bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Vibrio Cholerae Neuraminidase on the Generation of Cell-Mediated Cytotoxicity in Vitro
Authors: Ferguson Ronald , Anderson Susan M. , Schmidtke Jon R. , Simmons Richard L.
Published: December 2022

Vibrio cholerae neuraminidase (VCN)(12.5 units/2 × 106 cells/ml) continuously present for a standard 5-day MLC will significantly (p < 0.02) increase the cytotoxic activity generated by a given number of responding spleen cells without reducing the specificity. Heat-inactivated VCN produced no such augmentation. This augmented cytotoxicity could be reproduced by preincubating (1 hr) the responding spleen cells with VCN (25 units/2.5 × 106 cells/ml) before addition of stimulating spleen cells. Preincubating the stimulator spleen cells with VCN had no effect. VCN preincubation of target cells or presensitized effector cells produced no augmentation. The addition of soluble VCN to the killing assay also did not increase cytotoxicity. Thus, VCN acts only during the generation of specifically sensitized cytotoxic T cells. When the effect of VCN on MLC reactivity, cell recovery and total cytotoxicity (lytic units/106 cells) were compared, it became apparent that VCN increases the proliferation of responder cells after stimulation resulting in both an increased number of cells and also an increase in the proportion of specifically sensitized cytotoxic cells in the culture. VCN treatment of responder cell membrane apparently permits a more ready response to allogenic antigens in culture facilitating both increased proliferation and the increased development of specific cytotoxic killers.
Functional Characterization of Rat Mast Cell Arylsulfatase Activity
Authors: Orange Robert P. , Moore Elizabeth G.
Published: December 2022

Extracts of isolated rat peritoneal mast cells were demonstrated to contain appreciable quantities of arylsulfatase activity. The enzyme was inhibited by both phosphate and sulfate ions and demonstrated a pH optimum of 5.0. The enzyme was recovered in the eluate of DE-52 columns and appeared to have a m.w. of 150,000 on Sephadex G-200 gel filtration. These findings and the anomalous kinetic behavior of the enzyme suggest that at least part of the enzymatic activity is of the arylsulfatase IIA type. While spontaneous release of the enzyme was observed, challenge of isolated rat mast cells with a goat anti-rat IgE serum resulted in a significant increase in release of the enzyme. The arylsulfatase activity extracted from isolated rat mast cells demonstrated comparable activity in inactivating slow reacting substance of anaphylaxis (SRS-A) to that described for human eosinophil and lung arylsulfatase.
Cell-Mediated Destruction of Human Leukemic Cells by MHC Identical Lymphocytes: Requirement for a Proliferative Trigger in Vitro
Authors: Sondel Paul M. , O'Brien Carmeline , Porter Linda , Schlossman Stuart F. , Chess Leonard
Published: December 2022

These experiments have investigated cellular mechanisms involved in the generation of cellular immune responses to human acute leukemic blasts. Because normal human lymphocytes are not able to recognize immunologically, in vitro, lymphocytes from MHC identical siblings, the present studies have examined the in vitro proliferative and cytotoxic responses of normal lymphocytes to MHC identical AML and ALL blasts. In those cases where acute leukemic cells were unable to induce a proliferative response by MHC identical lymphocytes, the generation of effective anti-leukemic cytotoxicity required the addition of unrelated stimulating cells to the sensitization culture. In contrast, leukemic blasts that induced a proliferative response by MHC identical lymphocytes were also able to stimulate anti-leukemic cytotoxicity. This could be augmented by the addition of unrelated stimulating cells to the sensitization culture. The specificity of anti-leukemic cell cytotoxicity was demonstrated in all instances by simultaneous testing of putative killer cells on 51Cr leukemic blasts as well as 51Cr-labeled MHC identical phytohemagglutinin blasts or normal lymphocytes. Simultaneous sensitization to MHC identical leukemic blasts and unrelated stimulating lymphocytes did not invariably generate anti-leukemic cytotoxicity even when allogeneic cytotoxicity was observed; the absence of demonstrable suppressor activity in these nonreactive combinations suggested that some individuals may be specifically immuno-incompetent, and thereby unable to generate effective anti-leukemic CML.
Subpopulations of Chicken B Lymphocytes
Authors: Lifter John , Kincade Paul W. , Choi Yong Sung
Published: December 2022

Immunoglobulin-synthesizing cells from the spleen and bursa were fractionated by the 1 × G sedimentation velocity technique and characterized by their ability to synthesize immunoglobulin and by staining with fluorescent anti-light chain chain. Four subpopulations of immunoglobulin-synthesizing cells were identified. In the bursa, slowly sedimenting (S 2.3 mm/hr) and rapidly sedimenting (S greater than 3.5 mm/hr) subpopulations with surface immunoglobulin were present; in the spleen, a slowly sedimenting (S 2.3 mm/hr) subpopulation with surface immunoglobulin and plasma cells (S greater than 3.5 mm/hr) with large concentrations of intracellular immunoglobulin existed. The subpopulations differed most markedly in their ability to synthesize immunoglobulin (cpm Ig synthesized/106 Ig-positive cells); the rates of immunoglobulin synthesis were in the ratio 1:2:1:900. The slowly sedimenting B cells from the spleen and both subpopulations of B cells from the bursa released small amounts of immunoglobulin into the culture media, whereas, the plasma cells released immunoglobulin at a rate as much as 3700 times greater. Bursal B cells could be further distinguished from splenic B cells by a greater amount of DNA synthesis.
Hemolytic Plaque Inhibition: The Physical Chemical Limits on Its Use As an Affinity Assay
Authors: DeLisi Charles
Published: December 2022

An analysis of the fundamental physical chemical limits of hemolytic plaque inhibition as a method for obtaining thermodynamic and kinetic information is presented. It is shown that inhibition curve characteristics will be sensitive functions of reaction affinities only when the antibody inhibitor and antibody red blood cell reaction mechaisms are related in certain ways. It is further shown that conditions which are most sensitive to IgG affinity changes will generally not be the best for detecting changes in IgM affinity. The apparently conflicting reports on IgM maturation are completely explicable in terms of experimental requirements imposed by physical chemical characteristics of the reaction. Experiments in which maturation is observed are found to conform to the most sensitive conditions of the assay, whereas those in which it is not observed are found to conform to relatively insensitive conditions, and would therefore be capable of registering changes only when affinity shifts are large.
Chemical Treatment of Macrophages Increases Their Responsiveness to Migration Inhibitory Factor (MIF)
Authors: Remold Heinz G.
Published: December 2022

The response of guinea pig macrophages to migration inhibitory factor (MIF) is altered by several chemical treatments. Treatment of macrophages with the diazonium salt of sulfanilic acid (5 × 10-6 to 4 × 10-4 M) significantly increases the response of these cells to MIF. Treatment with acetic anhydride also augments the response of these cells to MIF. The latter finding suggests that alteration of amino, hydroxyl, or sulfhydryl groups is involved in this phenomenon. Treatment of macrophages with sodium periodate (2 × 10-5 to 10-3 M) which is known to oxidize cis-glycols and with hydroxylamine (2 × 10-5 to 2 × 10-3 M), which reacts with carbonyl groups also increases response to MIF. The following experiments suggest that the significant alteration occurs at the level of the cell surface. Incubation of macrophages with the diazonium salt of sulfanilic acid at 4°C, at which temperature pinocytosis is largely inhibited, is sufficient to increase the MIF response. The activity of the cytoplasmic enzyme aspartate aminotransferase, which in homogenates is susceptible to inactivation by low concentrations of the diazonium salt of sulfanilic acid, is not decreased when intact macrophages are incubated with high concentrations of the diazonium salt of sulfanilic acid. Cumulatively, these findings suggest that modification of different functional groups on the macrophage surface causes the same physiologic effect.
Passive Transfer of Systemic Tumor Immunity with Cells Generated in Vitro by a Secondary Immune Response to a Syngeneic Rat Gross Virus-Induced Lymphoma
Authors: Bernstein Irwin D.
Published: December 2022

Spleen cells taken from W/Fu rats 4 to 6 weeks after immunization with the syngeneic Gross virus-induced lymphoma, (C58NT)D cells, at a time when they lack detectable activity in a short-term 51Cr release assay, were previously shown to retain the capacity to generate cytotoxic activity upon reexposure to mitomycin C-treated lymphoma (C58NT)D cells in vitro. In the studies presented here, we evaluated whether in vitro sensitization of immune lymphoid cells before systemic transfer to a nonimmune recipient allows for more effective transfer of tumor immunity. The results show that the passive transfer of immune spleen cells after in vitro cocultivation with mitomycin-treated (C58NT)D cells allows for inhibition of growth of a subcutaneous inoculum of lymphoma cells. In contrast, spleen cells obtained 4 to 6 weeks after primary sensitization or after secondary in vivo sensitization did not effectively confer anti-tumor immunity. As few as 5 × 107 in vitro sensitized cells permitted complete inhibition of 106 (C58NT)D cells and also allowed for inhibition of the growth of 107 (C58NT)D-F cells, which was lethal to control animals. Immune cells sensitized with syngeneic thymocytes or normal spleen cells sensitized with (C58NT)D cells in vitro did not confer in vivo anti-tumor immunity. After systemic transfer of in vitro sensitized cells, delayed hypersensitivity occurred at the site of tumor inoculation and tumor growth was suppressed. Specificity of the passive immunity was shown by the failure to inhibit growth of a polyoma virus-induced sarcoma in rats which inhibited growth of the Gross virus-induced lymphoma cells. In vitro sensitized cells were more effective in the transfer of anti-tumor protection after 5 days, as compared to 2 days, of cocultivation with tumor. Results show that in vitro sensitized cells can effectively transfer systemic tumor immunity.
Reaginic Antibody Formation in the Mouse
Authors: Takatsu Kiyoshi , Ishizaka Kimishige
Published: December 2022

Attempts were made to increase the activity of suppressor cells in vitro. Antigen-specific suppressor cells were induced by i.v. injections of urea-denatured ovalbumin (UD-OA) into OA-primed mice. Nonadherent splenic lymphocytes from the UD-OA-treated mice were incubated at 37°C for 24 hr with either OA or OA-bearing macrophages and lymphocytes harvested from the culture were examined for the ability to suppress primary anti-hapten antibody response of nonirradiated mice to DNP-OA. The results showed that the suppressive activity of the lymphocytes increased after culture of the cells with OA or OA-bearing macrophages. Similar results were obtained when nylon column-purified T cell-rich fraction of the lymphocytes were similarly cultured. The suppressive activity was associated with ϑ-bearing lymphocytes and was specific for OA. Suppressor cells were not induced by the culture of OA-primed lymphocytes with OA. The helper function of splenic lymphocytes from both UD-OA-treated mice and OA-primed mice was enhanced by the culture of the cells with OA-bearing macrophages but not by culture with OA in the absence of macrophages.
Reversal of SV40 Tumor-Mediated Suppression of Spleen Cell Cytotoxicity by Antibody
Authors: Prather Suzanne O. , Lausch Robert N.
Published: December 2022

Spleen cells obtained from hamsters bearing PARA-7 tumors greater than 1.0 cm were not reactive in microcytotoxicity assays unless preincubated overnight. The events occurring during in vitro incubation which lead to reversal of tumor-mediated suppression of cellular immunity were investigated. After 24 hr of incubation, supernatants overlying spleen cells from tumor-bearing hosts contained a factor which blocked cytotoxicity of simian virus 40 (SV40)3-sensitized spleen cells at the PARA-7 target cell level but not at the effector cell level. The preparations did not mediate antibody-dependent cellular cytotoxicity (ADCC). Opposite results were obtained in assays of culture medium overlying spleen cells from hosts with a tumor burden less than 0.1 cm. Although ADCC activity was present, no significant blocking was detectable.

Treatment of inactive spleen cells with anti-hamster γ-globulin in the presence of complement (anti-HGG + C) prevented activation and formation of blocking factor but did not impair the cytotoxic activity of already activated cells. Addition of SV40 antiserum to anti-HGG + C-treated cells led to effector cell activation, whereas heterologous virus-immune sera did not. Control studies established that the antibody-mediated recovery of cytotoxicity was not due to arming. Further studies showed that PARA-7 tumor antigen extract blocked at the effector cell level, not at the target cell level. Addition of PARA-7 extract to spleen cell supernatants mediating ADCC resulted in formation of a factor which blocked at the target cell level but not at the effector cell level.

These data are compatible with the following interpretation. Spleen cell unresponsiveness is due to antigen blockade. Recovery of cytotoxicity occurs because antibody synthesized during the incubation period promotes elution of antigen from the effector cell surface. Thus, activation is accompanied by the generation of tumor antigen-antibody complexes.
Inhibition of the Mitogenic Response to Lipopolysaccharide (LPS) in Mouse Spleen Cells by Polymyxin B
Authors: Jacobs Diane M. , Morrison David C.
Published: December 2022

The addition of low doses of the cationic polypeptide antibiotic, polymyxin B (PB), to cultures of mouse spleen cells inhibits lipopolysaccharide-(LPS) induced DNA synthesis but not that stimulated by PPD, PHA, or Con A. Inhibition is stoichiometric; the mitogenic response is suppressed by 50% at a weight ratio of PB:LPS of 0.055 to 1. Furthermore, PB-LPS complexes have a much reduced mitogenic capacity. These complexes inhibit the mitogenic response of spleen cells to unmodified LPS but not to PPD, Con A, or PHA. The inhibitory activity of PB is less effective when added after LPS is mixed with responding cells, achieving 50% inhibition when addition is made at 4 to 6 hr. Time course experiments indicate that partial inhibition is a reflection of a lower rate of DNA synthesis. Thus, PB inhibition of LPS mitogenesis apparently occurs as a result of formation of PB-LPS complexes with reduced mitogenic capacity. Specific inhibition by the complexes of mitogenesis induced by native LPS suggests that the inactive complex may bind to B cells but is unable to trigger them.
Amyloid-Related Serum Protein SAA from Three Animal Species: Comparison with Human SAA
Authors: Anders Robin F. , Natvig Jacob B. , Sletten Knut , Husby Gunnar , Nordstoga Knut
Published: December 2022

The amyloid-related serum protein SAA has been isolated by gel filtration in 10% formic acid from three animal species: mink, mouse, and rabbit. Sera used in the isolation procedure were obtained from animals in which high concentrations of SAA had been induced by treatment with LPS.

The isolated SAA proteins had a subunit size similar to that of human SAA, with m.w. values ranging from 10,000 to 11,700 (estimated by gel filtration in 6 M guanidine-HCl) or 12,400 to 15,000 (estimated by SDS-PAGE). The m.w. studies and amino acid sequence data indicated that SAA and the amyloid fibril protein AA in the mouse, and probably also the mink, are related in the same way as in man, the two proteins having common NH2-terminal amino acid sequences and SAA being extended by 20 to 40 residues at the COOH-terminal end of the molecule.
The Active E-Rosette Test: A Sensitive in Vitro Correlate for Human Delayed-Type Hypersensitivity
Authors: Felsburg Peter J. , Edelman Robert
Published: December 2022

The “active” rosette test was adapted as an in vitro assay and correlated with human delayed cutaneous hypersensitivity (DCH) to two microbial antigens. Peripheral lymphocytes were purified from donors known to be responders or nonresponders to PPD-tuberculin or tularemia on the basis of prior DCH reactions. Skin test antigen, incubated with lymphocytes from antigen-sensitive donors, produced a significant increase (+2 S.D.) in the ability of the lymphocytes to form active rosette-forming cells (A-RFC) when compared to lymphocytes cultured without antigen. Skin test antigen incubated with lymphocytes from nonsensitive donors produced no increase in their A-RFC. The optimal dose of each antigen was approximately 100 ng/ml. The percentage of A-RFC rose to maximum levels between 3 and 4 hr after the addition of antigen to the lymphocytes incubated at 37°C. The assay appears to be specific for the antigen to which the individual demonstrates DCH. This assay may provide a new in vitro method for investigating mechanisms of cell-mediated immunity and a rapid diagnostic test for sensitization to microbial antigens.
Structural Differences between Parent and Mutant H-2K Glycoproteins from Two H-2K Gene Mutants: B6.C-H-2ba (Hzl) and B6-H-2bd (M505)
Authors: Brown J. Lynne , Nathenson Stanley G.
Published: December 2022

The H-2K glycoproteins from two mouse mutants B6.C-H-2ba (Hzl) and B6-H-2bd (M505) were isolated, and by tryptic peptide mapping techniques, were compared with each other and with the H-2K glycoprotein from the parent strain C57BL/6 (H-2b). The elution profile of the acid-soluble tryptic peptides from the H-2Kba glycoprotein showed that at least two to three arginine-labeled peptides and one lysine-labeled peptide were different from the H-2Kb product of the parent. These peptides were distinct from the soluble peptides which constitute the difference which was found between the H-2K products of H-2bd and the H-2b parent strains. The comparative peptide maps thus demonstrate small but significant primary structural differences among the H-2Kba, H-2Kbd, and H-2Kb glycoproteins. The findings are in agreement with the suggestion that alterations in the H-2K products from these two mutants are sufficient to permit their cells to stimulate each other and the parent cells in the mixed lymphocyte reaction (MLR) and to serve as target cells for the cell-mediated lymphocytotoxicity (CML) test. Because there is a significant reactivity in MLR and CML between H-2Kb, H-2Kba and H-2Kbd cells whereas there is no serologic reactivity, our findings are consistent with the notion there is a separation of the site(s) on the H-2 molecule having serologic reactivity and the site(s) involved in the MLR and CML reactivities.
Biologic and Chemical Characterization of HLA Antigens in Human Serum
Authors: Allison J. P. , Pellegrino M. A. , Ferrone S. , Callahan G. N. , Reisfeld R. A.
Published: December 2022

HLA antigens of both the A and B loci were shown to be associated with the high density lipoprotein fraction of serum prepared by ultracentrifugal flotation. HLA-A9 antigens were purified 100-fold with essentially complete recovery by a simple procedure of high density lipoprotein preparation involving precipitation with polyanions and ultracentrifugal flotation. The purified lipid-associated antigen was immunogenic since it elicited the formation of cytotoxic xenoantibodies in rabbits. Serum HLA-A9 antigens were found by immunoprecipitation and gel electrophoresis to consist of a 45,000 m.w. heavy chain associated with β2-microglobulin. The size of the HLA-lipid complex (<190,000 m.w.) and of the HLA-deoxycholate complex (<102,000 m.w.) suggests that HLA antigens are shed into plasma as a complex of a single HLA molecule and a single β2-microglobulin chain, associated with boundary lipid.
Receptors for IgM-Coated Erythrocytes on Chronic Lymphatic Leukemia Cells
Authors: Pichler Werner J. , Knapp Walter
Published: December 2022

Our experiments show that lymphocytes of CLL patients, having typical B cell characteristics, form rosettes with IgM-coated bovine erythrocytes. Of 18 investigated patients, 3 to 78% (mean 29%) of the isolated lymphocytes reacted with EA-IgM. With mixed rosette assays, EA-IgM bound to cells bearing receptors for IgG as well, but not to SRBC receptor-bearing lymphocytes.

Rosette formation could be completely blocked by addition of IgM at concentrations as low as 0.17 mg/ml. Ten milligrams per milliliter of aggregated human IgG had no effect on the rosette formation with EA-IgM but completely abolished the binding of EA-IgG. Adult human or rabbit serum blocked the EA-IgM binding, whereas cord blood serum and FCS had no effect.

These inhibition data indicate that EA-IgM binding does not occur via a somewhat altered IgG-Fc receptor but reacts with different membrane structures. That EA-IgM receptor can be cleaved off with trypsin and can be reconstituted after overnight cultivation, also supports this viewpoint.

In contrast to the situation in normal subjects, in CLL patients the IgM receptors are demonstrable before overnight cultivation and are found on cells with B cell characteristics.
Properties of tRNA-Specific Antibodies from NZB/NZW Mice
Authors: Eilat Dan , DI Natale Paola , Steinberg Alfred D. , Schechter Alan N.
Published: December 2022

Antibodies that bind tRNA are produced spontaneously in New Zealand Black/New Zealand White (NZB/NZW) F1 hybrid female mice. An assay for the detection of these antibodies has been developed by using gel filtration and radioactive tRNA. This assay was found superior to the widely used ammonium sulfate precipitation assay because of the nature of the interaction between the protein and the tRNA. The antibodies bound native tRNA preferentially to tRNA denatured by cross-linking with formaldehyde. This conformational specificity was confirmed in competition experiments. The antibodies to native tRNA had an average association constant of 5 × 107 liter/mole at 4°C and could bind to more than one site per tRNA molecule. Experiments with immunoglobulin class-specific anti-mouse antisera, in solution and by radioimmunoelectrophoresis, showed that the antibodies were heterogeneous, but were predominantly of the IgG class.

These antibodies may be useful for detection, localization, and conformational analysis of tRNA in solution as well as for understanding the pathogenesis of the lupus-like syndrome in these mice.
Structure and Biologic Functions of Human IgD
Authors: Ruddick John H. , Leslie Gerrie A.
Published: December 2022

Chicken anti-human IgD antiserum (anti-δ) has demonstrated an antigenically cross-reactive homologue on rat lymphocytes. IgD and IgM are the only cell surface immunoglobulins detectable by the lactoperoxidase radiolabeling technique employed. The results indicate that, although rat surface IgD is antigenically distinct from rat IgM, the respective H chains co-electrophorese in 10% polyacrylamide-SDS gels. Rat δ-chain has an apparent m.w. of 73,000 daltons and exhibits a minor 65,000 dalton component which probably represents a partially degraded δ-chain. The ontogenic emergence of rat IgD occurs ∼3.5 weeks after birth whereas IgM, in contrast, is apparent by 6 days of age. Thus, as in the human, IgM develops before IgD. IgD receptors are undetectable in the thymus but are present in increasing levels in spleen, blood, lymph nodes, and Peyer's patches.
Immunogenicity of Human B Cell Antigens Solubilized from Cultured Human Lymphoid Cells
Authors: Ferrone S. , Pellegrino M. A. , Reisfeld R. A.
Published: December 2022

Antigens solubilized from cultured human lymphoid cells WI-L2 and RPMI 1788 were partially purified by ultracentrifugation on a KBr gradient. These antigens injected into rabbits produced xenoantibodies which after absorption with melanoma cells became specific to B cell antigens. Three such xenoantisera were submitted to the Second Histocompatibility Workshop of the Americas and reacted much like alloantisera to B cell antigens against a large panel of B peripheral lymphocytes and cells from patients with chronic lymphocytic leukemia. Xenoantisera to B cell antigens inhibited the mixed lymphocyte reaction, but did not affect the mitogenic activity of phytohemagglutinin or the functional properties of C3 receptors, monkey red blood cell receptors, or T cell receptors.
Quantitative Studies on the Precursors of Cytotoxic Lymphocytes
Authors: Teh H.-S. , Harley E. , Phillips R. A. , Miller R. G.
Published: December 2022

A microculture system for estimating the frequency of cytotoxic lymphocyte precursors (CLP) to alloantigens is described. Cytotoxic T lymphocytes (CL) were generated by culturing limiting numbers of RNC (H-2k)-nu/+ lymph node (LN) cells with irradiated C3D2F1 (H-2k/d) spleen cells in the presence of RNC-nu/nu spleen cells for 7 days in microtiter trays. At the end of the culture period, individual wells were assayed for cytotoxic effector cells directed against 51Cr-P815 (H-2d) target cells. The effector cells generated under these conditions are sensitive to killing by rabbit anti-mouse brain serum and guinea pig complement. The process of differentiation from precursors to CL is radiation sensitive (Do ∼ 180 rads). The frequency of precursors for H-2d was calculated by fitting the proportion of nonresponding cultures to the zero order term of the Poisson distribution, Po = e-vN, where Po = per cent nonresponders, v = precursor frequency, and N = number of LN cells cultured per well. The frequency of precursors in the RNC-nu/+ LN population responding to the H-2d haplotype was found to be 1 in 776. Under conditions where no backstimulation is possible, the frequency of precursors responding to the H-2d haplotype is 1 in 885 for B6-nu/+ LN and 1 in 2550 for B6-nu/+ spleen, respectively. In combination with a visual assay for individual CL, it was found that the average clone size per precursor after 7 days in culture is about 1040. Our assay could detect clones with as few as 40 CL/well. Since the average clone size in 26 times the detection limit, we conclude that the assay for CLP is highly sensitive and does not represent a minimum estimate.
The Amino Acid Sequence of Duck Amyloid A (AA) Protein
Authors: Gorevic Peter D. , Greenwald Mark , Frangione Blas , Pras Mordechai , Franklin Edward C.
Published: December 2022

AA protein constitutes 50 to 60% of the amyloid fibrils from livers of ducks developing spontaneous amyloidosis and has a m.w. of about 12,000. The sequence of the first 73 residues was established by automatic Edman degradation of the whole molecule and isolated cyanogen bromide fragments, and corroborated by placement of tryptic peptides. The sequence from position 76–80 was tentatively derived from a peptide that was homologous to that region of human and monkey AA proteins. Carboxypeptidase digestion showed the carboxy terminal sequence to be Ala, Arg, with some heterogeneity (Ser. Arg). Compared to human AA protein, there were five additional residues at the amino terminus. In view of the m.w. and the existence of additional peptides an extension at the C-terminal end seems likely. Sequence homologies to other AA proteins are discussed.
Effect of Levamisole on the Human Monocyte IgG Receptor
Authors: Schreiber Alan D. , Parsons Janey N.
Published: December 2022

Levamisole, an isomer of tetrahydro phenylimidazo thiazole, is a synthetic anti-helminthic which may have immunostimulating properties both in vivo and in vitro (1). Its mode of action is incompletely understood. There is evidence in murine systems that levamisole can increase the phagocytosis of heterologous cells by macrophages in vitro (2) and accelerate the clearance of carbon particles (3). Additionally, levamisole may stimulate delayed hypersensitivity in man (4, 5). These observations suggest that the macrophage may be a major site of levamisole's action. Macrophages have specific surface receptors which serve to bind IgG- or C3-coated cells. We theorized that levamisole might mediate its effect in part by acting on these cell surface receptors. In order to assess the effect of levamisole on human macrophage IgG receptor function, we employed a quantitative in vitro model, with human erythrocytes, human IgG anti-erythrocyte (anti-D) antibody, and monolayers of human peripheral blood mononuclear phagocytic cells.
Solubilization and Partial Characterization of Cell Membrane Fc Receptors
Authors: Anderson Clark L. , Grey Howard M.
Published: December 2022

We assayed detergent cell lysates and culture supernatant fluid of mouse cells for the presence of soluble FcR by means of a radioligand bioassay. Strict correlation was found between the amount of FcR present in soluble form and that found on intact cells. The soluble FcR of all cells tested, except mouse macrophages, behaved as lipoprotein by buoyant density centrifugation analysis and enzyme treatment. In the absence of detergent, soluble FcR sedimented as a 20S macromolecule; in detergent the material was 7 to 9S. It was readily bound by insolubilized IgG but not by insolubilized BSA or F(ab')2 fragments of IgG.
Identification of the Schwann Cell as a Peripheral Nervous System Cell Possessing a Differentiation Antigen Expressed by a Human Lung Tumor
Authors: Bell C. Elliott , Seetharam Shakuntla
Published: December 2022

Recent studies of the plasma membrane antigens of a human lung tumor (oat cell carcinoma) indicated that the tumor expressed at least two normal differentiation antigens undetectable in normal respiratory epithelium. One antigen was characteristic of certain endodermally derived epithelial cells of the digestive system; the other antigen was characteristic of certain neural crest-derived cells in the peripheral nervous system. The present studies were undertaken to identify the reactive cell type in the peripheral nervous system.

Since similar cells in the rat peripheral nervous system expressed a cross-reactive form of this antigen, and since pure cultures of different rat nerve cell types were available, the following approach was possible. Cultures of pure neurons, pure Schwann cells, pure fibroblasts, neurons and Schwann cells, and neurons, Schwann cells, and fibroblasts were assayed for this antigen with rabbit anti-oat cell carcinoma plasma membrane antiserum absorbed with normal lung and liver. The indirect immunofluorescence method on both whole, viable cell and fixed cell substrates was used. Only Schwann cells expressed the antigen; Schwann cells in the presence of neurons expressed the antigen much more strongly than did pure Schwann cells. It was concluded that the oat cell carcinoma of the lung expressed a differentiation antigen present on Schwann cells.
Quantitative Measurement of Mouse IgG Subclasses with the Use of Heteroantisera: The Importance of Allotype Considerations
Authors: Epstein Suzanne L. , Gottlieb Paul D.
Published: December 2022

Double antibody radioimmunoassays have been used to determine the quantities of IgG1, IgG2a and IgG2b in samples of normal serum IgG from BALB/cJ, AKR/J and C57BL/6J inbred mice. The assays employed subclass-specific goat antisera which had been prepared with BALB/c myeloma proteins as immunogens and as immunoabsorbents. 125I-labeled BALB/c myeloma proteins were used as probes.

Results indicate that partial resolution of mouse IgG subclasses was achieved by ion exchange chromatography on DEAE-Sephadex. Nearly all of the protein in BALB/cJ and AKR/J IgG fractions could be accounted for as IgG1, IgG2a and IgG2b, and IgG2a was the predominant species observed. However, considerably less protein in C57BL/6J IgG fractions of purity similar to the BALB/cJ fractions could be accounted for as these three subclasses, and virtually no IgG2a was detected. Furthermore, an IgG2a myeloma protein bearing the C57BL/6 allotype failed to inhibit the IgG2a-specific assay significantly. Thus the IgG2a-specific antibody in the goat heteroantiserum employed appeared to consist nearly exclusively of antibody to BALB/c Ig-1a allotypic determinants. These findings point to the importance of allotype considerations in the use of heteroantisera to quantitate IgG subclasses.
Induction of Cell-Mediated Immunity to Chemically Modified Antigens in Guinea Pigs
Authors: Dailey Morris O. , Hunter Robert L.
Published: December 2022

Bovine serum albumin (BSA) conjugated with a lipid, dodecanoic acid, is capable of inducing strong delayed-type hypersensitivity (DTH) in guinea pigs. This paper reports experiments on the nature and specificity of this hypersensitivity. The response to lipid-conjugated BSA (L-BSA) was found to be classical DTH, as evidenced by its ability to be transferred passively by immune cells, but not by serum. In addition, special histologic examinations of skin test sites demonstrated the characteristics of DTH rather than cutaneous basophil hypersensitivity. Similar results were obtained when lipid-conjugated purified protein derivative of tubercle bacilli (L-PPD) was used. The increased immunogenicity of L-BSA was not caused by the presence of protein aggregates, but seemed to be related to the hydrophobic nature of the conjugated side chains. A series of cross-reacting serum albumins was used for a study of the specificity of the antibody and DTH responses to BSA. It was found that the degree of enhancement of immunogenicity for DTH caused by lipid conjugation varied for different antigenic determinants on BSA.
In Vitro Induction of Tumor-Specific Immunity
Authors: Burton Robert C. , Chism Stanley E. , Warner Noel L.
Published: December 2022

Many recent studies have demonstrated that cytotoxic T lymphocytes (CL) activated to various antigens other than those of the H-2 complex, will lyse target cells only when H-2 compatibility exists between the CL and target cell. From these observations, it has been inferred that T lymphocytes might only be capable of responding to H-2 antigen or antigens that become associated with H-2 region gene products. Our results suggest that this is not the case, and that in some situations, cytotoxic T lymphocytes can specifically lyse target cells of different H-2 types. Two in vitro systems are described where primary induction of cytotoxic T lymphocytes to oncofetal and plasmacytoma antigens results in CL capable of lysing suitable targets bearing these antigens, of either syngeneic or allogeneic derivation. Thus it is proposed that although interaction antigens involving H-2 components may preferentially activate T lymphocytes, this does not imply a restriction on the recognition potential of T lymphocytes.
Human Bone Marrow Lymphocytes
Authors: Fauci Anthony S. , Pratt Karen R.
Published: December 2022

Lymphocytes from the bone marrow and peripheral blood of the same normal individuals were assayed simultaneously for blast transformation as well as polyclonal activation with differentiation to antibody-forming cells after stimulation with pokeweed mitogen. Blastogenic responses were measured by tritiated thymidine incorporation and antibody-forming cells were enumerated by a newly described hemolysis-in-gel plaque-forming cell assay. There was no significant difference between the blastogenic responses of lymphocytes in the peripheral blood compared to the bone marrow of the same individuals. However, differentiation to antibody-forming cells measured by the plaque-forming cell response was significantly greater in lymphocytes in the bone marrow as compared to peripheral blood of the same individuals. These studies demonstrate that the lymphocytes in human bone marrow are at a stage of differentiation whereby they can be readily induced to differentiation toward antibody production by polyclonal activation, even more so than peripheral blood lymphocytes. This supports the concept that the bone marrow is a major source of immunoglobulin production in man.
Generation of Cytotoxic T Lymphocytes during Coxsackievirus B-3 Infection
Authors: Wong Catherine Y. , Woodruff Judith J. , Woodruff Jack F.
Published: December 2022

This report describes studies characterizing the virus-specific cytotoxic effector cells which are present in the spleens of mice 7 days after infection with Coxsackievirus B-3. An in vitro 51Cr assay employing syngeneic virus-infected neonatal fibroblasts was used to measure cytotoxic activity. Treatment of immune cells with (anti-thy-1.2) and complement abolished their cytotoxic activity, but no reduction occurred when B cells were removed by incubation with anti-Ig and complement or macrophages eliminated by adherence depletion. The findings therefore imply that the cytotoxic reaction was mediated by sensitized T cells and that B cells and macrophages did not play an important role. Reciprocal assays performed with BALB/c and CBA/J cells showed that Coxsackievirus-immune spleen cells lysed infected syngeneic targets but not allogeneic targets, providing further evidence that cytotoxicity was mediated by effector T cells. In addition an in vitro assay system employing neonatal myocardial cells was developed and used to demonstrate that Coxsackievirus-infected myofibers were susceptible to destruction by immune spleen cells. The evidence suggests that mice infected with Coxsackie B viruses are able to mount a cell-mediated immune response with production of cytotoxic T cells which have the capacity to damage tissues infected with these agents.
Effects of Carrageenan on Immune Responses
Authors: Ishizaka Shigeaki , Otani Shuzo , Morisawa Seiji
Published: December 2022

Carrageenan, a sulfated polygalactose having macrophage toxic properties, elicited a marked suppression of IgM response to T cell-dependent antigens such as sheep red blood cells (SRBC), dinitrophenylated bovine serum γ-globulin (DNP-BGG), and trinitrophenylated concanavalin A (TNP-Con A). In contrast, carrageenan did not inhibit antibody responses to such T cell-independent antigens as trinitrophenylated DEAE-dextran (TNP-DEAE-dextran), trinitrophenylated polyvinyl pyrrolidone(TNP-PVP), and trinitrophenylated Ficoll (TNP-Ficoll). Compared to total spleen cells, spleen cells from which macrophages had been removed by adhesion to plastic Petri dishes had less effect on the production of antibody against T cell-dependent antigens, but no change or a rather stimulated effect was observed in in vitro antibody synthesis against T cell-independent antigens. These results strongly suggest that macrophages are involved in antibody responses to T cell-dependent antigens but not in those to T cell-independent antigens. However, the antibody response to trinitrophenylated lipopolysaccharide (TNP-LPS), a T cell-independent antigen, was inhibited by carrageenan treatment, suggesting that the response is macrophage dependent. Moreover, antibody response to higher doses of dinitrophenylated phytohemagglutinin (DNP-PHA), a T cell-dependent antigen, was shown to be macrophage independent by carrageenan treatment, although the antibody response to low doses of the antigen was macrophage dependent. Considering all these results, carrageenan treatment seems to be a very useful method to determine whether immune response to various antigens are macrophage dependent or not.
Stimulation of Mouse Migration Inhibitory Factor (MIF) Production from MSV-Immune Lymphocytes by Soluble Tumor-Associated Antigen: Requirement for Histocompatible Macrophages
Authors: Landolfo Santo , Herberman Ronald B. , Holden Howard T.
Published: December 2022

Spleen cells from C57BL/6 mice immunized with murine sarcoma virus (MSV) are capable of producing migration inhibition factor (MIF) in response to stimulation with a specific tumor-associated antigen prepared by solubilization with 3 M KCl. We have previously demonstrated that this response is T cell dependent. Further investigations into the effector cells involved in the production of MIF have revealed that spleen cells from mice immunized with MSV cannot produce MIF when stimulated with tumor extract if the population has been previously depleted of macrophages. However, the response can be restored by adding nonimmune syngeneic macrophages but not by allogeneic macrophages. The inability of allogeneic macrophages to provide this function was not due to their increased suppressor activity since in mixing experiments they did not interfere with the ability of immune spleen cells to produce MIF. Furthermore, they were not defective since they could supply this “cooperative function” to appropriate F1 mice. The results indicate that macrophages are required for stimulation of MIF by soluble tumor antigens and that for efficient interaction the macrophages and lymphocytes must share some genetic similarities.
Immunochemistry and Polypeptide Composition of Hepatitis B Core Antigen (HBc Ag)
Authors: Budkowska Agata , Shih J. Wai-Kuo , Gerin John L.
Published: December 2022

Core particles were isolated from a nuclear extract of a hepatitis B-infected liver, labeled with 125I by using chloramine-T and further purified by rate zonal sedimentation on sucrose gradients. Iodinated HBc Ag was used as a ligand in a sensitive double-antibody radioimmunoprecipitation (RIP) assay for antibody to HBc Ag. The specificity of the RIP reaction was evaluated using defined anti-HBc sera and paired sera from six well-documented cases of hepatitis B infection. The polypeptide composition of the iodinated antigen was examined by SDS-polyacrylamide gel electrophoresis of solubilized complexes of 125I-HBc Ag and anti-HBc. Two major polypeptides with apparent m.w. of 17,000 and 35,000 daltons were observed and designated as cP-1 and cP-2, respectively.
Antigen-Binding Lymphocytes in Guinea Pigs
Authors: Prange Charles A. , Green Christopher , Nitecki Danute E. , Bellone Clifford J.
Published: December 2022

The monofunctional antigen L-tyrosine-p-azophenyltrimethylammonium chloride, tyr(TMA), and the polyfunctional antigen, TMA-human γ-globulin (TMA-HGG), were used to investigate the antigen structural requirements necessary for clonal proliferation of B cells. This clonal expansion was characterized with respect to receptor immunoglobulin class and affinity maturation. Antigen-binding analysis revealed that inoculation of tyr(TMA), although only of m.w. 344, triggers clonal expansion of B lymphocytes 9-fold in the absence of any apparent antibody production. There does not appear to be any maturation with respect to antibody class since τ;90% of the tyr(TMA)-specific B cells bear the μ receptor in the nonimmune and immune state. However, the average avidity of the B cells for this antigen increases with time after immunization. In contrast, immunization with TMA-HGG results in an 18-fold increase in B lymphocytes with significant amounts of anti-TMA antibody production. With time after immunization, both maturation of average avidity and class of Ig receptor (μ → γ shift) occur. These findings indicate that the functionally T cell-specific antigen tyr(TMA) can trigger clonal B cell expansion and affinity maturation at the receptor level in the absence of detectable antibody production.
Use of Bovine Conglutinin for the Assay of Immune Complexes
Authors: Eisenberg Robert A. , Theofilopoulos Argyrios N. , Dixon Frank J.
Published: December 2022

We have used purified bovine conglutinin to develop a solid state radioimmune assay for immune complexes. Employing aggregated human IgG incubated with fresh normal human serum (complement) as a model for immune complexes, we have shown that our conglutinin assay is sensitive and highly specific for immune complexes that have fixed complement. The assay preferentially detects large complexes, is minimally influenced by monomeric IgG, and can be inhibited by high ionic strength, calcium chelation, and acetamido sugars. In addition, we have surveyed several hundred clinical sera from patients with various immunopathologic disorders and find evidence of immune complexes in some.
Morphology of Delayed-Type Hypersensitivity Reactions in the Guinea Pig Cornea
Authors: Friedlaender Mitchell H. , Dvorak Harold F.
Published: December 2022

Guinea pigs sensitized with several soluble protein antigens in incomplete Freund's adjuvant (IFA) and tested at 5 to 7 days developed basophil-rich corneal reactions characterized by minimal edema and fibrin deposition. These lesions resembled the skin reactions of cutaneous basophil hypersensitivity (CBH) in their immunization requirements, time course, and character, but differed in that basophils were relatively less frequent and eosinophils and neutrophils more numerous in ocular than in cutaneous reactions developing simultaneously in the same animal.

In contrast, animals immunized with antigens such as ovalbumin in complete Freund's adjuvant (CFA) developed much more severe delayed-type ocular reactions whether tested at early or late intervals after sensitization. These intensely injected, chemotic reactions exhibited an extensive cellular infiltrate of mononuclear cells as well as marked corneal edema accompanied by extensive fibrin deposition. Hence, these ocular reactions were similar to the classic indurated tuberculin-type delayed hypersensitivity (DH) skin reactions except that neutrophils were more numerous.

These data indicate that in the eye as in the skin two distinct patterns of delayed-onset, cell-mediated hypersensitivity may occur to the same antigen, depending on the mode of immunization and the time of testing. However, significant differences were found in the frequency with which the various granulocytes infiltrated reactions elicited simultaneously in the eye and skin. Therefore, the character of immune expression may vary in different parts of the body and local factors, such as the local microvasculature, evidently contribute to these differences.
Inflammatory Cells in Solid Murine Neoplasms
Authors: Russell Stephen W. , Gillespie G. Yancey , McIntosh Ann Tozier
Published: December 2022

Macrophages (Mø) were recovered from disaggregated, spontaneously regressing Moloney sarcomas for in vitro testing of their cytotoxic capabilities. At a 3:1 ratio, Mø in monolayers efficiently killed (51Cr release) a variety of tumor target cells without regard to antigenic specificity. Within 24 hr most of their cytolytic potential was lost. Killing could be restored, however, if Mø were replated at higher cell densities. A soluble, heat-stable, dialyzable inhibitor of thymidine incorporation, which did not interfere with cellular proliferation, also was produced by Mø recovered from Moloney sarcomas. The finding of such a competitive inhibitor, probably cold thymidine, in supernatants further opened to question the validity of radioisotope incorporation into DNA as a measure of Mø-mediated cytostasis. The data presented suggest that Mø may participate in the regression process by directly killing tumor cells, thus complementing the antigen-specific cytolytic capabilities of the T lymphocytes that are also found in large numbers within regressing Moloney sarcomas.
Identification and Genetic Control of the n83 and n84 Allotypes of Rabbit IgM
Authors: Gilman-Sachs Alice , Dray Sheldon
Published: December 2022

Two additional allotypes of rabbit IgM, n83 and n84, have been identified and characterized with antisera obtained by cross immunization of rabbits with IgM. These allotypic specificities were not detected on IgG or IgA by double diffusion in agar gel and by quantitative radioprecipitin analyses. This finding implies that the specificities reside in the CH region of the µ-chain. Most of the IgM (88%) from an n83 homozygote reacted with anti-n83 Ab and most of the IgM (79%) from the n84 homozygote reacted with anti-n84 Ab. The IgG or IgA from n83 or n84 rabbits did not precipitate to a significant extent (< 6%) with the anti-n83 or anti-n84 Ab, respectively. That the n83 and n84 specificities are controlled at the n locus was verified by genetic analysis. Thus, four alleles are now known at the n locus, n81, n82, n83 and n84. The n locus is closely linked to the other six defined loci in the heavy chain chromosomal region and the allelic alternatives for each of the seven loci are coinherited by gene combinations called allogroups. Ten such allogroups are now defined with respect to the allele at the n locus.