Precursors of C4 and Albumin by Isolated Guinea Pig m-RNA
Authors: Sakiyama Yukio , Hall Robert E. , Colten Harvey R.
Published: January 2023

Guinea pig RNA was isolated from liver homogenates by phenol-chloroform-ethanol extraction and applied to a Sigmacell type 50 column in Tris (40 mM) buffer containing KCl (1.0 M) and MgCl2 (0.4 mM). Messenger RNA was eluted from the column, precipitated in ethanol and NaCl (1.0 M) washed in the same solution and redissolved in distilled water. The m-RNA was then added to a mixture containing rabbit reticulocyte polysomes (in some experiments pretreated with a micrococcal nuclease) an energy source, rat t-RNA, hemin, 3H-labeled leucine, 19 unlabeled amino acids and optimal concentrations of K+ and Mg++. Total protein synthesis by micrococcal nuclease-treated reticulocyte lysate was increased nearly four-fold (3700 cpm to 13,260 cpm) in the presence of m-RNA. Under these conditions, single-chain precursors of guinea pig albumin (m.w. > 68,000) and of C4 (m.w. ≥ 205,000) were synthesized, each accounting for less than 0.5% of the newly synthesized total protein.
Modulation of the C3 Receptor by HLA Antibodies and C1, 4, 2, and 3 Components of Complement
Authors: Sargent A. U.
Published: January 2023

Recent studies have suggested linkage of genes regulating synthesis of C2 and genes of the MHC complex with M.S. and certain autoimmune diseases. As Ig-secreting plasma cells are related ontogenically to a subpopulation of B lymphocytes which selectively express the C3 receptor, these findings suggest linkage of C2, CRL cells and membrane products of MHC genes, with pathogenic mechanisms mediating diseases of humoral immunity. The following studies were initiated to determine whether a functional relationship could be demonstrated between antibodies to membrane products of MHC genes, the activation unit of the classical complement pathway and C3 receptors. Monospecific HLA-A2 antibody and functionally pure human C1, C4, and C2 were used to initiate the assembly of C3 convertase on membranes of enriched populations of human B lymphocytes. The sensitized lymphocytes were then incubated with EAC1-3 cellular intermediates and formation of C3 rosettes was determined. Formation of C3 rosettes with the sensitized lymphocytes was reduced by over 50% from values obtained with unsensitized controls.
Presence of a C4-Binding Protein (C4-bp) in Human Serum
Authors: Scharfstein Julio , Ferreira Arturo , Gigli Irma , Nussenzweig Victor
Published: January 2023

As published elsewhere (Ferreira et al., J. Exp. Med., in press) mouse serum contains a 10S β-globulin which specifically binds to fluid phase activated Ss (C4) protein. This new protein was named Ss-bp or C4-bp. A search was made in human serum for the presence of a protein with similar properties. Human serum was incubated at 37°C with washed immune precipitates and centrifuged. Activated C4 was isolated from the supernatant by precipitation with a monospecific antiserum to C4, and injected into rabbits. The resulting antiserum was tested against whole human serum by crossed-immunoelectrophoresis. It reacted only against C4 and an additional serum component with properties very similar to those of mouse C4-bp.

Human C4-bp is a heat stable β-globulin which is found in the exclusion volume after filtration of serum or EDTA-plasma through a Sephadex G-200 column. It has a sedimentation coefficient of 13S as determined by sucrose gradient ultracentrifugation of serum from a patient with hereditary angioedema (HAE) with low levels of C4.
Release of 86Rb+ and 14C-Phosphatidyl Choline Markers from Liposomes, and of Hemoglobin from Erythrocytes, by Activated Complement: Effect of Length and Unsaturation of Acyl Chains and of Cholesterol/Phospholipid Ratio
Authors: Shin Moon L. , Paznekas William A. , Abramovitz Aaron S. , Mayer Manfred M.
Published: January 2023

The cytolytic action of complement (C) is believed to be due to the formation of trans-membrane channels by insertion of peptides from C5b-9 into the lipid bilayer. We have previously reported that phospholipid (PL) is released from the liposomal bilayer by activated C, along with channel formation. Since channel formation and PL removal involve lipid-protein interactions, the chemical properties of the lipids in the membrane would be expected to influence these processes. In the present work, we have studied the C-mediated release of 86Rb+ from the aqueous compartment and 14C-phosphatidyl choline (PC) from the lipid bilayer of liposomes containing different amounts of cholesterol, and liposomes made from PL differing with respect to acyl chain length and unsaturation. Effect of acyl chain length. Liposomes prepared from C(14:0)2, C(16:0)2 and C(18:0)2 lecithins, with a cholesterol/PL ratio of 0.75, were treated with antibody (Ab) and C.
Occurrence of Complement Activation Specific Neonantigens on Peripheral Blood Leukocytes
Authors: Sundsmo J. S. , Kolb W. P. , Curd J. G. , Müller-Eberhard H. J.
Published: January 2023

Human terminal C complexes C5b-6, C5b-7, C5b-8, and C5b-9 express neoantigenic determinants (neoAg; Kolb, W. P. and Müller-Eberhard, H. J., Proc. Nat. Acad. Sci. 72, 1687, 1975). Complex-specific antiserum (anti-neoAg) detected neoAg on 27 ± 11% of viable human peripheral blood leukocytes (PBL) when prepared from defibrinated blood by Ficoll-Hypaque sedimentation. Specificity of anti-neoAg was established by immunodiffusion, by agglutination of EAC1-7, but not EA, E, or EAC4,2,3b, and by inhibition of EAC1-7 agglutination by C5b-9 complex. Specificity of the fluorescent staining reaction was established by inhibition of staining with C5b-9, but not with fresh serum, and by fluorescent staining of EAC1-7, but not EAC4,2,3b, EA, or E. In addition to neoAg, the individual components, C5, C6, C7, C8, and C9 were detected on PBL and the coincidence of C5 and neoAg on the same cells was 83%, and of C8 and neoAg 79%.
Characterization of the Primary IgM Response to Gat and GT: Conditions Required for the Detection of IgM Antibodies
Authors: Waltenbaugh Carl , Dessein Alain , Benacerraf Baruj
Published: January 2023

Primary IgM antibody responses to synthetic linear copolymers of L-glutamic acid, L-tyrosine, and L-alanine were investigated. The appearance of primary IgM anti-GAT antibodies was detected in BALB/c mice by using a solid phase radioimmunoassay (SPRIA) procedure. This finding was verified for GAT in responder mice and GAT-MBSA and GT-MBSA in nonresponder mice in an indirect plaque forming cell (PFC) assay by using a rabbit antiserum directed against the μλ myeloma protein, MOPC 104E. Facilitated IgM PFC could be inhibited by a purified μκ myeloma protein, TEPC 183. Maximal facilitated IgM plaque response was found to precede the IgG response by several days. A direct plaque assay was developed for the detection of IgM anti-GAT plaques using poly-L-lysine (PLL) to couple GAT to sheep erythrocytes (SRBC). GAT-SRBC coupled by the PLL method optimally couple 4 to 5 times less antigen to the indicator cell surface than does the CrCl3-coupling method routinely employed in our laboratory. These findings were extended to a conventional antigen, chicken gamma globulin (CγG). We found that a less dense epitope coat on the indicator cell surface favors detection of direct IgM PFC, whereas a more densely coated indicator cell favors the detection of facilitated IgM and IgG PFC responses.