bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Size and Subunit Structure of the Fourth Component of Guinea Pig Complement
Authors: Hall Robert E. , Colten Harvey R.
Published: December 2022

The fourth component (C4) of human complement is a glycoprotein with a m.w. of 209,000 daltons and consists of three subunits of m.w. 93,000, 78,000, and 33,000 daltons linked by disulfide bridges (1, 2). The size and structure of the corresponding complement protein in guinea pig serum has not yet been elucidated, although the sedimentation coefficient of guinea pig C4 (8S20,w) is known to be similar to that of human C4 (3). The m.w. of guinea pig C4 has been estimated by gel filtration to be 180,000 (4).

We now report isolation of C4 from guinea pig serum by a one-step immunoabsorbent method. Its molecular size and subunit structure were similar to that of human C4. Normal guinea pig liver in culture synthesized and secreted C4 with size and subunit structure identical to serum C4.

Materials and Methods. A 14C-labeled l-amino acid mixture consisting of equal amounts of 14C-leucine (270 mCi/mmol), 14C-valine (225 mCi/mmol), 14C-isoleucine (270 mCi/mmol), and 14C-lysine (270 mCi/mmol) was obtained from New England Nuclear, Boston, Mass.
The Role of Neutrophils in Antiviral Defense-in Vitro Studies on the Mechanism of Antiviral Inhibition
Authors: Rouse Barry T. , Wardley Richard C. , Babiuk Lorne A. , Mukkur T. K. S.
Published: December 2022

Highly enriched populations of bovine neutrophils were added, in the presence of antiviral antibody, to herpesvirusinfected bovine cell cultures. A cell dose-dependent reduction in virus-induced cytopathology was observed. The mechanism of inhibition was presumed to be mediated by a subcellular neutrophil product and not the result of either direct cytotoxicity or antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, inhibition of comparable magnitude was observed when neutrophils and virus-infected cells were separated by cell impermeable membranes. In addition, plaque reduction occurred when antiviral immunoglobulins or fragments unable to mediate ADCC were used in the assays. Killed neutrophils and sonicates were unable to mediate plaque inhibition. Speculations were made as to the origin of the virus-inhibitory substances and the role that neutrophils might assume in mediating recovery from virus infection.
Interspecies Neural Membrane Antigens on Cultured Human and Murine Neuroblastoma Cells
Authors: Akeson Richard , Seeger Robert C.
Published: December 2022

Cell-surface antigens (CSA) of cultured human and murine neuroblastoma cells were studied serologically to determine if any were common to both species and if any were associated with normal adult neural tissue. Antisera were obtained after immunization of rabbits with: i) cultured murine neuroblastoma cells, ii) cultured human neuroblastoma cells, and iii) human followed by murine neuroblastoma cells. In microcomplement-fixation tests with cultured human cells as targets, some unadsorbed antisera to human or murine neuroblastoma cells had greater reactivity with neuroblastoma than with non-neuroblastoma cell lines. Booster immunization with murine neuroblastoma cells of all rabbits initially immunized with human neuroblastoma cells produced sera with higher titers and greater specificity for human neuroblastoma cells. One serum obtained after such sequential immunization with human and murine neuroblastoma cells was adsorbed sufficiently with non-neural cells and tissues so that it reacted only with neuroblastoma cells in a 125I-protein A-binding test. Anti-human neuroblastoma antibodies in this serum were specifically adsorbed by both some human and some murine neuroblastoma cells and also by the particulate fractions of human and murine brain. Anti-murine neuroblastoma antibodies in this serum were specifically adsorbed by the particulate fraction of human brain. These data define new interspecies cell typespecific CSA present on some cultured human and murine neuroblastomas and on normal adult brain of both species. Antisera to interspecies neural antigens can be readily prepared by sequential immunization with human and then murine neuroblastoma cells.
Biphasic Effect of Cyclic AMP on IgG Production and on the Changes of Non-Histone Nuclear Proteins Induced with Anti-Immunoglobulin and Enhancing Soluble Factor
Authors: Kishimoto Tadamitsu , Nishizawa Yoshio , Kikutani Hitoshi , Yamamura Yuichi
Published: December 2022

The relationship between the changes of non-histone nuclear proteins and the differentiation of B cells to IgG-producing cells was studied by analyzing the effect of exogenously administered dibutyryl cyclic AMP. Stimulation of B cells with anti-Ig or incubation of anti-Ig-stimulated cells with enhancing soluble factor (SF) induced an increase in the amounts and phosphorylation of non-histone nuclear proteins without any concomitant increase or decrease of histones.

The addition of 5 × 10-4 M dibutyryl cyclic AMP or 5 × 10-5 M isoproterenol at 2 hr after anti-Ig stimulation showed the maximum augmentation of IgG production induced by the sequential stimulation of B cells with anti-Ig and SF. The addition of the same concentration of dibutyryl cyclic AMP within 24 hr after the initiation of the second stage of culture with SF inhibited the induction of IgG. The same biphasic effect of dibutyryl cyclic AMP was observed in the changes of non-histone nuclear proteins induced with anti-Ig or SF. 5 × 10-4 M dibutyryl cyclic-AMP augmented an increase of amounts or phosphorylation of non-histone nuclear proteins induced with anti-Ig, whereas an increase of amounts or phosphorylation of non-histone nuclear proteins induced with SF was suppressed by the addition of the same concentration of dibutyryl cyclic AMP. The same biphasic effect of dibutyryl cyclic AMP on IgG production and on the changes of non-histone nuclear proteins strongly suggests the intimate involvement of non-histone nuclear proteins in the differentiation process of B cells.
Induction of Guinea Pig Antibody Responses in Vitro
Authors: Cohen Philip L. , Ellner Jerrold J. , Shevach Ethan M.
Published: December 2022

Guinea pig spleen cells cultured together with peritoneal exudate lymphocytes (PEL) were found to generate large numbers of antibody-forming cells (AFC) in vitro in response to hapten-protein antigens. Neither cell type cultured alone yielded appreciable responses. Strain 13 or F1 (Strain 2 × Strain 13) lymphocytes, but not those from strain 2 animals, are able to respond to the genetically controlled antigen, DNP-guinea pig albumin (DNP-GPA). Antisera directed against responder (strain 13) parent Ia antigens selectively blocked the generation of AFC by F1 (strain 2 × strain 13) spleen-PEL mixtures in response to DNP-GPA. Both allogeneic (strain 2) and syngeneic macrophages functioned equally well in presentation of DNP-GPA to strain 13 lymphocytes.
The Subunit Composition and Sedimentation Properties of Human C1
Authors: Ziccardi Robert J. , Cooper Neil R.
Published: December 2022

An equimolar ratio of C1q, C1r, and C1s was found in normal human serum by radial immunodiffusion when 410,000, 190,000, and 174,000 daltons were used as their respective m.w. These values for C1r and C1s represent the sum of the weights of the two noncovalently linked subunits of C1r and the sum of the two calcium-linked subunits of C1s. Up to 95% of the serum C1 hemolytic activity applied to sucrose gradients formed in buffers of physiologic ionic strength was recovered from the gradients in a single peak which sedimented with a rate of 16S. After pooling, dialysis, and concentration, this peak was analyzed by single radial immunodiffusion and was found to contain equimolar concentrations of C1q, C1r, and C1s. The specific activity of this peak of C1, when expressed as effective molecules per microgram of C1q, C1r, and C1s was identical to that found in serum. C1 reassembled from C1q, C1r, and C1s sedimented as a 15 to 16S macromolecule and also contained equimolar concentrations of C1q, C1r, and C1s. With dilution of serum before centrifugation, the recovery of C1 from the gradients dropped dramatically and only 18% of the C1 activity was recovered from a 1/10 dilution. Under these conditions, minor peaks of C1 activity representing only a few percent of C1 activity became evident at 12S and 19S. Under low ionic strength conditions C1 aggregated. C1t antigen was found to behave independently from C1 in these studies. The number of moles of C1t in serum varied from one-third to two-thirds of that observed for C1q, C1r, and C1s. In sucrose gradients all of the C1t sedimented separately from C1 with a rate of <13S. Isolated C1t did not substitute for any C1 subunit or enhance C1 activity or the sedimentation rate of C1. Thus, no role for C1t in C1 activity or structure was evident in these studies.

This work indicates that C1 is composed of equimolar concentrations of C1q, C1r, and C1s assembled in a macromolecule with a m.w. of 774,000 daltons which sediments in sucrose gradients with a rate of 16S.
Immunosuppressive Factor(s) Specific for L-Glutamic Acid50-L-Tyrosine50 (GT)
Authors: Waltenbaugh Carl , Debré Patrice , Thèze Jacques , Benacerraf Baruj
Published: December 2022

The random synthetic copolymer of L-glutamic acid50-L-tyrosine50 (GT) fails to elicit a GT-specific antibody response in all inbred strains of mice tested. Preimmunization with GT specifically inhibits a GT-MBSA response in certain, H-2d,k,s, but not other, H-2a,b,q, nonresponder mice. This unresponsiveness is mediated by GT-specific suppressor T cells. Extracts prepared from lymphoid cells of GT-primed suppressor haplotype mice inhibit the development of primary GT-specific antibody responses to GT-MBSA in normal syngeneic mice. Nonsuppressor haplotype mice do not produce GT-specific suppressor factor. The GT-suppressive extract has affinity for antigen and a m.w. of less than 50,000 daltons, thus, resembling antigen-specific immunosuppressive factors already described. However, the GT-suppressive extract does not appear to have H-2 restrictions since it works across allogeneic barriers. Evidence is presented that two genes are required for factor-mediated suppression.
The Response of Recombinant Inbred Strains of Mice to Bacterial Lipopolysaccharides
Authors: Watson James , Riblet Roy , Taylor Benjamin A.
Published: December 2022

Fourteen recombinant inbred strains of mice have been produced by the inbreeding of the F2 generation of a cross between C57BL/6J and C3H/HeJ progenitor mice. The responses of these BXH strains to bacterial lipopolysaccharides (LPS) have been characterized. Four BXH strains are high LPS responders and nine strains are low LPS responders. One BXH strain shows intermediate responsiveness which may reflect residual heterozygosity. F1 hybrid mice from low × high responder strains were intermediate in their response to LPS suggesting additive genetic control. The LPS responses in backcross mice from the F1 × low LPS responders showed segregation consistent with LPS responsiveness being determined by a single gene. In 13/14 BXH strains, there was concordant inheritance of LPS responsiveness and the major urinary protein locus Mup-1b. The association of the expression of the Mup-1 alleles with LPS responsiveness in the BXH strains suggests that the defective LPS response gene in C3H/HeJ mice is located on chromosome 4.
Rapid in Vivo Destruction of Semi-Syngeneic and Allogeneic Cells by Nonimmunized Mice as a Consequence of Nonidentity at H-2
Authors: Carlson George A. , Wegmann Thomas G.
Published: December 2022

By monitoring the death and metastatic distribution of 131I-iododeoxyuridine (IUdR) prelabeled H-2d leukemia cells, hybrid and allogeneic resistance were studied in vivo. All mice tested were able to recognize and kill leukemia cells which were not H-2 identical with self. Resistance was found, not only against completely allogeneic leukemia cells, but also against semi-syngeneic parental strain cells in F1 hybrid mice. This resistance against semi-syngeneic and allogeneic cells was manifest in two ways. First, the total survival of incompatible cells as determined by whole-body γ counting was less than the survival in H-2 identical hosts, with the killing of the grafted cells apparent within 24 hr of injection. Second, colonization of the spleen by the H-2 homozygous leukemia cells was inhibited in H-2 heterozygous and H-2 incompatible mice when measured as early as 3 days after injection. This splenic rejection of grafted leukemia cells was organ specific because the sharp reduction of radioactivity seen in the spleen was not paralleled by a concomitant loss in other organs such as the liver. Differences at either the K through I-B regions of the H-2 gene complex or the D region alone were sufficient to initiate rejection.

Although all nonirradiated mice showed early recognition and rejection of cells which were not H-2 identical with self, irradiation abolished rejection in some strains of mice whereas in others the rejection process was unaffected. While differences at H-2 provided the targets for recognition, non-H-2 genes determined the susceptibility of the rejection process to irradiation.
Activation of Human B Lymphocytes
Authors: Haynes Barton F. , Fauci Anthony S.
Published: December 2022

Con A-induced suppression of the direct PFC response to polyclonal stimulation in human B cells has been described. Two types of experiments are presented. First, Con A was added directly to PWM-stimulated PB or tonsil cells resulting in a dose-dependent suppression of the PFC response, with maximal suppression occurring at a Con A concentration of 10 µg/ml. This suppression is completely removed by the simultaneous addition of αMM to the cultures. Secondly, Con A stimulation of tonsil or PB lymphocytes generated a population of cells which when added to autologous lymphocyte cultures induced a marked and reproducible suppression of the PFC response. The generation of suppressor cells is dependent on cell division and is blocked by αMM. Once generated the process of suppression is independent of the presence of Con A itself and is mediated by an activated lymphocyte population. These studies demonstrate a simple and reproducible model for the generation of a population of suppressor cells capable of inhibiting the direct PFC response to PWM-induced polyclonal activation of normal human B lymphocytes.
Similar Cell Surface Antigens on Hamster Cells Transformed by Different Papovaviruses
Authors: Molinaro Giuseppe A. , Major Eugene O. , Bernhardt Gregory , Dray Sheldon , di Mayorca Giampiero
Published: December 2022

Transformed baby hamster kidney (BHK) cells were tested for surface antigens by an immunocytoadhesion method. The cells were sensitized with rabbit antisera to cell clones transformed by polyoma or by BK virus and then rosetted with erythrocytes coated with antibody to rabbit immunoglobulin. These antisera detected common antigens on BHK cells transformed by either of three papovaviruses, polyoma, BK, or SV40, but apparently not on normal BHK cells.
Hydrolysis of a Synthetic Amide Substrate by Human C1 Esterase (C1s̄)
Authors: Morgan Paul H. , Nair Indira G.
Published: December 2022

C1 esterase (C1s̄), the enzymatically active form of the C1s subunit of the first component of complement, activates the classical complement pathway by limited proteolysis of complement components C2 and C4. In searching for chromogenic amide substrates which would reflect the physiologic function of C1s̄, it was found that preparations of C1s̄ slowly hydrolyzed N-benzoyl-L-phenylalanyl-L-valyl-L-arginine p-nitroanilide (BzPheValArgNA). Chromatographic, electrophoretic, and inhibition studies confirmed that the BzPheValArgNA-hydrolyzing activity of these preparations was attributable to C1s̄. Although C1s̄ is known to cleave a number of N-acylated α-amino acid esters, no synthetic amide substrate has been reported previously for this enzyme.

Kinetic analysis of the hydrolysis of BzPheValArgNA by C1s̄ yielded an apparent Km of 0.36 mM and a Kcat of 5.7 min-1. Enzymatic activity toward BzPheValArgNA appeared to be controlled by two ionizable groups with pKs of approximately 6.5 and 8.9. In view of the sequence homology between C1 esterase and the pancreatic serine proteases, it is suggested that these groups correspond respectively to the β-carboxyl group of aspartic acid 102 and the α-amino group of isoleucine 16 in α-chymotrypsin.

Although C1s̄ hydrolyzed BzPheValArgNA, it did not cleave N-benzoyl-DL-arginine p-nitroanilide. This finding suggests that substrate binding at the subsites occupied by the valyl and/or phenylalanyl residues is critical to the amidase activity of C1s̄.
Precipitation of Radiolabeled Antigen-Antibody Complexes with Protein A-Containing Staphylococcus Aureus
Authors: Brunda Michael J. , Minden Percy , Sharpton Thomas R. , McClatchy J. Kenneth , Farr Richard S.
Published: December 2022

The feasibility of using protein A-containing Staphylococcus aureus to measure antibodies in sera from several mammalian species was studied. A variety of unrelated radiolabeled antigens were tested, including components of bovine serum, DNA, and bacterial and tumor-associated extracts. The use of S. aureus was found to be a reliable way to detect and measure the primary interactions between many of the antigens and antibodies tested. Results were equivalent under many circumstances to those obtained with the ammonium sulfate and heterologous anti-immunoglobulin methods. However, some of the limitations noted were that certain antigens bound directly to S. aureus and that all classes of human immunoglobulins tested, in particular IgG3 and IgA1, were not precipitated by S. aureus. If these limitations are taken into consideration, the use of S. aureus can be of value in studying immunochemical reactions with other antigens.
In Vivo Immune Response Suppression by the Supernatant from Concanavalin A-Activated Spleen Cells
Authors: Reinertsen James L. , Steinberg Alfred D.
Published: December 2022

Supernatants from concanavalin A- (Con A) activated murine spleen cells have been shown to suppress the in vitro plaque-forming cell (PFC) response to sheep red blood cells (SRBC). The present study examined the effect of such Con A-activated spleen cell supernatants (herein termed CONS) on the in vivo immune response to SRBC in C57BL/6, BALB/c and CDF1 mice. CONS derived from BALB/c spleen cells suppressed direct PFC 4 and 8 days after immunization with 2 × 108 SRBC. CONS also suppressed indirect PFC 8 days after immunization, as well as serum hemagglutinins to SRBC. The PFC response of C57BL/6 (H-2b) mice was suppressed as much as that of BALB/c (H-2d) by CONS derived from BALB/c mice, indicating a lack of H-2 specificity of the CONS.

In addition to suppression of the antibody response to SRBC, in vivo CONS administration resulted in reduction in spleen cell number. This reduction was not sufficient to explain the decreased PFC response.

When the CONS was separated into <10,000 m.w. and ≥ 10,000 m.w. fractions, the immunosuppressive activity was found in the <10,000 m.w. fraction. This observation suggests that intact interferon, SIRS, and MIF were not responsible for the results obtained.
Nature of the Antigenic Complex Recognized by T Lymphocytes
Authors: Thomas David W. , Yamashita Uki , Shevach Ethan M.
Published: December 2022

We have previously demonstrated that nonimmune guinea pig T lymphocytes could be specifically sensitized with TNP-modified allogeneic macrophages after eliminating the alloreactive T cells with bromodeoxyuridine (BUdR) and light treatment. This procedure allowed the unique opportunity to use anti-Ia sera directed against the Ia antigens of only the stimulator macrophages or responder T cells to determine against which cell type anti-Ia would block TNP-specific stimulation. It was found that the TNP-specific DNA synthetic response of BUdR and light-treated T cells stimulated with TNP-modified allogeneic macrophages was totally eliminated by anti-Ia sera directed solely against the allogeneic stimulator macrophage. In contrast, anti-Ia sera directed only against the responder T cells had no effect on their response to TNP-modified allogeneic macrophages. These findings indicate that macrophage Ia antigens are required for efficient T cell-macrophage interactions and raise the possibility that T cell Ia antigens may not be required for collaboration with macrophages. This latter possibility was substantiated by experiments in which we show that treating T cells with anti-Ia sera and complement to remove the Ia-positive cells either before or after priming, or both, had no effect on their ability to be primed and restimulated with TNP-modified macrophages.
Delineation of Three Classes of CEA Antigenic Determinants: Identification of Membrane-Associated CEA as an Independent Species of CEA
Authors: Leung Julia P. , Plow Edward F. , Eshdat Yoval , Marchesi Vincent T. , Edgington Thomas S.
Published: December 2022

The immunochemical relationship between membrane-associated CEA (CEA-M) and isolated conventional whole CEA and the species CEA-S was investigated. These glycoproteins gave precipitin reactions of apparent identity by using antisera to CEA-M and CEA-S; however, the binding of each of these glycoproteins relative to one another by anti-tumor glycoprotein, anti-CEA-S, and anti-CEA-M anti-sera differed to a significant degree, indicative of some degree of nonidentity. Competitive inhibition radioimmunoassay with a 125I CEA-S ligand and a broadly reactive anti-CEA-S antiserum demonstrated that all three glycoproteins possessed in common antigenic determinants of the CEA group of glycoproteins but at apparent different concentration. These appeared to reflect the presence of CEA group-specific determinants. On the other hand, when 125I CEA-M and an anti-CEA-M antiserum were used in competitive inhibition assays, both CEA and CEA-S were antigenically deficient relative to CEA-M. After absorption of the anti-CEA-M with CEA, CEA-M produced complete inhibition, SDS-unfolded CEA produced 60% inhibition and CEA was unreactive. This antiserum was further absorbed with SDS-unfolded CEA and used in assay with 125I CEA-M. Although CEA-M remained reactive, CEA or CEA subjected to denaturants such as SDS, 2-mercaptoethanol, and SDS with 2-mercaptoethanol did not inhibit this assay system, indicating the absence of the remaining determinants present on CEA-M. Based on these observations we have proposed the existence not only of CEA group-specific determinants but also the existence of CEA cryptic determinants that may be generated from soluble forms of CEA and CEA-S by unfolding and exposure of cryptic sites. In addition, the existence of CEA species-specific determinants, which appear to be unique to a particular species of CEA, have been demonstrated. In a CEA-M-specific radioimmunoassay system with CEA-M absorbed antiserum the CEA-M species-specific determinants can be directly demonstrated in micro-some-rich homogenates of colonic adenocarcinomas. Thus, molecules bearing CEA-M species-specific determinants are produced by cells and are not an artifact of isolation of CEA-M. Demonstration of this third class of determinants on CEA-M in this study establishes that it is an independent species of CEA.
Migration Inhibition by an Agarose Microdroplet Assay: Monitoring of Tumor-Associated Antigens on a Simian Virus 40-Induced Sarcoma
Authors: McCoy J. L. , Padarathsingh M. , Dean J. H. , Henriksen O. , Natori T. , Law L. W.
Published: December 2022

Macrophage migration inhibition assays, with a direct agarose microdroplet method, were used to monitor TAA activity of preparations of SV-40-induced mKSA cells. These preparations included cell-free crude membranes, papainsolubilized and NP40 detergent-solubilized membrane extracts from mKSA tumor cells. The assay was extremely sensitive and could detect migration inhibition reactivity with all three types of antigenic preparations with concentrations as low at 250 pg protein/ml. The reactivities were quite reproducible from experiment to experiment using the same or different lots of these antigen preparations, and the reactivities were specific in that peritoneal exudate cells from BALB/c mice, immunized with antigenically unrelated but syngeneic plasmacytomas, were not inhibited by these antigens. The results demonstrated the usefulness of this assay in rapidly detecting small concentrations of partially purified TAA preparations by using small number of immune cells.
Association of a Low Molecular Weight Helper Factor(s) with Thymocyte Proliferative Activity
Authors: Koopman William J. , Farrar John J. , Oppenheim Joost J. , Fuller-Bonar Janet , Dougherty Suanne
Published: December 2022

Human mixed leukocyte supernatants contain thymocyte proliferative activity (TPA) and a low m.w. helper factor, designated HP-1, which is capable of partially restoring the antibody response of T cell-deficient adherent murine spleen cells to the thymic-dependent antigen, SRC. TPA and HP-1 appear to have a comparable m.w. (14,000 to 14,500 daltons) by Sephadex gel filtration column chromatography. Furthermore, HP-1 and TPA exhibit similar patterns of heterogeneity on DEAE-cellulose chromatography, elute together on CM-cellulose chromatography, and manifest identical patterns of migration on polyacrylamide gel electrophoresis. These data suggest that the TPA and HP-1 activities reside in either the same molecule(s) or in different molecules with identical charge/mass ratios. Furthermore, the results support the hypothesis that the helper activity of HP-1 is derived from its capacity to activate T and/or pre-T cells.
Release of Phospholipids from Complement-Mediated Lesions on the Surface Structure of Escherichia Coli
Authors: Inoue Kozo , Kinoshita Taroh , Okada Mariko , Akiyama Yoko
Published: December 2022

When varying numbers of sensitized, 14C-labeled bacteria were treated with a certain amount of complement, in a fixed reaction volume, 14C compounds were liberated into the surrounding medium in proportion to the number of the bacteria, whereas the amount of the phospholipids liberated was constant regardless of the number of the bacteria even in the range of relative excess of complement. Since it is conceivable that a certain amount of complement might form a fixed number of lesions on the surface of all the sensitized bacteria, the amount of the liberated phospholipids seems to be proportional to the number of complement lesions.

The 14C-materials released from complement-attacked bacteria were analyzed by isopycnic sucrose density gradient ultracentrifugation and they were mainly free phospholipids and other smaller molecules. A small amount of the smaller membrane proteins were also released as revealed by acid and SDS-polyacrylamide gel electrophoresis.

These results suggest that the release of phospholipids is due to the displacement of membrane lipids by the complexes of the late acting complement components during their insertion into the membrane lipid bilayer.
Inhibition of Human Lymphocyte-Mediated Mitogen-Induced Cytotoxicity of Murine L-929 Cells by Heterologous Anti-Human Lymphotoxin Antisera In Vitro
Authors: Hiserodt John C. , Granger Gale A.
Published: December 2022

Heterologous anti-human lymphotoxin (LT) antisera have been employed to investigate the role of LT in mitogen-(Con-A, PHA) induced destruction of murine L-929 cells by human lymphocytes in vitro. These various antisera will effectively neutralize human LT molecules associated with the stable (70 to 90,000 dalton) α-LT class of cytotoxin (anti-α-LT), the more unstable (35 to 50,000 dalton) β-LT class of cytotoxins (anti-β-LT), and antisera which will neutralize all classes of these cytotoxins in vitro, anti-whole supernatant (anti-W.S.). These anti-LT sera will greatly inhibit lysis of L-929 cells by using mitogen-activated human effector lymphocytes in vitro. This blocking was shown to be mediated by whole serum, purified IgG, or IgG-Fab fragments, which had been extensively absorbed with bovine serum, human serum, mitogens, and normal human lymphocytes. Inhibition of lysis was not apparently due to interference with either lymphocyte-target cell contact or lymphocyte activation step(s). The blocking effects of these sera were also shown to occur during the lymphocyte-independent phase of the lytic reaction. These data support the concept that the lymphocyte deposits an LT-like effector molecule on the target-L cell surface during the lymphocyte-dependent phase, which mediates cell lysis at a later time during the lymphocyte-independent phase.
Cyclic Nucleotide Changes in Human Neutrophils Induced by Chemoattractants and Chemotactic Modulators
Authors: Hatch Gary E. , Nichols William K. , Hill Harry R.
Published: December 2022

Two classes of agents have effects on the chemotactic response of human neutrophils. Chemoattractants such as activated complement components, E. coli culture filtrates (BF) and the recently discovered N-formylmethionyl peptides, initiate directed movement, whereas chemotactic modulators enhance or depress cell movement initiated by chemoattractants. We and others have previously shown that cyclic AMP (cAMP) depresses neutrophil migration toward BF whereas cyclic GMP (cGMP) enhances the same process. In the present study, we examined the effects of several chemoattractants and chemotactic modulators on neutrophil levels of cGMP and cAMP. Trypsinized human complement, the E. coli BF and the synthetic peptide, formylmethionylalanine stimulated the accumulation of neutrophil cGMP (40 to 70% average increase) but had little effect on cAMP concentrations. The enhancing chemotactic modulators — phenylephrine, prostaglandin F2α, carbachol, and phorbol myristate acetate — also produced small (19 to 34%) but significant increases in PMN cGMP levels. In contrast, two agents which inhibit the chemotactic response, isoproterenol and prostaglandin E1, significantly elevated cAMP concentrations in PMN.

These results suggest that the ability to elevate cGMP is inherent in the action of both chemoattractants and enhancing chemotactic modulators. Increases in cAMP appear to be correlated with inhibition of chemotaxis.
Murine Plasma Cells Secreting More Than One Class of Immunoglobulin Heavy Chain
Authors: Neiders Mirdza E. , Morse Herbert C. , Lawton Alexander R. , Asofsky Richard
Published: December 2022

SAMM 368, a plasmacytoma which produces IgA-κ and IgG2b-κ was established in vitro from primary explants or after animal passage. The 9 lines that were established produced both paraproteins. The production of both immunoglobulins by single cells was demonstrated by immunofluorescent staining and cloning. Continuous culture of 3 parent lines for 18 months and 11 cloned lines for periods from 5 to 7 months demonstrated that double production is a stable characteristic of this plasmacytoma.

Two single paraprotein-producing varients (IgG2b or IgA) were derived when cells were cultured in the presence of Fungizone.

Chromosomal analysis of SAMM 368 indicates that this double producing tumor has a modal number similar to those observed in myelomas producing a single immunoglobulin class.
A Possible Difference in the Heterogeneity of the Heavy and Light Chains from a Rabbit Anti-P-Azobenzoate Antibody Preparation
Authors: Wang Lom-Seng , Grzyb Kathleen T. , Friedenson Bernard
Published: December 2022

CNBr cleavage of rabbit heavy (H) chains leads to the formation of a fragment, C-1, which consists of the N-terminal half of the H chain. Fragment C-1 is cleaved at methionyl residues but held together by intrachain S-S bonds so that smaller fragments can be liberated by total reduction and alkylation. In the case of the C-1 fragment from an anti-p-azobenzoate antibody preparation, which has a light (L) chain of markedly restricted heterogeneity, total reduction and alkylation liberated seven major fragments in good yield. The N-terminus of two of these fragments corresponds to position 35 of the H chain but their N-terminal sequences are clearly different. The H chain regions represented by the other fragments implied that they were derived from H chains having different distributions of methionyl residues. This hypothesis was supported by isolating six different antibody components from the antibody preparation by isoelectric focusing and then digesting them with CNBr. Comparison of the products showed that the six components all appeared to behave differently. These results are interpreted as suggesting that the process whereby H and L chains are paired in vivo may not be completely specific and may provide a simple means of generating a significant contribution to antibody diversity.
The Interaction of Immunoglobulin Heavy and Light Chains in the Absence of the VH Domain
Authors: Levo Yoram , Recht Bernard , Michaelsen Terje , Franklin Edward C. , Frangione Blas
Published: December 2022

Recent studies of the µ- and κ-chains of the first patient (GLI) with µHCD indicated that the observed defect was the result of the failure of assembly of the intact κ-chain to the µ-chain, which lacked the VH domain but had the CH1 Cys normally linked to the light chain. To explore the possibility that the VH region is necessary for the formation of the HL disulfide bond, in vitro studies were performed with GLI µ- and κ-chains and with the CH1 domain and κ-chain derived from an IgG3 myeloma protein, KUP, which yields separate VH, CH1, and κ-chains after papain digestion and reduction. The proteins were reduced and allowed to reoxidize, and the combination products were assessed by gel chromatography under dissociating conditions by SDS-PAGE and by immunoprecipitation techniques. The results suggest that, although in vitro covalent and noncovalent combinations are possible between intact light chains and their autologous heavy chains even in the absence of the VH domain, the efficiency is less than that when the intact Fd region is used. Hence, it seems likely that lack of VH alone is not sufficient to explain the failure of assembly observed in µHCD.
Colonic Tumor Membrane-Associated Glycoprotein: Isolation of Antigenically-Active Peptides after Chemical Cleavage
Authors: Leung J. P. , Eshdat Y. , Marchesi V. T.
Published: December 2022

A membrane-associated glycoprotein fraction, referred to as CEA-M was isolated from human colonic tumor tissue by sodium dodecyl sulfate extraction of membrane fragments followed by wheat germ agglutinin affinity chromatography, Bio-Gel A-1.5 gel filtration and preparative slab gel electrophoresis. With a m.w. of approximately 200,000, isoelectric point of about 4.2 and carbohydrate:protein ratio of 2:1, this glycoprotein has physiocochemical and antigenic similarities to carcinoembryonic antigen, CEA. Immunochemical studies have shown that antiserum developed for this glycoprotein possesses relative specificity for human colonic carcinomas.

Chemical cleavage of this glycoprotein by 2-nitro-5-thiocyanobenzoic acid resulted in three major Coomassie Blue and two periodic acid Schiff stainable fragments (one of which stains with both). It was found that one of the glycopeptides, labeled as TA, isolated by affinity and covalent chromatography, contained 77% carbohydrates and possessed antigenic determinants recognized by at least 70% of the antibody population raised against the total glycoprotein fraction; purified antibodies to this region of the molecule seem promising for the development of a specific assay for gastrointestinal tumors.
Haplotype Preference in Lymphocyte Differentiation
Authors: Skidmore Barry J. , Katz David H.
Published: December 2022

An experimental protocol was designed to study the regulatory activity of carrier-specific F1 hybrid T cells prepared by immunization of unprimed thymocytes within the environment of one or the other of the parental haplotypes. Thus, normal (BALB/c × A/J) F1 (CAF1) thymocytes were transferred into irradiated CAF1 hybrids or parental A/J or BALB/c recipients and primed to KLH. The resulting KLH-specific activated T cells (ATC) were then co-transferred with DNP-specific B cells obtained from CAF1, BALB/c or A/J donors into irradiated CAF1 recipients which were then secondarily challenged with DNP-KLH. The result of this type of experiment revealed that KLH-primed CAF1 ATC primed in a CAF1 recipient provide comparable helper activity for DNP-primed B cells obtained from either parent or from F1 donors. In contrast, CAF1 ATC primed in parental A/J recipients were found to cooperate considerably better with B cells derived from A/J than from BALB/c donors, and vice-versa in the case of CAF1 ATC primed in parental BALB/c recipients. The crucial, and unexpected, observation was that CAF1 ATC prepared in either parental strain failed to provide adequate helper activity for CAF1 B cells.

These findings suggested the possibility that during the course of priming of F1 T cells in the environment of one of the parents: 1) helper T cells are generated capable of preferentially cooperating with B cells of the same parental haplotype as used for priming; and 2) suppressor cells are generated capable of preferentially interacting with lymphoid cells bearing the opposite parental haplotype. Indeed, strong evidence was found that suppressor cell activity directed against the opposite parental haplotype does exist in populations of CAF1 ATC prepared in either parental strain, as shown by the capacity of such ATC to inhibit the cooperative response between conventionally primed CAF1 spleen cells and DNP-primed B cells bearing the relevant parental haplotype. Moreover, CAF1 thymocytes exposed in situ to low-dose ionizing irradiation before priming manifested even greater suppressive activity measured in this manner.

These observations can be most readily understood by the concept that a population of lymphocytes heterozygous at the major histocompatibility locus consists, either before or after antigen encounter, of separate populations of regulatory lymphocytes, one of which is genetically restricted in its capacity to interact with lymphoid cells bearing one of the parental haplotypes, and a second which is genetically restricted to interact only with lymphoid cells possessing the opposite parental haplotype. The basis of this cellular dichotomy may involve the “restricted expression” of cell interaction (CI) genes and their molecular products in these respective lymphoid subpopulations.
Persistence of Antigen-Binding Cells with Surface Tolerogen: Isologous Versus Heterologous Immunoglobulin Carriers
Authors: Venkataraman M. , Aldo-Benson Marlene , Borel Yves , Scott David W.
Published: December 2022

Adult mice injected intravenously with fluorescein isothiocyanate (FL) conjugated to an isologous carrier (mouse IgG2a) (FL-MGG) or to a heterologous carrier (sheep γ-globulins) (FL-SGG) are rendered specifically unresponsive to the FL-hapten. Tolerance induced by either tolerogen persists upon adoptive transfer. Since cells with tolerogen on their surface are detectable by immunofluorescence in both experimental situations, we compared the persistance of such cells in the spleens of mice injected with either tolerogen or with an immunogen (FL-KLH). FL-binding cells were seen during the first 5 days after the injection of FL-SGG, but were undetectable by day 7 and thereafter. In contrast, fluorescent cells were detectable up to 20 to 30 days in some animals after FL-MGG, although the number decreased after day 11. FL-binding cells decreased significantly after day 1 in FL-KLH-injected mice. When the number of FL-binding cells in tolerant animals had reached baseline levels, the animals were reinjected with their respective tolerogen. Normal or slightly increased numbers of FL-binding cells were found in the spleens of FL-SGG injected mice, whereas decreased numbers were observed in the spleens of FL-MGG injected mice. This suggests that in tolerance induced by heterologous carrier, specific lymphocytes possess free receptors but cannot respond. On the other hand, with the isologous carrier-induced tolerance, free receptors are either not available (modulated?) or blocked with undectable amounts of tolerogen. Whether this difference in the kinetics represents a difference in the mechanism of tolerance is unknown.
Antibody Avidity and Allelic Selection for L Chain Allotypes in Rabbits Hyperimmunized with Streptococcal Vaccine
Authors: Sogn John A. , Hooke Phyllis Diane , Freedman Stephen M. , Kindt Thomas J.
Published: December 2022

An almost absolute selection in favor of allotype b4 occurs in the antibody response of heterozygous b4b9 rabbits immunized with Group C streptococcal vaccine. A possible explanation for this “allelic selection” in favor of b4 is that during the antistreptococcal response the favored allotype (b4) is associated with V region binding sites yielding higher avidity anti-streptococcal antibodies. In order to test this possibility, IgG from heterozygous, hyperimmune rabbits was fractionated by avidity on a Group C immunoadsorbent column and allotypes of the various fractions were quantitatively determined. Contrary to the predicted result, when secondary immunization sera were examined the fractions of highest avidity were enriched in the nonselected allotype. Antibodies from two primary immunization sera lacked the selection for the non-b4 allotype in high avidity fractions observed for the later sera. Thus, the uneven allotype ratios observed in fractions of different avidity from secondary immunization sera can be interpreted as the result of a secondary maturation effect.

The observations of allelic selection in heterozygous rabbits were extended to rabbits with allotypes b4b5 and b4b6. Although b4 predominated in immunoglobulin from these rabbits, the allotype ratios in anti-streptococcal antibody were the same as those in total IgG before immunization.
Antibody-Dependent Cellular Cytotoxicity against Murine Leukemia Viral Antigens: Studies with Human Lymphoblastoid Cell Lines and Human Peripheral Lymphocytes as Effector Cells Comparing Rabbit, Goat, and Mouse Antisera
Authors: Peters C. J. , Theofilopoulos Argyrios N.
Published: December 2022

A thymic lymphoblastoid cell line derived from a New Zealand Black mouse produces murine leukemia virus (MuLV) and was used as a target in model systems for the in vitro study of antibody-dependent cellular cytotoxicity (ADCC). Several human lymphoblastoid cell lines were investigated as potential effector cells. The most promising (Raji cells) bound to antibody-coated target cells but caused only modest levels of ADCC at 25:1 effector-to-target cell ratio with substantial lysis in the absence of antiserum. Human peripheral lymphocytes were active as effector cells in ADCC at a 5:1 ratio and produced no lysis in the absence of antibody. These cells were used to demonstrate that high dilutions of rabbit antisera to MuLV antigens p30, p15, p12, and p10 were capable of mediating lysis of MuLV-producing target cells but not of a virus-negative murine cell line.

A murine antiserum to Thy 1.2 and three caprine antisera to MuLV antigens that were active in complement-mediated cytotoxicity functioned poorly in inducing ADCC; however, rabbit antisera to similar antigens were 16- to 512-fold more efficient in cell-mediated than in complement lysis. The inefficiency of goat antisera was not due to shedding of cell surface antigens or generation of blocking factors but rather to lack of lytic interaction of antibody-coated targets with the effector cells.
Human Suppressor T Cells Induced by Concanavalin A: Suppressor T Cells Belong to Distinctive T Cell Subclasses
Authors: Sakane Tsuyoshi , Green Ira
Published: December 2022

Human peripheral blood lymphocytes were stimulated by concanavalin A (Con A) and then evaluated by their suppressive activity for thymus-derived (T) cell- and bone marrow-derived (B) cell-proliferative responses to mitogen and allogeneic cells. Con A-activated T cells markedly suppressed these responses, but Con A-activated B cells failed to demonstrate suppressor activity. Discontinuous bovine serum albumin (BSA) density gradient separation of T cells which had been activated by Con A demonstrated that a fraction containing blast cells as well as fractions containing unproliferated cells manifest the same degree of suppressor capabilities. However, when density gradient separation of T cells followed by subsequent incubation with Con A was performed, fractions of proliferating cells of low density exhibited no suppression; a fraction containing high density T cells produced marked suppression, but this fraction incorporated only little thymidine in response to Con A.

Thus, these studies indicate that Con A-induced suppressor T cells belong to a distinctive subpopulation which has already been programmed to express this function before exposure to Con A and that cell proliferation may not be a prerequisite for the development of such suppressor T cells.
Immunogenetic Analysis of H-2 Mutations. VII. Cells Responding to H-2 Kk Gene Products Give Rise to an Allogeneic Supernatant
Authors: Kettman Jack , Klein Jan , Forman James
Published: December 2022

The mixed lymphocyte reaction (MLR) in the mouse is a response by thymus-derived (T) cells against genetically determined alloantigens (1). Two genetic regions have been shown to control the elicitation of strong MLR responses: the I region of the H-2 complex and the Mls region (2–4). Genes in these regions presumably control the expression of cell surface antigens that are recognized by a large number of responding T cells (5). In addition to the cell proliferation that results from the coculture of cells differing at these loci, a factor, termed allogeneic supernatant (AS),2 is also generated very early during the MLR in the culture supernatants. The AS enhances the response of normal spleen cells to T-dependent antigens (6–8), and has the ability to substitute for T cells in the splenic B cell humoral response to the same class of antigens.
The Effects of Extracellular K+, Na+ and Ca++ on Lysosomal Enzyme Secretion from Polymorphonuclear Leukocytes
Authors: Showell Henry J. , Naccache Paul H. , Sha'afi Ramadan I. , Becker Elmer L.
Published: December 2022

Release of the lysosomal enzymes, lysozyme and β-glucuronidase, from rabbit peritoneal polymorphonuclear leukocytes induced by three kinds of secretogogues, chemotactic factors in the presence of cytochalasin B, the K+ ionophore, valinomycin, and the Ca++ ionophore, A23187 is profoundly affected by the presence and level of K+, Na, and Ca++ in the extracellular medium. K+ approximately doubles the ability of all chemotactic factors to release in the presence of cytochalasin B. Ouabain, except in a minority of instances, does not prevent enhancement by K+ suggesting that the enhancement is not due to activation of the Na+,K+ pump. Other monovalent cations can substitute for K+ but with less efficiency. Removing Na+ from the medium increased spontaneous release in the presence of Ca++ but to a much lesser extent in its absence. Chemotactic factor-stimulated release is depressed in the absence of Na+. The K+ ionophore, valinomycin, has no effect on chemotactic factor-stimulated release; however, it is capable of inducing release by itself. The secretogogue action of valinomycin requires the presence of Ca++ and is enhanced by cytochalasin B or K+. Nigericin, another K+ ionophore, in noncytotoxic concentrations is without effect on release. The enhancing effect of Ca++ on release by chemotactic factors and cytochalasin B was confirmed. La+++ has two effects on release; at sufficiently low concentrations it inhibits the Ca++-dependent release induced by chemotactic factors. At concentrations above 10-5 M, it can sustain chemotactic factor-dependent release in the presence or absence of Ca++. The release induced by the Ca++ ionophore A23187 requires Ca++ and is enhanced by cytochalasin B and by K+.

The results obtained attest to the central role of external Ca++ in the induced secretion of lysosomal enzymes from the neutrophil. It is suggested that all three kinds of secretogogues induce release by causing an influx of Ca++ into the cytoplasm of the PMN. Cytochalasin B and K+ are believed to exert their effects by increasing the rate or extent of the hypothesized Ca++ influx.