bluuebunny/crossref_metadata_2025 huggingface dataset:
Possibility of utilizing Inter Simple Sequence Repeat regions, bark powder morphology and floral morphometry to characterize the Cinnamomum species in Sri Lanka
Authors: Hathurusinghe H. A. B. M. , Bandusekara B. S. , Pushpakumar D. K. N. G. , Ranawaka R. A. A. K. , Bandaranayake P. C. G.
Published: January 2023

The genus Cinnamomum of the family Lauraceae is an economically important crop. The Ceylon cinnamon (Cinnamomum verum syn. C. zeylanicum) has an exceptional position in the global cinnamon market. In addition to the cultivated species, Sri Lanka is home to seven endemic wild Cinnamomum species, C. capparu-coronde, C. citriodorum, C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum, and C. sinharajaense. Nevertheless, the species delimitation has not been successful with some morphometric and molecular traits. Therefore, this study focused on molecular characterization with an Inter Simple Sequence Repeats (ISSR) nuclear marker, floral morphometric characteristics, and microscopy of the powered bark of Cinnamomum species reported in Sri Lanka. According to the results four polymorphic ISSR regions resulted in an average of 83.7% polymorphism among all collected species, suggesting considerable polymorphism. The bark fiber size of the cultivated species is different from the studied wild species and could be used as a key to identify adulterants during export. The cultivated cinnamon, C. verum and two wild species (C. sinharajaense, and C. capparu-coronde) have considerably larger flowers compared to other species. Moreover, floral traits such as flower colour and shape could differentiate species C. sinharajaense and C. capparu-coronde. The Scanning Electron Microscopy (SCM) of pollen showed that the pollen size, spine length, interspinal distance, and spine ornamentation significantly contribute to species variation, and such variations need to be studied comprehensively. Nevertheless, the phylogeny of Cinnamomum species could not be completely resolved using ISSR regions, bark powder morphology, and floral morphological traits assessed and suggested for further studies.
In Vivo Protection Against Syngeneic Gross Virus-Induced Lymphoma in Rats: Comparison with in Vitro Studies of Cell-Mediated Immunity
Authors: Glaser Moshe , Lavrin David H. , Herberman Ronald B.
Published: January 2023

Spleen cells at various times after inoculation of W/Fu rats with a syngeneic Gross virus-induced lymphoma, (C58NT)D, were tested for their in vivo activity in adoptive transfer experiments and for their in vitro reactivity in a 4-hr 51Cr release cytotoxicity assay and in a mixed lymphocyte-tumor cell interaction assay. In adoptive transfer, the best protection against tumor growth was observed with immune spleen cells taken at 30 days after tumor cell inoculation (the peak of reactivity in the mixed lymphocytetumor cell interaction assay) whereas cells taken at 10 days (the peak reactivity in the 51Cr release cytotoxicity assay) gave only partial protection. The protection detected in the adoptive transfer experiments was specific for (C58NT)D associated antigens, and this correlated well with the specificity observed in the in vitro cell-mediated immunity assays. T cells, but not complement receptor-bearing cells or macrophages, were essential for the protection against tumor growth in vivo, and also for the in vitro reactivity in the 51Cr release cytotoxicity and the mixed lymphocytetumor cell interaction assays.
Cholesterol-Dependent Nonimmune Human Complement Activation Resulting in Damage to Liposomes
Authors: Alving Carl R. , Guirguis Adel A.
Published: January 2023

Liposomes containing varying concentrations of cholesterol (CHOL) were prepared in order to investigate the role of CHOL in complement (C)-mediated immune damage. The liposomes were comprised of dipalmitoyllecithin, CHOL, dicetyl phosphate, and either contained or lacked galactocerebroside as antigen. At high concentrations of liposomal CHOL each of the preparations (either containing or lacking galactocerebroside) released trapped glucose spontaneously in C in the absence of antiserum. This did not occur at low concentrations of CHOL. This effect was observed in the presence of human C from 8 of 12 individuals studied, but was not found with guinea pig C. It was eliminated after inactivation of C by a) preheating the serum at 56° for 30 min, b) addition of Mg2EDTA, or c) preabsorbing the serum with immune complexes (egg albuminanti egg albumin). Furthermore, preabsorbing the serum with high CHOL liposomes caused a drop in the hemolytic C titer, whereas low CHOL liposomes did not affect the hemolytic titer.
Complement-Dependent Lysis of RNA Tumor Viruses
Authors: Cooper N. R. , Welsh R. M. , Jensen F. , Oldstone M. B. A.
Published: January 2023

Although RNA tumor viruses (oncornaviruses) infect and are associated with malignancies in numerous species, they have been difficult to detect in human cancers. We found that fresh normal human sera inactivate Moloney (MLV), Rauscher (RLV), and AKR leukemia viruses, Moloney sarcoma virus, feline leukemia virus and simian sarcoma virus (Welsh, Cooper, Jensen, and Oldstone, Nature 257:612, 1975). Inactivation was due to viral lysis as RNA dependent DNA polymerase (RDDP) and RNA were released during the reaction. Fresh guinea pig and rabbit C and heated human sera did not have these properties. Antibody (Ab) did not seem to be involved since 1) agammaglobulinemic sera released RDDP, 2) cytolytic potential was not removed by absorption with cells expressing MLV, and 3) heated sera did not sensitize MLV for lysis by fresh guinea pig C. Viral lysis did not occur in human sera deficient in C2 or C4.
Genetic Control of C3 Levels in Mice
Authors: Ferreira Arturo , Nussenzweig Victor
Published: January 2023

At 2 to 3 weeks of age, but not at 9 weeks, AKR/J (H-2k) mice have higher C3 levels than DBA/2J (H-2d) and the F1 hybrids have intermediate levels. We recently completed analysis of linkage tests between C3 levels and H-2 genotypes in newborn mice. Screening of 202 mice of backcrosses (AKD2F1 × DBA/2J and AKD2F1 × AKR/J) and of F2 generation (AKD2F1 × AKD2F1) at 21 days of age confirmed a previous finding of significant differences (p < 0.001) between C3 levels of mice with different H-2 genotypes and, in addition, strongly suggests that very few independently segregating pairs of alleles are involved. Measurements of C3 levels of congenic lines of mice which bear five standard, independent H-2 haplotypes showed that they can be classified into at least two groups: C3-high (H-2f, H-2k, and H-28) and C3-low (H-2d and H-2b).
Isolation of Soluble C3 Receptor Activity from Rabbit Platelets
Authors: Henson Peter M. , Neshyba Janice
Published: January 2023

Interaction of the C3 receptor on rabbit platelets with C3-bound EA or zymosan (ZC3) results in adherence of the platelets and the initiation of an active secretory response. The secretion apparently involves the activation of a serine esterase as an early or initial step. Experiments were undertaken to isolate and characterize the C3 receptor in order eventually to determine its relationship to the esterase. Washed rabbit platelets were disrupted in sucrose by either homogenization or sonication. Platelet membranes (identified by 5′nucleotidase or previously bound 125I-antiplatelet antibody) were isolated on continuous or discontinuous sucrose gradients. Alternatively, the homogenate was passed through Sepharose-4B and the material excluded (containing the membranes) was employed.

Two assays were used to detect C3 receptor activity. Both, in addition, serve to define the nature of the C3 receptor. The assays are quantitative and reproducible and comprise: 1) inhibition of the binding of 51Cr-platelets to ZC3 or 2) inhibition of the ZC3-induced secretion from platelets.
Activation of Complement Through the Alternate Pathway by Microbial Glucans
Authors: Inai S. , Ebisu S. , Kato K. , Kotani S.
Published: January 2023

Complement and its components levels in the normal human serum treated with glucans of oral streptococcus and of the other microbials were studied. It was found that the enzymatically synthesized water-insoluble glucan of Streptococcus mutans OMZ 176, containing consecutive α-1.3 (60%) and branched α-1.6 (35%) linked glucose units, was a potent activator of the alternate pathway. CH50 in the serum treated with more than 200 µg/ml of OMZ 176 glucan was less than 5 units, and no significant reductions in the early acting components were observed, but considerable decrease in C3 and almost complete reduction of C5 were found. These complement component profiles were similar to that of serum treated with zymosan. Glucan of S. mutans OMZ 65, which is considered to have almost the same structure to OMZ 176 glucan, could also activate the alternate pathway. S. mutans Ingbritt insoluble glucan which contained less α-1.3 linkages than that of OMZ 176 glucan was less active than OMZ 176 glucan.
Two Mechanisms for the Activation of Serum Complement by Bacterial Lipopolysaccharides: A Role for Lipid A in Only One of Them
Authors: Morrison David C.
Published: January 2023

Preparations of lipopolysaccharide (LPS) from different bacterial sources vary significantly in their relative content of lipid A (LA) and polysaccharide (PS). As a number of biologic properties of LPS have been attributed to the LA region of the molecule, and as isolated LA has been demonstrated to activate complement, the relationship between the LA content of LPS and its anticomplementary activity was examined. Activation was determined by a) reduction in hemolysis of sensitized erythrocytes, b) alteration of the electrophoretic mobility of C3 in normal (NHS) and C2-deficient (C2-def) human serum, and c) consumption of early complement components C1, C4, and C2. Two antigenically similar preparations of LPS from Escherichia coli 0111:B4, which vary significantly in their relative ratio of LA:PS (LPS-I, low LA:PS and LPS-II, high LA:PS) were used. In addition, LPS from E. coli 055:B5, which is structurally similar but antigenically unrelated to LPS-I; LPS from Salmonella minnesota R595, which contains only LA and a trisaccharide; and purified LA itself were also examined.
Normal Values of CH50 and Individual Complement Components
Authors: Opferkuch W.
Published: January 2023

Sera were obtained from 605 clinically healthy human individuals. On these sera the following studies have been performed: determinations of the CH50 and C1 inactivator activity and molecular titrations of C1, C4, and C2. The protein concentration of C4, C3, C1 inactivator and C3-proactivator in the sera was determined by means of the Mancini technique. At the same time the reproducibility of these tests was investigated. It was found that the standard error of each individual test was about 7 to 15% of the mean value. Only in the determinations of the C1-inactivator activity there was a standard error of about 25%.

Comparing the male and the female populations, no statistically significant difference in their mean values and distribution curves could be observed. With increasing age, however, a statistically significant increase in the Ch50 titer, levels of C1, C4, C2 activity and C4 and C3 serum protein concentrations could be found in the female population.
Role of Complement in Enhanced Tumor Growth in Nude Mice
Authors: Shearer W. T. , Atkinson J. P.
Published: January 2023

Traditionally, the role of complement (C) in the regulation of malignant cell growth has been thought to be one of cell destruction by cytolysis. A new perspective was achieved when it was discovered that C markedly augmented the phenomenon of immunologic enhancement in vitro. Thus, anti-L cell antibody exerted a biphasic effect upon L (transformed mouse fibroblast) cell growth with high concentrations inhibiting growth and low concentrations stimulating growth. Complement augmented both cytotoxic and cytostimulatory effects of antibody (Ab) upon L cells in vitro. This report represents evidence to suggest that C exerts a synergistic effect with Ab in the stimulation of tumor growth in vivo.

Congenitally athymic (nude) mice were depleted of C3–C9 C components by the repeated i.p. injections of Cobra venom factor (CVF) and given 40 µl of either control rabbit serum (CR) or rabbit anti-L cell antiserum (AL) on day -1.
Linkage of C4 Deficiency to the Major Histocompatibility Locus in the Guinea Pig
Authors: Shevach E. , Green I. , Frank M. M.
Published: January 2023

C4 deficient (C4D) guinea pigs have a deficiency in C4 synthesis that appears to be due to a structural gene defect. Previous work has demonstrated that inheritance of this defect follows simple Mendelian laws. This report demonstrates linkage between C4D and an antigen controlled by a gene in the major histocompatibility complex of the guinea pig. Recent studies have defined a major histocompatibility locus (MHL) in the guinea pig (B) which appears to be equivalent to the D or K loci in the mouse, as well as a second region which resembles the mouse Ia. Four alleles of the B locus have been identified: B.1, B.2, B.3, B.4. Preliminary screening of the NIH multipurpose colony (the colony in which C4D was first observed) showed that 5 to 10% of the animals were homozygous for the B.1 antigen; the remaining animals had detectable B.3 antigen by serologic testing.
Purification, Subunit Structure, and Isolation of Peptides from Limit Tryptic Digests of Human C3
Authors: Tack B. F. , Prahl J. W.
Published: January 2023

A method for the preparative level purification of human C3 from plasma has been developed. Initial fractionation of plasma with polyethylene glycol (PEG) yields a protein fraction precipitating between 5 and 12% (w/v) PEG that contains 81% of the total C3 together with most plasma β-globulins. Chromatography of this fraction on Whatman DE52, Sepharose 6B, and hydroxylapatite completes the purification of C3. A specific elution of C5, IgG, and IgA from hydroxylapatite is obtained with 2.0 M KCl before the elution of C3 with 100 mM K2HPO4 (pH 7.4). A constant ratio of 14,000 C3 CH50 units/mg C3 was observed throughout the purification, with quantitative immunoprecipitant and hemolytic titration assays indicating a 32% recovery of C3.

The reduction (0.02 M dithiothreitol, pH 8.6) and alkylation (0.042 M iodoacetamide) of C3 in 6.0 M guanidinium chloride (GuCl) results in the disassociation of two polypeptide chains: an α-chain and a β-chain with respective molecular weights of 120,000 ± 12,000 and 75,000 ± 7500.
Demonstration of a Non Osmotic Swelling and of a Three Step Mechanism During the Immune Lysis of Erythrocytes by Antibody and Complement
Authors: Valet G. , Opferkuch W.
Published: January 2023

E to EAC1-9 cell intermediates were sized electrically with a hydrodynamic focusing system which is a modified Coulter counter method that avoids the methodologically right skewed volume distribution curves of the original method. No changes in the volume distribution curves of the erythrocytes were observed up to the EAC1-8 state. On addition of C9, a dose-dependent part of the cells did swell and three steps could be distinguished in the lytic mechanism: 1) fixation of C9 to EAC1-8 cells, a process which proceeds also well at 0°C; 2) a temperature-dependent swelling of the erythrocytes to their maximal volume in an all or nothing fashion; and 3) lysis of the cell. The swelling of the erythrocytes to spheroids is of a non-osmotic nature because it occurs also in highly concentrated 36% albumin solutions where the colloid osmotic forces of the intracellular protein are balanced on a molar basis. It persists into the ghost phase.
Human Platelet-Initiated Formation and Uptake of the C5-9 Complex of Human Complement
Authors: Zimmerman T. S. , Kolb W. P.
Published: January 2023

We have studied the interaction of radiolabeled complement components with normal human platelets, platelets from a patient with paroxysmal nocturnal hemoglobinuria, and rabbit platelets in the absence of known complement activators or in the presence of cobra venom factor (CVF). When unwashed platelets in platelet rich plasma, or washed platelets suspended in serum or autologous plasma, were incubated for 30 min, C3 and terminal components (C5, C8, and C9) were found to bind to them. The terminal components were shown to be bound as the C5–9 complex, rather than as individual proteins, by eluting them from the platelet membrane and examining their behavior on ultracentrifugation. They co-sedimented at a rate characteristic of the stable C5–9 complex (22S). The complex so formed did not differ by ultracentrifugational criteria from that binding to rabbit platelets after CVF activation of complement. Although C3 was not included in the complex, it did not appear to be bound by nonspecific absorption.
Normal Mouse Serum Immunosuppressive Activity: Action on Adherent Cells
Authors: Martineau Ronald S. , Johnson John S.
Published: January 2023

The immunosuppressive activity of normal mouse serum (NMS) was evaluated by enumeration of primary in vitro plaque-forming cell response produced by normal spleen cell suspensions in response to sheep erythrocytes. The site of action of the inhibitor(s) was shown to be at the level of adherent cells since incubation of this cell type with NMS could reproducibly inhibit responses, whereas, in appropriate experiments, incubation of nonadherent cells with NMS had no effect. Furthermore, addition to normal spleen cells of excess spleen-adherent cells, normal peritoneal adherent cells, or of 2-mercaptoethanol could abrogate the inhibitory ability of NMS. Addition of excess nonadherent cells under the same conditions had no influence on inhibition. NMS was also shown to decrease the number of cell clusters, hemolytic clusters, and the number of nucleated cells recovered upon culture termination. It is suggested that NMS, acting on the adherent cell may hinder cell-to-cell contact and interactions.
Rabbit Antiserum against Daudi-Cell Membrane Fractions Detects Cell Surface Antigens Present on Subpopulations of Human Lymphocytes
Authors: Carrel S. , Gross N. , Salvi S.
Published: January 2023

An antiserum was produced by immunization of rabbits with membrane preparations of a human lymphoblastoid B cell line, Daudi. After absorption with a human endometrial carcinoma cell line, this antiserum appeared to be specific for antigen(s) present on adult and fetal thymocytes as well as on tonsillar lymphocytes but absent, or present in very small amounts, on normal or phytohemagglutinin- (PHA) stimulated peripheral blood lymphocytes (PBL). When T and B cell-enriched fractions from tonsillar lymphocytes were tested with the anti-Daudi serum, the reactivity was equally distributed in each population. Among 13 human lymphoblastoid cell lines tested, reactivity was demonstrated on three out of four T cell lines, and on four out of nine B cell lines. The positive reacting B cell lines were derived from two African and two American Burkitt lymphomas. The antigen(s) described does not seem to be related either to human Ia-type antigens or to Epstein-Barr virus-associated antigens because these antigens are not present on fetal or adult thymocytes. Reciprocal absorption experiments indicate that this anti-Daudi serum detects the same antigenic structures present on certain subpopulations of T and B lymphocytes.
Rous Sarcoma Virus-Transformed Avian Cells Express Four Different Cell Surface Antigens that Are Distinguishable by a Cell-Mediated Cytotoxicity-Blocking Test
Authors: Ignjatovic Jagodina , Rübsamen Helga , Hayami Masanori , Bauer Heinz
Published: January 2023

Japanese quails bearing avian sarcoma virus-induced tumors develop immune spleen cells that are cytotoxic in vitro against virally and chemically transformed cells, as well as against embryonic cells. The cell-mediated cytotoxicity can be blocked by soluble antigens extracted from in vitro cultured cells. The existence of partial as well as total blocking effects in tests with extracts from various transformed and untransformed virus-producing cells makes it possible to distinguish up to four different kinds of antigens expressed on sarcoma virus transformed cells: a) a subgroup-specific determinant of the virus-envelope glycoprotein gp85 (s-gp85) is expressed at the surface of productively infected, transformed as well as untransformed cells; b) a group-specific determinant of gp85 (g-gp85) that is only expressed on the surface of virus-transformed cells; c) embryonic antigens, also detectable on chemically transformed as well as on primary normal embryonic cells, and finally; d) a sarcoma virus transformation-specific antigen (TSSA) that is not a structural constituent of the virus.
Inhibition of Lymphoma Growth in the Spleen and Liver of Lethally Irradiated Mice
Authors: Iorio Anna , Campanile Franca , Neri Mariella , Spreafico Federico , Goldin Abraham , Bonmassar Enzo
Published: January 2023

The growth of murine lymphomas carrying the H-2d (LSTRA, P388), the H-2k (6C3HED), and the H-2a (LAF-17) haplotypes was studied in spleen and liver of lethally irradiated (850 to 1000 R) compatible or allogeneic mice, with the 125I-5-iodo-2′-deoxy-uridine uptake method. According to the genetic pattern of the Hh (hemopoietic-histocompatibility) system, recipient hosts were either susceptible or resistant to bone marrow graft carrying the same haplotypes as those of the lymphomas studied. The results showed that the tumor cell growth was impaired in the spleen of irradiated Hh-incompatible resistant hosts (e.g., P388 into B10, B10.BR and CBA), suggesting that lymphoma and bone marrow cells could share common Hh antigens. However, inhibition of lymphoma growth occurred also in the spleen of irradiated Hh-susceptible mice, either Hh-compatible (e.g., P388 into B10.D2; 6C3HED into AKR or CBA) or Hh-incompatible, but genetically unable to respond against bone marrow cells (e.g., LSTRA or P388 into C3H, or 6C3HED into BALB/c or SJL). In almost all cases growth inhibition was absent or weak in the liver. These results suggest that lymphoma cells could express antigens not detectable on normal bone marrow cells, capable of eliciting radioresistant inhibition of tumors (RIT) predominantly in the spleen of mice susceptible to marrow graft of the same genetic origin as that of the lymphoma tested. At the present time no direct data are available to determine whether natural cytotoxicity would play a role in RIT responses.
Binding and Activation of Human C1 by Immune Complexes Effect of Inhibitors
Authors: Arlaud G. J. , Sim R. B. , Reboul A. , Colomb M. G.
Published: January 2023

Human serum (pH 7.0) was incubated for 30 min at 0°C with ovalbumin-IgG anti-ovalbumin complexes in the presence of 50 µg/ml soybean trypsin inhibitor. After centrifugation and three washes at 0°C in a buffer containing 5 mM CaCl2 and 40 µg/ml STI (pH 7.0), C1̄r and C1̄s were resolubilized. From the C1̄s esterase activity and the SDS-polyacrylamide gel electrophoresis pattern, extracted C1̄r and C1̄s were found to be 75% activted. Complete activation was obtained by incubation for 30 min at 30°C before extraction.

Extraction of C1q, C1r and C1s in the presence of calcium. At pH 7.5, C1̄r and C1̄s were both readily extracted by increasing relative salt concentrations above 0.15. In contrast, C1q extraction started at RSC = 0.30 and increased more slowly.

C1̄r extraction paralleled C1̄s extraction as a function of pH, with a minimum at pH 7.0 and maxima at extreme pH values.
C1s Bound in Macromolecular C1 Exists in Equilibrium with C1s Free in Serum
Authors: Bartholomew R. M. , Esser A. F.
Published: January 2023

We have established the existence of an equilibrium between free C1s and C1s bound in macromolecular C1 in human serum. Untreated human sera were incubated for 35 hours at 4°C with 125I-C1s to allow the exchange between free and bound C1s to reach equilibrium. The C1 complex thus labeled was separated from most of the serum proteins and from excess 125I-C1s by centrifugation in linear 10–30% sucrose gadients. 125I-C1s became incorporated into macromolecular C1, as demonstrated by specific cleavage of all the labeled C1s to C1s̄ after incubation with known activators of the classical pathway of complement. Thus, C1 fractions containing 125I-C1s can be used directly and conveniently to assay C1 activation.

Incubating for more than 35 hours at 4°C neither increased nor decreased the amount of 125I-C1s in serum C1. Furthermore, excess 125I-C1s remained in the free protein pool of samples incubated more than 35 hours and entered C1 upon incubation with additional fresh serum.
Ss, The Murine Homologue of C4
Authors: Carroll Michael C. , Capra J. Donald
Published: January 2023

Serum substance (Ss), a serum protein, is known to be linked to the major histocompatibility locus of the mouse. Until recently the function of Ss was unknown but several lines of evidence, i.e. serologic, functional, and structural suggest Ss is murine C4.

The Ss protein has been isolated in pure form from murine ascites fluid and has been shown to have functional C4 activity. Purification was monitored by antigenic reactivity with a specific antiserum to Ss as well as with a hemolytic assay using genetically C4-deficient guinea pig serum.
Interactions Between Complement Proteins and Cell Membranes: Reduction in Membrane Fluidity Due to the Perturbation of the Lipid Bilayer by the Terminal Complex
Authors: Dahl Charles E. , Levine R. Paul
Published: January 2023

We have used 5, 12, and 16-nitroxyl stearic acid (5, 12, and 16-NSA) to spin label erythrocytes in order to investigate complement-induced changes in the organization of lipid bilayers. These changes are characterized by measurements of the hyperfine coupling, 2T11, a sensitive indicator of the motional freedom of the spin label and hence a measure of membrane fluidity. When 5, 12, or 16-NSA-labeled E or EA are lysed in hypotonic solution the value of 2T11 decreases indicating an increase in the fluidity of the bilayer. Upon resealing the lysed E or EA in the presence of Mg2+, 2T11 returns to its normal value. The fluidity of EAC ghosts, as measured by 2T11 is comparable to that of hypotonically lysed E or EA. When EAC are resealed in Mg2+ there is a significant decrease in membrane fluidity, for 2T11 increases to values greater than those obtained for resealed ghosts of E and EA.
Dependence of Complement Rosettes on a Nonenzymatic and an Enzymatic Step
Authors: Dierich M. P. , Landen B.
Published: January 2023

Rosette formation between typical EAC14oxy23 (human complement components; 1.2 hemolytically active C2 sites and 1 × 104 C3 molecules) and Raji cells can be reduced by 20 to 30%, of 0.5 mM diisopropylfluorophosphate (DFP) are present during the rosette assay. Reduction of rosette formation is only slightly less pronounced, if EAC14oxy23 are pretreated for 10 min at 23°C with 0.5 mM DFP. To elucidate the possible contribution of the C42Ѕ enzyme to the formation of rosettes, EAC1423 were prepared with nonoxidized C2. These EAC1423 were kept at 37°C for 30 min to decay the C2 and to obtain EAC143. Such cells formed about 10 to 15% rosettes. By treating the EAC143 with increasing amounts of oxidized C2 to form EAC14oxy23, rosette formation was increased up to 40%. This C2 dependent increase in rosette formation could be suppressed by DFP or by performing the rosette assay at 4°C.
The Morphology, Lifetime, and Pore Size of Complement Lesions on Erythrocyte Membranes
Authors: Esser A. F. , Halverson C. A. , Müller-Eberhard H. J.
Published: January 2023

The action of complement on sensitized erythrocytes (EA) results in release of hemoglobin (Hb) with concomitant formation of cell ghosts (EAC · gh). Prior cross-linking of EA with 0.025% glutaraldehyde (EA · X) prevents release of Hb upon incubation with C but not of trapped ions such as 86Rb indicating a limiting pore size of the lesion. The mechanism or entity that allows Hb release from EA, however, remains only transiently functional since EAC · gh reseal again. Using a simple sucrose-step-gradient centrifugation assay we found that more than 80% of EAC·gh are impermeable to molecules of the size of sucrose. In contrast, resealed ghosts, prepared from EA by hypotonic lysis and subsequent incubation with isotonic buffer (EA · gh), that have been cross-linked (EA · gh · X) remain permeable after incubation with complement (EA · gh · X · C). The successful introduction of C dependent pores into EA · gh · X depends on the amount of cross-linking.
C3b Inactivator: The Effect on Cell Bound C3b and a Simplified Functional Assay
Authors: Gaither Thelma A. , Gadek James , Frank Michael
Published: January 2023

The activity of C3b inactivator on the cellular intermediate site EAC43b was compared in three test systems (a functional hemolytic assay, an immune adherence assay, and a functional radioimmunoassay). 125I-C3 was incubated with EAC142 to form EAC1423*. C1 was removed with EDTA and C2 was allowed to decay at 37°C C/1 hr. When this intermediate was incubated with whole human serum or with partially purified C3b inactivator, there was a rapid loss of hemolytic C3 activity but very little specific removal of radioactive C3. A two-step assay was devised in which dilutions of normal human serum in low ionic strength buffer at pH 6.0 (.0375 µ dextrose-Veronal buffered saline, with 0.01 M EDTA, EDTA-VBS) were added EAC43b*. On exposure to trypsin (2.5 µg/ml) in .060 µ EDTADVBS for ten min at 30°C, radioactive C3 was released from the intermediate. This release of radioactivity correlated with the loss in hemolytic activity on exposure of EAC43b to C3b inactivator alone.
The Size of the Lesion Induced by the Alternative Complement Pathway on Human Erythrocyte Membranes
Authors: Giavedoni Elda B. , Dalmasso Agustin P.
Published: January 2023

By using a direct approach we have demonstrated previously that the classical C pathway produces on sheep erythrocyte membranes functional lesions of about 4 nm diameter. We have now investigated the size of the lesions induced on human erythrocyte membranes by activation of the alternative C pathway. Human red cells were treated with 2-aminoethylisothiouronium bromide (AET) to increase their reactivity with C. They were then lysed hypotonically in the presence of radiolabelled marker molecules of different size which were incorporated into the membranes ghosts during resealing. The membranes were finally treated with human serum together with inulin particles to activate the alternative pathway. This resulted in identical % release of 3H-sucrose from membrane ghosts and of hemoglobin from AET-treated intact erythrocytes. No release of 125I-ovalbumin or 125I-BSA was obtained from membrane ghosts. The release of soluble 3H-inulin was slightly slower and that of 125I-RNase markedly slower than that of 3H-sucrose.
The Role of Complement in Host Defense Against Bacteremia
Authors: Hosea Stephen W. , Frank Michael M.
Published: January 2023

In order to further study the role of complement in host defense, a model of pneumococcal bacteremia was developed in guinea pigs and mice. Type 4 pneumococci grown overnight in trypticase soy broth were injected intravenously, and clearance rates and organ distribution were studied by colony counts and 59Fe labeling of bacteria. C4-deficient (C4D) and normal NIH guinea pigs (GP) had similar patterns of rapid clearance. This was associated with activation of approximately 20,000 C3 molecules per bacterium. However, cobra venom factor- (CVF)treated animals had significantly delayed clearance of bacteria which was followed by overwhelming sepsis and death. Immunization produced enhanced clearance in normal GP but not in C4D animals, suggesting the requirement for an intact classical complement pathway for efficient immune clearance. Actively immunized CVF animals continued to exhibit delayed clearance and subsequent death.

Sequestration of bacteria occurred in the liver, spleen, and lung in the normal animal.
Mechanism of Endotoxin-Initiated Complement-Dependent Platelet Lysis
Authors: Morrison David C. , Oades Zenaida G.
Published: January 2023

We demonstrated earlier that bacterial endotoxins (lipopolysaccharides, LPS) have the capacity to activate both pathways of complement, with the polysaccharide portion of the molecule activating the alternative pathway and the lipid A region being responsible for classical pathway activation (Morrison, D. C. and Kline, L. F., 1977, J. Immunol. 118:362). We further showed that only preparations of LPS which initiate alternative pathway activation are capable of LPS-initiated complement-dependent rabbit platelet lytic responses. (Morrison, D. C. et al., 1976, Fed. Proc., 35:516).

Although the lipid A region of the LPS molecule is, itself, incapable of initiating lysis, our current experiments provide strong evidence that it does, in fact, play a critical role in the rabbit platelet response to LPS. Treatment of LPS with mild alkali (using conditions known to enhance binding of LPS to both erythrocytes and lymphocytes) significantly enhanced the extent of the complement-mediated platelet lysis.
C3bINA and its Macromolecular Weight Co-factor; Purification and Characterization
Authors: Nagasawa Shigeharu , Stroud Robert M.
Published: January 2023

The complement system is proteolytically modulated by the C3bINA-C4bINA system which is generally believed to cleave C3b into two fragments, C3c and C3d. However, little is known as to the exact mechanism of the proteolytic action of C3bINA on C3b and C4b. Recently, we documented the requirement of a macromolecular weight co-factor for the proteolytic function of C3bINA (Immunochemistry, in press). On the other hand, it was recently reported that β1H plays a role also as a co-factor for the proteolytic activity of C3bINA.

In the present study, we have purified the macromolecular weight co-factor and characterized it as a new serum protein. Also, we have highly purified the C3bINA by affinity chromatography with C3b-Sepharose, in order to study the cleavage mechanisms.

Purification and characterization of the macromolecular weight co-factor.
Analysis of the Peptide Maps of Human C2 and Factor B: Evidence for Structural Homology and Common Ancestry
Authors: Ngan Bo-Yee , Minta J. O.
Published: January 2023

C2 and Factor B are heat-labile β-globulins in normal human serum and are the precursors of the C3 and C5 convertase enzymes of the classical and alternative complement pathways, respectively. Genes controlling C2-deficiency and polymorphism and Factor B polymorphism have been linked to the HLA locus. In this study, proteolysis of C2 and Factor B have been compared by a peptide mapping technique on SDS polyacrylamide gel electrophoresis. C2 and Factor B were purified from the 20% Na2SO4 supernatant of human plasma by sequential column chromatography and by preparative gel electrophoresis in barbital-glycine buffer, pH 8.8. The highly purified C2 and Factor B sedimented at 5.2S and 5.0S, respectively, and were hemolytically and esterolytically active. The m.w. for C2 and Factor B estimated by SDS polyacrylamide gel electrophoresis in the presence or absence of reducing agents were 117,000 and 93,000 daltons, respectively, suggesting that each protein is comprised of a single polypeptide chain.
The C2 Polymorphism: Its Genetics and Presentation of Typing Technique Modifications
Authors: Olaisen B. , Teisberg P. , Thorsby E. , Gedde-Dahl T.
Published: January 2023

The originally described C2 typing technique has been modified to allow typing on ordinary electrofocusing equipment, without the use of specific C2-deficient serum. The modifications include prolonged electrofocusing time, iodine treatment of gels after focusing, and the use of low concentration normal human complement as “C2-lacking complement.”

In a Norwegian population sample C2 gene frequencies were: C21: = 0.97, C22: = 0.03, these frequencies fit well with those reported in other Caucasian populations.

C2-linkage relations and haplotype associations have been examined in an extensive Norwegian family material. No recombinations were found in 22 informative C2/HLA-B meioses and the rare C22 allele was accompanied by the HLA-Bw15 allele in 6 out of 7 instances. Our data therefore confirm the linkage of C2 to the HLA region loci, and we conclude that C2 is situated in close proximity to the HLA-B locus.
Influence of Lymphokine on Complement Components Synthesis by Guinea Pig Spleen Cells In Vitro
Authors: Peltier A. P. , Cambier A.
Published: January 2023

A large part of complement components are synthetized by cells of the macrophage-monocyte type which are under narrow control by lymphokines. Preliminary results obtained in our laboratory have shown that addition of phytohemagglutinin M to a culture of whole normal guinea pig spleen cells decreased the amount of C2 detectable in the culture supernatant at the same time where it strongly stimulated lymphocyte proliferation, as shown by a high rate of cellular 3H-thymidine incorporation. One possible explanation for this phenomenon would be the inhibition by some lymphokine of C2 synthesis by macrophages.

In order to substantiate this hypothesis, a two step experiment was designed. Whole normal guinea pig spleen cells (0.5 ml of 20 × 106/ml suspension) were first cultivated for 24 hours in the presence of an optimal 50 µg dose of insoluble concanavalin A. The supernatant obtained was assayed for its lymphokine activity by a classical Macrophage Inhibition Test.
A Specific Indicator for the C3b Receptor on Mouse B Cells and Altered States of C3b Reactivity
Authors: Ranken Raymond , Linscott William D.
Published: January 2023

Mouse B lymphocytes have separate surface receptors for mouse C3b and C3d, but study of their function has been hampered by a lack of indicator particles completely specific for each receptor type.

We report here that mouse spleen lymphocytes will bind to guinea pig (GP) C3b but not to GP C3d, so any possible contamination of the indicator cells with C3d should be of little consequence. There was no binding to GP C4. Indicator cells were prepared from EAC142 GP and purified GP C3. They were maximally reactive in I-A, unreactive with bovine conglutinin, and rosetted with about 27% of mouse and GP spleen cells. When these EAC43b were incubated for 40 min at 0°C in EDTA with heated GP serum, there was little or no change in their I-A activity, but they lost completely the ability to rosette with mouse (but not GP) lymphocytes; their C3 hemolytic activity was markedly reduced; and they gained the capacity to be agglutinated by bovine conglutinin.
On the Perturbation of Phospholipid Bilayers by Complement: The Use of Inside-Out Vesicles from Complement-Lysed Erythrocytes for the Demonstration of Perturbating Peptide Chains
Authors: Rauterberg E. W.
Published: January 2023

It has been postulated by different groups that late complement components may partly insert into the phospholipid bilayer of complement lysed erythrocytes and form a trans-membrane channel.

The present report examines whether complement component antigens can be detected at the inner surface of complement lysed membranes by the use of inside-out vesicles. Insideout and right-side-out vesicles from osmotically or complement lysed sensitized sheep erythrocytes (EA or EAC1-9) were obtained by an incubation in a low ionic strength phosphate buffer, pH 8.0, followed by a gentle homogenization to liberate the entrapped vesicles.

Inside-out and right-side-out vesicles were separated on linear gradients of Dextran T 110 by centrifugation to equilibrium. Inside-out vesicles from EAC1-9 (IO-EAC) as well as those from osmotically lysed sensitized sheep erythrocytes (IO-EA) revealed both a very low binding of 3H-Ouabain in comparison to the respective right-side-out vesicles (RO-EAC and RO-EA). The four types of vesicles were radioiodinated by lactoperoxidase and glucose oxidase.
Structural Variability in Human C4: Methodological and Genetic Data
Authors: Teisberg P. , Olaisen B. , Gedde-Dahl T. , Thorsby E.
Published: January 2023

We have previously demonstrated a genetic polymorphism of human C4. Modification of the methods employed in the separation and detection of C4 proteins has shown that further molecular variability exists in C4. By employing prolonged high voltage electrophoresis in agarose and a sensitive hemolytic detection system, three common C4 alleles and one less common have been detected. The following gene frequencies have been found in the Norwegian population: C4F: 0.46, C4F1: 0.20, C4S: 0.32, C4M: 0.02.

In 165 informative meioses not a single recombinant has been observed between C4 and HLA-B. In three known cross-over events between HLA-A and B, C4 in each instance follows B. The short map distance between C4 and HLA-B is further substantiated by haplotype association data. All 12 HLA-B8 chromosomes carry C4S. In seven out of eight instances HLA-B7 follows C4F1.

In 53 informative meioses no cross-over event has been observed between C4 and Bf (locus for factor B).
Hydrolytic Activation of the Fifth Component of Complement (C5) by C3 Convertases Depends on its Binding to Surface-fixed C3b; Specific Interference with Binding, by Properdin, Factor B, or Propamidine, Leads to Inhibition of C5 Cleavage and Utilization
Authors: Vogt W. , Schmidt G.
Published: January 2023

The C3 convertases of the classical and alternative pathways, and C3bBb (P) are capable of cleaving C5 only in the presence of C3b fixed to a solid surface (S-C3b) immediately subsequent to its release at that surface (e.g., on red cells, agarose, zymosan). The fact that fluid-phase and to a lesser degree soluble , attack C5 in the presence of S-C3b in EDTA medium, indicates that S-C3b interacts with and modulates the substrate, C5, rather than the convertase. In fact, reversible binding occurs between S-C3b and C5. Soluble C3b shows no indication of C5 binding, and does not support C5 cleavage by or C3b Bb. Cobra venom factor (CVF) which supports a B-dependent C5-cleaving enzyme, CVFBb, combines with C5 even in solution. While CVFBb cleaves C5 efficiently only when substrate (C5) and enzyme (Bb fragment) are bound to the same CVF molecule (free CVF added to a mixture of CVFBb and C5 is inhibitory), S-C3b has to be free of other ligands in order to function as a C5-binding site.
Modulation of Generation of the Amplification Convertase C3b,Bb by Heparin
Authors: Weiler J. M. , Yurt R. W. , Austen K. F.
Published: January 2023

Commercial heparin, a degraded mixture of hog glycosoaminoglycans, inhibits generation of the amplification convertase, C3b, Bb, on a cellular intermediate or in the fluid phase. Heparin is most active in inhibiting convertase generation on cellular intermediates formed with the lowest C3 input and developed with the highest B concentration, suggesting that the point of heparin action is the receptor site for B on C3b. Inhibition is observed also when C3b,Bb generation takes place on cellular intermediates in the presence of P or C3NeF, which yield stabilized forms of the convertase. This inhibitory action of heparin is not due to chelation of magnesium. Fifty times the concentration of heparin required to inhibit convertase generation does not accelerate the decay of the unstabilized or the C3NeF-stabilized convertases and has only a modest effect on the P-stabilized convertase. These studies indicate that in addition to the previously reported sites of heparin inhibition on classical pathway function noted by others, heparin acts to prevent effective interaction of C3b and B, most likely by a reversible action on C3b.
Activation of the Alternative Pathway by Pneumococcal Cell Wall Teichoic Acid
Authors: Winkelstein Jerry A. , Tomasz Alexander
Published: January 2023

Previous studies on the interaction of pneumococci and the alternative pathway have shown that the cell wall, rather than the cell membrane or capsule, plays a major role in the activation of the alternative pathway. The present study was performed in order to identify the biochemical component of the pneumococcal cell wall responsible for activating the alternative pathway.

Teichoic acids were extracted from purified pneumococcal cell walls by three different procedures (0.05 M periodate at 37°C; 100% formamide at 170°C; 5% TCA at 4°C). In each case the teichoic acids were able to consume C3 in C4-deficient guinea pig serum (C4D GPS) at concentrations as low as 3 to 10 µg/ml. In contrast, the residual cell walls, which contained the peptidoglycan, lost their activity in proportion to the amount of teichoic acid that had been extracted. Notably, cell walls extracted with periodate lost over 98% of their teichoic acid and the residual cell wall peptidoglycan was unable to consume C3 in C4D GPS at concentrations as high as 1 mg/ml.
Biochemical Relationship between Murine Immune Interferon and a Killer Cell Helper Factor
Authors: Simon Philip L. , Farrar John J. , Kind Phyllis D.
Published: January 2023

Concanavalin A and alloantigen-stimulated mouse spleen cells release into the supernatant at least two distinct antigen-nonspecific factors that enhance the generation of cytotoxic cells in vitro. As previously reported, analysis of the supernatant by ammonium sulfate fractionation, gel filtration, hydroxylapatite chromatography, and hydrophobic chromatography showed one of the two killer cell helper factors (KHF) to be associated with thymocyte mitogenic factor. This report demonstrates the second KHF to be separable from thymocyte mitogenic factor but inseparable from type II (immune) interferon. In addition, this KHF exhibits the same sensitivity to exposure to pH-2 buffer as does immune interferon. The KHF activity of an unfractionated supernatant, which is greater than that of either the TMF-associated or interferon-associated KHF alone, is the result of the additive activities of the two independently acting helper factors.
Antigen-Binding T Cells: Dose Response and Kinetic Studies on the Development of Different Subsets
Authors: Eardley Diane D. , Shen Fung W. , Cone Robert E. , Gershon Richard K.
Published: January 2023

We have described a number of the parameters involved in the in vitro induction of specific SRBC-binding T cells (T rosette-forming cells, T-RFC). Although T-RFC precursors pass through nylon, most of the induced cells do not; nor do detectable numbers of Ly 1+2, 3- cells bind antigen with sufficient stability to form rosettes. The ratio of Ly 2,3:Ly 1,2,3 T-RFC varies with time after immunization and with the dose of antigen used for stimulation. Relatively high or low doses of antigen selectively induce Ly 1,2,3 T-RFC. Ly 2,3 T-RFC, when they appear, follow Ly 1,2,3 T-RFC. Pretreatment of T cells with anti-Ly sera before RFC induction prevents formation by Ly 2+ T-RFC.

Since anti-Ly 1 treatment blocks RFC formation and since Ly 1,2,3 T-RFC always precede the appearance of Ly 2,3, T-RFC, our results suggest that some Ly 1+ cells (Ly 123 at least, but perhaps also Ly 1) may act as inducers, precursors, and/or amplifiers for Ly 2,3 RFC as they appear to do for Ly 2,3 suppressor and killer cells. Thus, our results confirm and extend the observed similarities between T-RFC and other Ly 2+ cells such as killer and suppressor cells as well as their differences from Ly 1+ helper cells.
Characterization of an Antiserum Specific for the Fabα Fragment
Authors: Doe William F. , Spiegelberg Hans L.
Published: January 2023

An antiserum to the Fabα fragment of human IgA was prepared by injecting κ- and λ-type IgA1 myeloma protein Fab fragments in complete Freund's adjuvant into a goat. After appropriate absorptions, the antiserum reacted specifically with Fabα fragments, normal IgA, and κ- and λ-type IgA1 and IgA2 myeloma proteins. This antiserum contained predominantly antibodies to determinants shared by different IgA myeloma proteins and expressed on isolated α chains. These determinants were presumably located on the first constant domain of the α chain (Fd fragment). When incorporated into agarose, the anti-Fabα antiserum is a potentially valuable reagent for screening human sera for α-heavy chain disease proteins by an immunoelectrophoretic method of immuno-selection.