bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement Depletion in Cryptococcal Sepsis
Authors: Macher Abe M. , Bennett John E. , Gadek James E. , Frank Michael M.
Published: January 2023

Marked depletion of serum opsonins and complement (C) components was documented during cryptococcal septicemia in three patients. Phagocytic indices (PI) obtained with normal (nl) neutrophils and sera from these patients were <0.2 (nl 1.8 to 2.3). Serum total hemolytic C (CH50) was <10% of normal; C3 levels by immunoassay were depressed in all three patients (values were 23, 37, and 24 mg%; nl 80 to 140). Serum C4 levels by bioassay were normal in two patients and depressed in a single patient whose serum contained IgG antibody to Cryptococcus neoformans (CN) (C4 level 2956; nl 70,000 to 193,000). Cleavage products of factor B in serum were demonstrated in the three patients by immunoelectrophoresis. Ability of these sera to mediate C3 binding to CN was severely impaired. Direct immunofluorescence assay detected no C3 fixation to CN. Radioimmunoassay (RIA) of two patients' sera diluted 1/20 detected 36 and 26% normal binding of C3 to CN. Fluorescent and RIA assays with CN-absorbed sera and MgEGTA sera suggest an important role for the alternative C pathway in fixation of opsonic C3 fragments to the CN surface. No depletion of serum opsonins or C components was found in six other patients with cryptococcosis who were not fungemic at time of study.

Guinea pigs given 6 × 108 CN developed fungemia and died within 2 weeks. Their sera revealed depletion of serum opsonins and hypocomplementemia. Mean serum PI was 1.0 (nl 2.2 to 3.3) and mean CH50 was 99 (nl 150 to 300 units/ml). Heat-killed CN in intracardiac doses of 2 × 109 and 5 × 109 cells produced the same phenomenon acutely. Sera obtained at 60 min had PI of 0.7 and 1.1 and CH50 of 119 and 103.

Depletion of serum C components accompanied CN fungemia in both man and experimentally infected guinea pigs. Yeast within the bloodstream may be the direct cause of hypocomplementemia. The resulting loss of serum opsonins may further impair the host's ability to contain the infection.
Metabolic Study of Human IgE: Evidence for an Extravascular Catabolic Pathway
Authors: Iio Atsushi , Waldmann Thomas A. , Strober Warren
Published: January 2023

Immunoglobulin E (IgE) is catabolized at a greater rate than any of the other immunoglobulins. To determine if this is due to an additional pathway of catabolism operating partly in the extravascular compartment, metabolic studies of radioiodinated IgE along with the other four major immunoglobulins were performed in 26 patients with nonallergic diseases and in two normal volunteers. The data of IgE and IgG metabolism were analyzed first by using computer fitting to compartmental models with and without an extravascular catabolic pathway. It was found that the IgE data could be best fit to the former model, whereas the data of IgG metabolism could be best fit to the latter model. The data of IgE and other immunoglobulins were then analyzed with the method of Nosslin. This method provides an intercept value from plots of calculated values derived from the amount of radioiodinated immunoglobulin in the serum and radioiodide in the urine at different time points after injection of labeled immunoglobulin. If the intercept value is equal to 1, there is no extravascular catabolism. The mean intercept value of IgE was 0.68 ± 0.15, which was significantly less than 1 (p < 0.001); similarly, the mean intercept value of IgD was 0.80 ± 0.11, which was also significantly less than 1 (p < 0.01). In contrast, the mean intercept values of IgG, IgA, and IgM were 0.92 ± 0.05, 1.14 ± 0.12, and 1.20 ± 0.31, respectively, which were not significantly different from 1 (p > 0.01). The results indicate that there is definite extravascular catabolism of IgE and possible extravascular catabolism of IgD, whereas there was no significant extravascular catabolism of the other three major immunoglobulin classes (IgG, IgA, and IgM). It is reasonable to propose that this extravascular catabolism is part of a unique catabolic mechanism specific for IgE which is related to unique interactions of IgE with basophils and mast cells.
Misuse of the C3H/HeJ Mouse in the Determination of Endotoxin Contamination of Various Biologic Preparations
Authors: Walker Sharyn M. , Weigle William O.
Published: January 2023

Much interest was generated when the C3H/HeJ mouse was considered to be refractory to endotoxin, a biologically active product released from Gram-negative bacteria (1, 2). This interest was partly because such a mouse would permit a simple, indirect assay for possible endotoxin contamination in biologic preparations exhibiting the endotoxin-like activity of B cell mitogenesis. For example, if C3H/HeJ splenic B cells with a presumed inability to respond to endotoxin proliferated upon exposure to a certain substance, the mitogenic effect would presumably be due to that substance, not to endotoxin contamination. A precedent for use of the C3H/HeJ mouse in this manner was set with purified protein derivitive (PPD) (1). It was shown that PPD caused proliferation of splenic mouse B cells, including those of the C3H/HeJ to levels equaling that of other mice. Since the C3H/HeJ was considered a low responder to endotoxin, endotoxin contaminants in PPD were excluded as the source of B cell mitogenicity.
Enhancement of Human Eosinophil Complement Receptors by Pharmacologic Mediators
Authors: Anwar A. R. E. , Kay A. B.
Published: January 2023

Human eosinophils and neutrophils were shown to have receptors for rabbit and human IgG, and for the human complement components C4, C3b, and C3d (A. R. E. Anwar and A. B. Kay (1977), J. Immunol. 119: 976, 1977. We now report that agents which have a selective effect on human eosinophil motility in vitro, such as the ECF-A peptides and histamine, also enhance the number of receptors for C3b and C4, but not for IgG and C3d. The ECF-A tetrapeptides (Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu) and histamine increased the percentage of eosinophils forming rosettes with EAC3b by approximately 90%, an effect that was observed in a dose- and time-dependent fashion. Neutrophil and monocyte C3b receptors were unaltered by this treatment and other pharmacologic agents such as bradykinin and the prostaglandins E1, E2, and F2α were without effect on any of the cell types when tested in the dose range 10-4 to 10- mole/1.
Evidence for a Linkage Group of the Polymorphic Complement Proteins C4, C2 and Factor B Related to the MHC of the Guinea Pig(GPLA)
Authors: Bitter-Suermann D. , Krönke M. , Hoffmann T. , Geczy A. F. , Hadding U.
Published: January 2023

Plasma probes of outbred and inbred strains of guinea pigs were investigated by agarose high voltage electrophoresis and isoelectrofocusing in PAGE for polymorphism of the complement proteins C4, C2 and factor B of the alternative pathway. In breeding experiments mode of inheritance and linkage to the GPLA system was studied. The polymorphic complement proteins C4 (with the allelic structural genes C4F(fast), C4S(alow), C4S1 and C4o(deficient)) factor B (with the common alleles BfF and BfS) and C2 (with the frequent alleles C2B(basic), C2A(acidic), and C2A1) are closely linked together. In inbred strains haplotypes of the composition: C4F-BfS-C2A1 or alternatively C4S or S1-BfF-C2B were found. The mode of inheritance of C2A and its linkage to C4 and Bf is a matter of further breeding studies. All three complement proteins are linked to the GPLA system as shown by family studies with well characterized and GPLA-typed inbred strains and outbred animals.
The Activation of the Complement System by Paracoccidioides brasiliensis in Vitro. Its Opsonic Effect and Possible Significance for an in Vivo Model of Infection
Authors: Calich Vera L. G. , Kipnis Thereza L. , Mariano M. , Dias Da Silva W.
Published: January 2023

The yeast phase of P. brasiliensis is poorly phagocytosed by “stimulated” mouse peritoneal macrophages in culture. However, either heterologous (sera from patients with South American Blastomycosis) or homologous antibodies (sera from mice chronically infected with P. brasiliensis) greatly enhanced the phagocytosis of P. brasiliensis organisms by macrophages. The same effect was observed when guinea-pig, mouse, or human sera were added to the fungus-macrophage cultures. Heating of the sera at 56°C or the addition of EDTA to the system completely abolished this effect. Conversely, EGTA had no effect. These data indicate that the alternative pathway of complement activation is involved in the expression of this phenomenon. This conclusion is further supported by the fact that Cobra venom Factor (CoF) treated serum, properdin, or factor B depleted serum had no opsonic properties. When yeast cells of P. brasiliensis were incubated with fresh normal serum, the third component of the complement system changed its electophoretic mobility of β1C to β1A.
Brazilian Snake Serum and Venom: Studies of the Alternative Pathway and C3 in Man and Serpent
Authors: Calich Vera L. G. , Kipnis Thereza L. , Dias da Silva W. , Alper Chester A. , Rosen Fred S.
Published: January 2023

It was previously found that antiserum to the anti-complementary protein (CoF) in Naja naja venom cross-reacted with a cobra serum protein with many of the characteristics of C3. Cobra serum but not human C3b inactivator destroyed the activity of CoF.

In the present study, the serum and venom of several types of Brazilian snakes were investigated with respect to 1) reactivity with anti-CoF, 2) ability of the venoms to convert human serum C3, 3) ability of the serum to inactivate CoF, and 4) serum alternative pathway activity.

The serum, but not the venoms, of Bothrops jararaca, B. jararacussu, B. moogeni, and Crothalus terrificus contained a protein reactive with anti-CoF but failed to destroy the alternative pathway activity of CoF for human serum. The anti-CoF-reactive serum protein in each of the Brazilian snake serums converted to a more cathodal form on incubation of the serum with Escherichia coli 055:B5 endotoxin (ET), CoF, zymosan (Z), and BSA-rabbit anti-BSA-specific precipitates (AgAb) but not on heating at 52°C for 30 min nor incubation with hydrazine.
Initial Characterization of the Human Polymorphonuclear Leukocyte (PMN) C5a Receptor
Authors: Chenoweth D. E. , Hugli T. E.
Published: January 2023

It has been postulated that the potent chemotactic properties of the complement-derived anaphylatoxin C5a are due, at least in part, to the interaction of the peptide with a specific PMN receptor. We have examined the nature of this receptor by two techniques. In the first case, the receptor has been directly demonstrated by examining the cellular binding of 125I-labeled human C5a. Kinetic studies showed that uptake of the labeled peptide was essentially complete within 1–3 min. The C5a binding data obtained under equilibrium conditions, over the concentration range of of 10-11 to 10-7 M, indicated the ED50 for specific binding was 2–6 × 10-9 M. This value is in close agreement with an ED50 of 2–8 × 10-9 M determined for PMN chemotaxis. A total of 1–3 × 105 binding sites per cell was estimated from the uptake results. Because C5a-induced release of lysosomal enzymes from cytochalasin B-treated PMNs closely parallels the binding of C5a, enzymatic release provides direct measure of the C5a-cellular interaction.
Absence of non-DFP-Inhibitable Activities of Factor D̄
Authors: Dieminger L. , Vogt W.
Published: January 2023

Factor D is an active serine esterase capable of specifically cleaving factor B in the presence of C3b and thereby generating the active C3- and C5-convertase C3bBb. In the absence of C3b when nephritic factor (NF) or activated properdin (P̄) is available, D̄ appears to have a different function. Native C3, B, and NF or P̄ form a complex which, dependent on traces of D̄ has C3 cleaving, C3b generating activity. B seems to remain in this complex in its uncleaved form [Schreiber et al., J. Exp. Med. 142, 760 (1975)]. This suggested to us that D̄ might have other than hydrolytic activities, the more so as we found it difficult to completely block its functional activity in the generation of B-dependent enzymes by the esterase inhibitor, DFP.
Single-Chain C4 from Human Plasma
Authors: Gorski J. P. , Müller-Eberhard H. J.
Published: January 2023

Serum C4 has been shown to be composed of three disulfide-linked nonidentical subunits. Recently, Hall and Colten (PNAS 74, 1707, 1977) achieved cell-free synthesis of guinea pig C4 and thereby demonstrated the existence of a single-chain form of C4 (Pro-C4). We now wish to report the isolation of Pro-C4 from human plasma.

Plasma was obtained from fresh, unclotted blood and plasminogen removed by affinity chromatography. Crude total serum C4 was obtained by adsorption to and elution from anti-C4 Sepharose 4B. Gel filtration on Sepharose 6B in 6 M guanidine hydrochloride (GuHCl) and 0.6 M ε-aminocaproic acid removed contaminants. Subsequent filtration on Sepharose 6B in 6 M GuHCl and 15 mM Dithiothreitol (DTT) separated single-chain C4 from the three C4 subunits.

Identification of Pro-C4 rests upon its similarity to three-chain C4 with respect to molecular size, amino acid composition and antigenicity. Specifically, comparison of the amino acid composition of both forms revealed them to be indistinguishable.
Biological Activities of the Purified Ba Fragment Derived from Guinea Pig Factor B of the Alternative Pathway
Authors: Hadding U. , Hamuro J. , Bitter-Suermann D.
Published: January 2023

While the central function of the Bb fragment as C3 activating enzyme is well recognized, there are up to now no reports concerning the smaller fragment Ba. To ascertain whether Ba exhibits biological functions the following studies were performed.

Factor B was purified from EDTA-plasma using a seven-step-procedure. The last step was an affinity chromatography at low ionic strength (1 mM MgCl2) on a sepharose 4B column, to which cobra venom factor was coupled. The final product was homogeneous on SDS-PAGE and showed only one precipitation line after immunoelectrophoresis using two sera: goat anti-whole guinea pig serum and goat anti-guinea pig factor B.

Factor B was cleaved by immobilized cobra venom factor and D̄. Fragment Ba was recovered from the filtrate of the reaction mixture while fragment Bb was eluted by 2M NaSCN. The m.w. for B, Bb and Ba as determined by the SDS-PAGE method was 106,000, 65,000 and 53,000, respectively. Reduction did not change the picture, indicating a single chain structure.
The Physiological Breakdown of C3b
Authors: Harrison R. A. , Lachmann P. J.
Published: January 2023

The requirements for and products of C3b inactivator (KAF) action on C3b have been studied. C3 was purified from human serum. Final traces of contaminating proteins were removed by passage through a Sepharose “anti-impurity antiserum” column. C3b was generated by using insolubilized cobra venom factor convertase, and separated from C3a by chromatography on hydroxyl-apatite.

The KAF-dependent cleavage of fluid phase C3b was found to have an absolute requirement for β1H (or possibly a contaminant of β1H preparations); neither KAF nor β1H alone had any discernable proteolytic action on C3b. The initial pattern of cleavage was found to be the same with purified KAF and β1H or with serum (as a source of these factors). The α-chain of C3b was cleaved into two polypeptide chains, an α1a chain of about 68K and an α1b chain of about 46K. The α1b chain was rapidly ‘trimmed’ to about 43K (α1b1). All of these three α-chain fragments were still covalently bonded to the β-chain.
Deviated Lysis: Generation of the Activity by Physicochemical Means
Authors: Hänsch Gertrud , Rother Ursula , Rother K.
Published: January 2023

The term deviated lysis (d.l.) describes a particle bound or fluid phase C function, which lyzes unsensitized cells (E) in the presence of EDTA. Its generation via the classical or the alternative pathway has been described. The activity was stable in the fluid phase. It was found in the region of 180,000–220,000 daltons or 9.7S, respectively, and consisted of C5, C6, C7, C8 and C9. Once generated, the activity could be blocked by the respective antibodies, but not by anti-C3 or anti-B.

We now report on a similar activity generated by exposing human serum either to acid pH or low ionic strength. As in C activation by inulin, the factors C5–C9 are involved. Although factor B is converted when normal human serum is brought to pH 6.4, C3 is not. C3, factor B, or properdin, respectively, are not needed. The activation takes place at 0°C within seconds.
The Inhibition of B Lymphocyte Stimulation by C3b Inactivator
Authors: Hartmann Klaus-Ulrich
Published: January 2023

Purified human C3b added to mouse lymphocyte suspensions triggers stimulation of up to 50% of the B lymphocytes; the stimulation was detected by increased incorporation of thymidine, blast cell formation, and antibody secretion (K.-U. Hartmann and V. A. Bokisch, J. Exp. Med. 142, 600, 1975). Purified human C3 does not stimulate these lymphocytes. Addition of purified C3b inactivator (C3bINA) (M. K. Pangburn, R. D. Schreiber and H. J. Müller-Eberhard, J. Exp. Med. 146, 257, 1977) — 1 to 2 µg C3bINA to 100 µl lymphocyte cultures — strongly inhibited the stimulation of B lymphocytes induced in the presence of C3b or LPS. This inhibition was not dependent or enhanced by the addition of purified human β1H-globulin. The inhibition caused by the addition of C3b INA was reduced or prevented by the presence of FCS. Preincubation of C3b with C3bINA did not inactivate the activity, leading to stimulation in the presence of FCS. These experiments suggest an activity of C3bINA directed toward the cells involved.
Influence of Antibody on the Rate of Alternative Pathway Activation
Authors: Johnston Richard B. , Polhill Rutherford B. , Newman Simon L. , Pruitt Kenneth M.
Published: January 2023

Using a kinetic assay, we have examined the role of antibody in the lysis of rabbit erythrocytes (RaRBC) through the alternative complement pathway. Sera from 7 of 11 hypogammaglobulinemic (Hγ) humans and all agammaglobulinemic chickens tested had subnormal activity in the assay. γ-Globulin injections restored normal activity to the two Hγ patients studied. Heated normal human or chicken sera, but not heated Hγ sera, normalized activity in deficient Hγ serum and initiated hemolysis in the presence of rabbit serum as complement source. Absorption of heated normal human serum with RaRBC, but not with sheep erythrocytes or zymosan, removed its ability to reconstitute deficient Hγ serum. Normal hemolytic activity could be restored to Hγ serum with human IgM, IgG, or colostral IgA, with small amounts of goat anti-RaRBC IgG, or with an eluate from serum-sensitized RaRBC, but not with myeloma IgA. Restoration of hemolytic activity to Hγ serum could be achieved in a dose-dependent fashion with the F(ab′)2 fragment of IgG.
Cytotoxic Effect of Normal Human Sera on Murine Lymphoma (EL4) Cells. Evidence for a Role of Activation of Complement System
Authors: Kuo Chao-Ying
Published: January 2023

Normal human sera have been shown to kill murine target cells, including both normal lymphoid and tumoral cells. In this study, the evidence of activation of complement system for this heterocytotoxicity is presented. Chemical carcinogen-induced EL4 lymphoma cells were used as target cells. The EL4 cells were obtained either from the ascitic fluid of tumorous mice or from tissue culture. The cytotoxicity of human serum was determined by microscopic examination of viability of target cells after incubation with serum samples. Viable cells were determined by exclusion of trypan blue dye. The hemolytic activity of serum complement was quantitated by microtiter plate assay with EA cells.

The evidence for the activation of complement system responsible for the heterocytotoxicity of human serum to EL4 cell was obtained from decomplementary experiments. 1) Both the cytotoxic effect and hemolytic activity of human serum was totally abolished after heating at 56°C for 30 min.
Analysis of the Mechanism of VSV Inactivation by Human Serum
Authors: Mills B. J. , Cooper N. R.
Published: January 2023

Vesicular stomatitis virus (VSV), a mammalian RNA virus enclosed by a lipid bilayer membrane of host cell origin, is inactivated by normal human serum. Several possible mechanisms of this inactivation were examined. 1) Neutralization by antibody alone. This possibility was ruled out by showing that virtually all (>90%) of the neutralizing activity was lost after heating the sera. Also most preparations of human IgG, IgA, or IgM failed to neutralize VSV. 2) Neutralization by a combination of low levels of antibody together with complement. Anticellular antibody is unlikely since human sera effectively neutralized VSV grown in cell lines from multiple species even after absorption with uninfected cells in which the virus had been grown. Similarly, anti-viral antibody appears unlikely since normal human serum effectively neutralized after absorption with cells expressing VSV antigens. Furthermore, purified human IgG did not potentiate the neutralizing ability of normal serum.
Effect of Lipids on the Cytolytic Action of Complement (C)
Authors: Ohanian Sarkis H. , Schlager Seymour I. , Borsos Tibor
Published: January 2023

Several recent reports raised the possibility that the target for the action of C (particularly the late acting components) in the cell membrane may be lipids or phospholipids. We have postulated that under certain conditions (e.g., after partial activation) C action could be inhibited by lipids found in cell membranes. The following lipids were tested: phosphatidyl glycerol (PG), cardiolipin (CL), cholesteryl esters (CE), phosphatidyl serine (PS), phosphatidyl ethanolamine mammalian (PEm) or bacterial (PEb), sphingomyelin (SP), cholesterol (CH), triglyceride, or phosphatidic acid. Lipids were mixed into VBS-gelatin and sonicated until a stable suspension was obtained. From dose response and kinetic studies we determined that about 10 min incubation of C at 0°C with 30 µg of lipids was appropriate for screening for inhibiting activity. The following pattern of inhibition of lysis emerged: 1) lysis of EA by limiting HuC and GPC: human C, no inhibition; GPC: whole C and C1̄ by PG only; C8, C9, no inhibition.
The Structural C6 Polymorphism: Nonlinkage Between C6 and Chromosome 6 Marker Loci
Authors: Olving J. H. , Olaisen B. , Teisberg P. , Gedde-Dahl T. , Thorsby E.
Published: January 2023

The complement factor 6 polymorphism has been examined in a Norwegian family material by two techniques: either high voltage agarose gel electrophoresis or isoelectric focusing in polyacrylamide gel slabs. Proteins with C6 activity were visualized by a specific, hemolytic assay. Agarose electrophoresis gave good separation of common allele products and easy discrimination between the common C6 types: C6A, C6AB, and C6B. Prolonged isoelectrofocusing gave better band separation, but the discrimination between the common C6 types is not noticeably better than with the agarose technique. For detection of rare alleles isoelectrofocusing is presumably the method of choice.

In a Norwegian population sample, C6 gene frequencies were: C6A: 0.61 and C6B: 0.39.

The data on the linkage relationship between the HLA region loci and C6 show that C6 complement locus is not within measurable distance to the HLA region. Linkage studies also indicate nonlinkage between C6 and another chromosome 6 marker locus, PGM3. C6 has therefore been excluded from a substantial portion of the short arm of chromosome 6.
Interaction Between Lymphocytes and Fluid Phase Murine and Human C3b
Authors: Papamichail M. , Pepys M. B.
Published: January 2023

C3 participates in lymphocyte cooperation leading to antibody formation and in antigen-localization by germinal centers. These functions probably involve the adherence of fixed C3b to complement receptors on lymphocytes and possibly macrophages. During investigation of the role of C3 in antibody formation in human and murine in vitro culture systems, the interaction of purified fluid phase C3b with lymphocyte complement receptors was studied by immunofluorescence. Human peripheral blood and tonsil complement receptor lymphocytes (CRL) were stained by incubation with human or murine C3b followed by specific F(ab)2 anti C3-FITC. Murine CRL failed to stain with these same reagents, despite the fact that murine C3b blocked formation of complement-dependent rosettes. Binding of mouse C3b to murine CRL was directly demonstrated using C3b itself labeled with fluorescein and could be blocked by unlabeled mouse C3b. It affected the same cells as formed C3-dependent rosettes, none of which were T lymphocytes.
Stable Evolutionary Conservation of Complement-Activating and Related Pentagonal Serum Proteins: C-reactive Protein and Protein SAP
Authors: Pepys M. B. , Dash A. C. , Richardson N. , Munn E. A. , Feinstein A. , Fletcher Thelma C.
Published: January 2023

Human CRP efficiently activates the classical complement pathway via C1q. Protein SAP (amyloid P-component) is a normal plasma protein which resembles CRP structurally, but differs in not being an actue phase reactant. Both proteins can interact with cell membranes but their in vivo functions are not known; they are presumably important since, as we now report for the first time, very similar molecules are also present in the sera of diverse species including elasmobranchs, teleosts, amphibia, birds and non-primate mammals. CRP and protein SAP were isolated by calcium-dependent affinity chromatography on insolubilised pneumococcal C-polysaccharide and agarose respectively, followed by gel filtration. The different proteins all had pentagonal disc-like molecules in the electron microscope and there were marked similarities between the CRP and SAP of such diverse species as the plaice and man. The pure proteins each yielded homogeneous subunits in SDS-PAGE with molecular weights of between 20–30,000 in different species.
A New “Inhibitor” of Complement (C5)-Derived Chemotactic Activity
Authors: Daniel Perez H. , Manning Donna , Lipton Mark , Goldstein Ira M.
Published: January 2023

In the course of examining polymorphonuclear leukocyte (PMN) chemotaxis in patients with systemic lupus erythematosus (SLE), we have found a previously undescribed serum “inhibitor” of complement (C5)-derived chemotactic activity. Serum from a 25-year-old Black female with untreated SLE, when activated with zymosan, failed completely to attract either her own or normal PMN (measured by the “leading front” method of Zigmond and Hirsch). Incubation of normal PMN with the patient's serum (PS) did not affect random motility or chemotaxis towards normal zymosan-treated serum (ZTS). Whereas levels of C3 were modestly low in PS, no gross abnormalities involving alternative complement pathway activation could be detected. Furthermore, treatment of PS with zymosan resulted in the generation of normal amounts of C5-derived PMN lysosomal enzyme releasing activity (attributable to “C5a”). Incubation of normal ZTS or column (Sephadex G-75)-purified “C5a” with PS did not affect lysosomal enzyme releasing activity but did result in a significant dose-dependent diminution of chemotactic activity.
SC5b-9: Regeneration of the Ability to Interact with Lipid by Selective Removal of the S-Protein
Authors: Podack E. R. , Halverson C. , Esser A. F. , Kolb W. P. , Müller-Eberhard H. J.
Published: January 2023

The various complexes were prepared by mixing isolated C5b-6 in the presence of detergents or phospholipids with equimolar amounts of C7, C7 and C8, or with C7, C8 and C9 in molar ratios of 1:1:1:3. C5b-9 was also prepared by treating the SC5b-9 complex purified from inulin activated serum with 6 mM Na deoxycholate (DOC) at pH 8.1 and subjecting it to gel filtration on Sepharose 6B in the presence of DOC. This treatment resulted in selective dissociation of the S-protein, in exchange for binding of DOC by C5b-9.

Detergent-binding studies showed that C5b-6, C5b-7 and C5b-8 bind 26, 28 and 65 mole 14C-DOC per mole of complex, respectively, as measured by equilibrium dialysis. C5b-9 (prepared from SC5b-9) binds 86 mole DOC per mole of complex, measured by gel filtration in the presence of 3H-DOC. DOC binding as well as cetyl trimethyl ammonium bromide (CTAB) binding of all complexes could also be demonstrated by charge shift electrophoresis.
Characterization of the Ss and S1p Genetic Variants of Mouse C4
Authors: Roos Maria H. , Atkinson John P. , Shreffler Donald C.
Published: January 2023

The genetic control of quantitative (Ss) and qualitative (S1p) variations in two closely related mouse serum proteins has been localized to the S region of the mouse major histocompatibility complex (H-2). These serum proteins were found to be immunochemically and probably functionally homologous to human C4. Further biochemical and genetic characterizations of the Ss and S1p proteins have been done by immunoprecipitation techniques. Adherent peritoneal exudate cells from mice of strain B10.W7R, which express both the Ss and the S1p proteins, were cultured in vitro in medium containing 14C-labeled amino acids. These cells synthesized both Ss and S1p, as revealed by immunoprecipitation with anti-Ss and anti-S1p from the 14C-labeled, 24 hr macrophage culture supernatant. After SDS solubilization and 2-ME-reduction of the precipitates and separation on an SDS polyacrylamide slab gel, autoradiography revealed three major bands of activity with each antiserum. With anti-Ss, these polypeptides had m.w. of 98,000, 75,000, and 33,500, comparable to the α β and γ chains of human C4.
Murine Factor B (Bf): Electrophoretic Polymorphism and Linkage to the H-2 Region
Authors: Rubinstein Pablo , Day Noorbibi K.
Published: January 2023

Mouse Bf measured by indirect lysis with glutathione-treated cells was shown to be present in high quantities in chemically induced mouse ascites fluid (MAF). Functional MAF Bf has physical properties identical to mouse serum Bf. It is heat labile, Mg++ dependent, and is activated by purified CVF. Purification was carried out with Bio-Rex 70, CM 32, and DE 52 column chromatography. The optimum isoelectric pH was previously determined by isoelectric focusing. Fractions for Bf were tested by immunochemical means by using cross-reacting goat antihuman Factor B and by functional assays. Antiserum to purified Bf was prepared in the goat. By immunoelectrophoresis and rocket electrophoresis MAF Bf and serum Bf migrated anodally. Cleavage of a cathodal migrating Bf fragment was observed with purified CVF. Bf phenotypes were typed by the immunofixation procedure. Sera from male and female mice of 16 inbred strains were examined (A/J, A.KR, CBA, C3H, B10.BR, C58, B10, B10.A, C57L, C57 BL/Ks, B10.D2, DBA/2, DBA/1, BALB/C, 129/J).
Nephritic Factor (TA): A Homogeneous Immunoglobulin Directed Toward the Complex of C3 and Factor B of Human Complement
Authors: Schreiber R. D. , Muller-Eberhard H. J.
Published: January 2023

Nephritic factor (NF) was isolated from serum T.A., NF(TA), as we have previously described, including selective binding to and decay-release. After two such cycles, 90% of the protein bound to compared to 2% specific binding of partially purified material consisting only of IgG. Purified NF(TA) did not bind to EC3b in absence of B and D, nor to C3bINA and β1H-treated EC3b in presence of B and D. The chain structure of NF was examined by SDS PAGE of the reduced protein after iodination using the chloramine-T (CT) or the Bolton Hunter (BH) method. According to the distribution of radioactivity, the subunit size of CT labeled NF was 60,000 daltons (92%) and the subunits of BH labeled NF were 60,000 (67%) and 25,000 daltons (33%). IgG iodinated by either method showed subunits of 52,000 (67%) and 25,000 daltons (33%).
Studies of a Polysaccharide in Ant Venom (Pseudomyrmex sp.) that Activates the Classical Complement (C) Pathway
Authors: Schultz D. R. , Arnold P. I. , Wu M-C , Volanakis J. E.
Published: January 2023

A polysaccharide (PS) from the venom of a tropical ant interacts with a human nonimmunoglobulin protein(s) in serum, causing C1 activation and the consumption of the functional activity of C4 and C2 (D. R. Schultz, P. I. Arnold, J. Immunol., in press). We now describe these studies in more detail.

The PS had a heterogeneous size in native ant venom. The smallest moiety that activated serum C1 had a m.w. of ca. 3,000 as determined by gel chromatography. The PS was not precipitated by barium salts, showing no apparent sulfate groups. The following sugars and the molar ratios were found by gas chromatography: mannose (7.7), fucose (1.8), N-acetylgalactosamine (1.6), galactose (1.4), glucose (1.0), and N-acetylglucosamine (1.0).

Evidence showing that a serum β-lipoprotein (B-LP) participates with venom PS to cause activation of C1 includes: 1) The PS caused a visible precipitation when incubated with normal human serum or its highly purified B-LP at 37°C for 12 hr.
The Immunoglobulin Nature of Nephritic Factor: NEF Activity Is Found in Fab and Fab′2 As well As in Intact IgG
Authors: Scott D. M. , Amos N. , Sissons J. G. P. , Peters D. K.
Published: January 2023

In earlier studies1 we were unable to separate Nephritic Factor (Nef) from IgG by a variety of immunochemical techniques, indicating that Nef shares antigenic determinants with IgG. Purified Nef was found to bind to Protein-A-Sepharose, and could be eluted with acid. This binding was complete in three samples of Nef from patients with PLD or MPGN, but only partial in the fourth.

We have now investigated the effects of pepsin and papain digestion on Nef structure and activity. Digestion of Nef with pepsin caused virtually no loss of activity. Before pepsin digestion this Nef preparation bound completely to Protein-A-Sepharose. After pepsin treatment 40 to 65% of the Nef was now recovered in the breakthrough peak and only 7% was eluted in the acid eluate. Digestion of Nef with papain caused some loss of activity. Approximately 30 to 80% of the activity was recovered.
Complement Metabolism in Sickle Cell Disease (SCD)
Authors: Sisson J. G. P. , Wilson W. A. , Thomas J. , Frampson G.
Published: January 2023

Individuals with homozygous Sickle Cell Disease (SCD) are prone to bacterial infection. SCD sera have previously been shown by others to have a defective capacity to opsonify pneumococci—this has been attributed to impaired alternative pathway function, which was postulated to be due to defective synthesis of factor B in such cases.

Sera from 100 cases of homozygous SCD have now been studied. There was reduced alternative pathway activity in one-third of sera (using a haemolytic plate assay dependent on Mg++ and agarose to activate the alternative pathway), and haemolytic factor B activity was significantly lower in this group than in the other sera. C3 concentrations were also reduced in one-third of sera.

The metabolism of purified radiolabeled factor B and C3 was studied in 12 cases to determine whether diminished synthesis or increased catabolism of these proteins occurred in SCD. Synthesis of B and C3 was normal or increased in all cases.
Utilization of Properdin in Children with Vasculitis and Milk Allergy
Authors: Spitzer R. E. , Stitzel A. E. , Urmson J. R.
Published: January 2023

Patients with various types of vasculitis, both nonspecific and that associated with more definitive syndromes such as Henoch Schönlein Purpura (anaphylactoid purpura), have serologic findings which indicate that properdin is consumed during disease activity. In all of 22 patients with documented Henoch Schönlein Purpura, the level of serum properdin was reduced more than three standard deviations below the mean in the first week of the disease. Those patients with predominant signs of abdominal pain and glomerulonephritis had more pronounced reductions in serum properdin than did those with arthritis as a presenting symptom. All other serum C components and alternative pathway proteins including C1–C9, factor B, and factor D were normal. Serum levels of properdin convertase were reduced in 12 of the 22 patients. As the disease subsided, levels of properdin and properdin convertase returned to normal. Properdin has also been found deposited in the glomerulus of these patients by immunofluorescent studies of renal biopsies.
Studies on the Liberation and Cleavage of Lipids from the Erythrocyte Membrane During Complement Lysis
Authors: Valet G. , Kahle H. , Scherer R.
Published: January 2023

Inoue et al. (J. Immunol. 116, 1737, 1976) and Shin et al. (Fed. Proc. 36, 1209, 1977) have reported that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are liberated from liposomes during C lysis. We have investigated if this occurs also in erythrocyte membranes. Radiolabeled PC, PE, glycerinetrioleate (TO), glycerinetripalmitate (TP), or free fatty acids (FFA) were incorporated in separate experiments into the membrane of sensitized (EA) and unsensitized (E) sheep erythrocytes by incubation at 4°C. After several washes with isotonic buffer, the erythrocytes were lyzed with distilled water. This procedure removed the nonmembrane-bound lipids from the exterior and interior of the cells. EA ghost membranes prepared in this way are still reactive to the C action (Valet et al., J. Immunol. 115, 1028, 1975). Half of the EA ghosts were incubated with fresh, and the other half with 56°C inactivated guinea pig serum for 2 to 5 min at 37°C.
Identification of C3 Nephritic Factor
Authors: Williams D. Gwyn
Published: January 2023

The serum from two patients—one with mesangiocapillary glomerulonephritis (MCGN) and one with partial lipodystrophy and MCGN-with C3 nephritic factor (C3NeF) was separated chromatographically on DEAE, CMS, and Sephadex G-200. In each case only IgG was detectable on double immunodiffusion in the final C3NeF-containing fractions. IgG from these fractions was adsorbed onto a CNBr-anti-IgG column, and on elution the IgG was shown to have C3NeF activity. Removal of IgG from the fractions by immunoadsorption with solidified monospecific anti-IgG resulted in removal of C3NeF activity. Analysis by double immunodiffusion of the IgG with C3NeF activity in each case showed it to contain more than one subclass of IgG, and that both κ- and λ-chains were present. It is concluded that in these two patients the C3NeF activity resided in nonrestricted IgG.
Activation of Phospholipid Metabolism During Mediator Release from Stimulated Rat Mast Cells
Authors: Kennerly Donald A. , Sullivan Timothy J. , Parker Charles W.
Published: January 2023

Phospholipid metabolism was studied during mediator release from highly purified rat mast cells. The incorporation of 32PO4 into individual phospholipids was determined after isolation of phospholipid classes by two-dimensional thin layer chromatography. Various stimulators of histamine release (anti-IgE antibody, concanavalin A [Con A], compound 48/80, or the ionophore A23187) increased 32PO4 incorporation into phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylcholine (PC) 4- to 10-fold within 15 min. No change in 32PO4 incorporation into phosphatidylserine (PS), phosphatidylethanolamine, or sphingomyelin was detected. Anti-IgE antibody caused significantly increased labeling of PA within 8 sec and of PI and PC within 30 sec. The concentrations of anti-IgE and Con A causing threshold, half maximal, and maximal phospholipid labeling were slightly less than those required for comparable changes in histamine release. Both phospholipid labeling and mediator release in response to anti-IgE or Con A were enhanced 3- to 4-fold by PS. The addition of 50 mM α-methylmannoside to mast cells 10 min after challenge with Con A rapidly halted both ongoing mediator release and PA labeling. The results of these studies indicate that marked and selective changes in phospholipid metabolism occur before as well as during mediator release from mast cells and that these reactions may be an intrinsic part of the biochemical mechanisms that control mediator release.
Slow Reacting Substances: Comparison of Some Properties of Human Lung SRS-A and Two Distinct Fractions from Ionophore-Induced Rat Mononuclear Cell SRS
Authors: Bach Michael K. , Brashler John R. , Brooks Carter D. , Neerken A. John
Published: January 2023

When mononuclear cells from the peritoneal exudates of rats are challenged with the ionophore A 23187 they produce a slow reacting substance (SRSri), and production of this activity is markedly enhanced if 0.01 M L-cysteine is also added to the incubations. Previous work from this laboratory had shown that the properties of SRSri resembled those that had been described for slow reacting substance of anaphylaxis (SRS-A) in the literature. We have now directly compared SRSri to SRS-A generated immunologically in chopped human lung. A high performance liquid chromatography separation of SRSri on Florisil has been developed by using a linear gradient of water into methanol for elution. SRSri is completely resolved into two peaks of activity, comprising approximately 33 and 67% of the total activity. SRS-A elutes at a postion that is identical to that for the smaller of the SRSri peaks. Although both peaks of SRSri activity are stable to boiling in alkali and are destroyed upon boiling in 0.1 N HCl, there are quantitative differences in the rate of destruction of the two activities and of SRS-A. Similarly, the susceptibility of the three preparations to inactivation by sulfatase from Patella vulgata was different. In addition, inhibition of contractions caused by these three preparations by the end organ antagonist of SRS-A, FPL 55712, also suggested differences between the preparations. Thus, the results suggest the existence of a family of substances that, loosely, satisfy the criteria to be referred to as SRS-A, although they differ in certain details.
Induction of a Fibrin-Gel Investment: An Early Event in Line 10 Hepatocarcinoma Growth Mediated by Tumor-Secreted Products
Authors: Dvorak Harold F. , Orenstein Neil S. , Carvalho Angelina C. , Churchill W. Hallowell , Dvorak Ann M. , Galli Stephen J. , Feder Joseph , Bitzer Ann M. , Rypysc Jane , Giovinco Patricia
Published: January 2023

Within a few hours of implantation in the subcutaneous space of strain 2 guinea pigs syngeneic line 10 tumors became invested in a fibrin-gel. We hypothesized that tumors might induce and modulate this fibrin-gel by secreting molecules capable of activating the host's clotting and fibrinolytic systems. To test this possibility, line 10 tumor cells, and several non-neoplastic control cells, were cultured for 2 to 4 hr in serum-free medium and the culture supernatants were assayed for their capacity to activate host inflammatory pathways. Four distinct mediator activities were recognized in line 10 tumor cell culture supernatants: a vascular permeability factor (VPF), a procoagulant (PC), a plasminogen activator (PA), and a macrophage-migration inhibitory activity (MIF). Nonmalignant control cells secreted none of these activities with the exception of rabbit kidney cells that not unexpectedly released a PA activity, presumably urokinase.

Based on conditions of culture, physical and chemical properties, inhibitor studies, and kinetics of secretion, it is likely that four distinct molecules are responsible for these activities. The complexity of the culture medium was not critical but low pH was an important variable favoring PA secretion. Secretion of all mediators was inhibited by cold and that of VPF and PA by puromycin. Cytochalasin B and EDTA caused significant increases in PA secretion. The line 10 PA, like that from other sources, was irreversibly inactivated by diisopropyl fluorophosphate and is probably a serine esterase.

The capacity to secrete mediators that activate important host inflammatory pathways provides tumors with a mechanism for stroma induction and may be an important factor in determining a tumor's histologic pattern and degree of malignancy.
Augmentation of Mouse Natural Killer Cell Activity by Interferon and Interferon Inducers
Authors: Djeu Julie Y. , Heinbaugh Jeffrey A. , Holden Howard T. , Herberman Ronald B.
Published: January 2023

Interferon (IF), in addition to its anti-viral capacity, is increasingly being found to be a regulator of cell division, cell surface antigens, and cell function. To determine whether IF also plays a role in the regulation of natural killer (NK) cell activity in mice, the in vivo and in vitro effects of IF and IF inducers on NK activity were studied. We observed that pyran, lipopolysaccharide, and polyinosinicpolycytidylic acid (poly I:C) as well as crude and purified IF preparations significantly elevated splenic NK levels in normal mice within 3 to 24 hr of i.p. administration. Normal spleen cells treated with poly I:C or IF in vitro also had augmented NK activity. Poly I:C and IF were themselves not cytotoxic and their presence was not required during the lytic process, indicating that IF acts on lymphocytes to activate NK function. The addition of anti-IF in the incubation medium completely blocked the boosting of NK activity by poly I:C or IF. The characteristics of the effector cells activated by IF were consistent with those of NK cells rather than macrophages, since the boosted effector cells were not retained by a rayon column or removed by carbonyl iron. Moreover, they were resistant to treatment with anti-Thy 1.2 serum plus complement, which eliminated mature T cells.
Role of Macrophages in the Augmentation of Mouse Natural Killer Cell Activity by Poly I:C and Interferon
Authors: Djeu Julie Y. , Heinbaugh Jeffrey A. , Holden Howard T. , Herberman Ronald B.
Published: January 2023

Mouse natural killer (NK) cell activity is readily augmented by interferon (IF) and IF inducers. To further our understanding of the regulation of NK activity by IF and poly I:C, we have investigated the cell types involved in in vivo and in vitro augmentation by these agents. Removal or inactivation of macrophages by silica, carrageenan, passage through rayon columns, or by the carbonyl iron-magnet technique before poly I:C treatment significantly depressed the augmentation of NK activity. In parallel, such treatments also decreased the level of IF induced by poly I:C. These observations were made in both conventional and nude mice. After induction of NK activity and IF by poly I:C, the removal of macrophages had no effect on the outcome of the activity. When a macrophage cell line, PU5 1.8, was treated with poly I:C, the culture fluid contained high concentrations of IF that were able to boost substantially NK activity in fresh spleen cells above the control level. Moreover, normal macrophages pretreated with poly I:C were able to produce IF by themselves and boost NK activity in fresh spleen cells. All these data taken together indicated that the requirement for macrophages in the boosting of NK activity by poly I:C was related to the need for these cells for the production of IF.

In contrast, removal or inactivation of macrophages had no effect on the induction of NK activity by IF. Therefore, it appears that IF acts directly on NK cells to increase their function and that macrophages may play a secondary role, by production of IF, in response to inducers such as poly I:C.
Lymphocyte Binding and T Cell Mitogenic Properties of Group A Streptococcal Lipoteichoic Acid
Authors: Beachey Edwin H. , Dale James B. , Grebe Stephen , Ahmed Aftab , Simpson W. Andrew , Ofek Itzhak
Published: January 2023

We previously reported that lipoteichoic acid (LTA) of group A streptococci binds spontaneously to mammalian cell membranes via lipid moieties ester-linked to the LTA molecule. We now describe biochemical and immunologic evidence that LTA binds to human and murine lymphocytes as an early event in the induction of mitogenesis in T lymphocytes. The biochemical studies showed that binding of radiolabeled LTA to lymphocytes was lymphocyte-concentration, and temperature dependent, and it reached a maximum in 15 min. Binding was reversible and specific with a dissociation constant of 89 μM for adult lymphocytes and 57 μM for cord blood lymphocytes. Immunologic studies showed that the LTA was mitogenic only for T lymphocytes. Dose response curves of lymphocyte mitogenesis induced by LTA and the binding of LTA to intact lymphocytes were shown to be related. The results suggest that LTA binds to specific receptor sites on T lymphocytes to trigger the mitogenic response.