bluuebunny/crossref_metadata_2025 huggingface dataset:
An Improved Primary Response from Mouse Spleen Cells Cultured in Vivo in Diffusion Chambers
Authors: Goodman S. A. , Chen M. G. , Makinodan T.
Published: December 2022

The optimum conditions for the hemolytic plaque-forming cell response of mouse spleen cells to sheep red blood cells (SRBC) in Millipore diffusion chambers were determined because of the wide applicability of the technique as an in vivo but closed system for the study of the immune response. The effect of pretreatment of chamber recipients with x-rays or cyclophosphamide, as well as the effects of chamber volume, culture medium, chamber detoxification and sterilization techniques, were investigated. The improvement in response over that previously reported by this laboratory was attributed to larger chamber volume, chamber detoxification twice over a 24-hr period, culture medium supplemented with horse serum, and lethally irradiated or cyclophosphamide-treated recipient C31F1 mice. With the improved conditions the peak primary response of mouse spleen cells to SRBC (104 direct plaque-forming cells per 106 nucleated cells harvested) was almost 20-fold greater than that obtained in the cell transfer system and about 5-fold greater than the best response obtained both in vitro and in situ. The burst size was also significantly larger than that obtained in the other systems, and culturing efficiency was twofold greater than previously reported from this laboratory for the diffusion chamber technique. The improved technique was used to determine the response of mouse spleen cells to SRBC in both allogeneic (across H-2 and non-H-2 barriers) and xenogeneic hosts. The enhanced response obtained by the present method is attributed to increased culturing efficiency and burst size, but not increased efficiency of interaction of the cells initiating the response.
Tissue Localization of Two Populations of Human Lymphocytes Distinguished by Membrane Receptors
Authors: Silveira Nelson P. A. , Mendes Nelson F. , Tolnai Marina E. A.
Published: December 2022

Lymphocyte suspensions from man and primates show a high proportion of cells adhering to sheep erythrocytes (E) forming rosettes (1–3). The binding of E to lymphocytes is dependent on temperature and is probably not immunologic in nature because 20 to 40% of normal human peripheral blood lymphocytes form rosettes when incubated with E (1). It appears as if the lymphocytes which have receptors for E constitute a subpopulation of lymphoid cells which are thymus dependent or derived (T) since almost 100% of human thymocytes form this type of rosette (1).

A number of reports validate the use of certain membrane markers for the characterization of lymphocyte populations. Distinct markers for “T” and bone marrow-derived (“B”) type cells have been described on the membrane of mouse lymphocytes: the theta (θ) alloantigen (4–6), the receptors for a modified complement component (C3) (7–9) and the membrane-bound immunoglobulins (8, 10, 11).
Measurement of Mycoplasma Pneumoniae Mycoplasmacidal Antibody in Human Serum
Authors: Brunner Helmut , James Walter D. , Horswood Robert L. , Chanock Robert M.
Published: December 2022

Investigation of the conditions which affect the mycoplasmacidal action of antibody and complement upon Mycoplasma pneumoniae indicated the importance of a filtered suspension of predominantly single organisms, and an appropriate concentration of cations. The mycoplasmacidal effect of antibody and complement resembled the hemolytic reaction in that variation in concentration of either antibody or complement yielded an S-shaped curve. The antigens involved in the mycoplasmacidal reaction appeared to be the glycolipids of the organism which had previously been shown to be of major importance in the complement fixation and metabolism inhibition reactions. The standardized mycoplasmacidal antibody assay proved to be considerably more sensitive than the conventional assay systems for measurement of M. pneumoniae antibody in human serum.
The Production of Anti-Dnp Antibody in the Bullfrog, Rana Catesbiana
Authors: Rosenquist Grace L. , Hoffman Ronald Z.
Published: December 2022

Bullfrogs injected with dinitrophenyl keyhole limpet hemocyanin (DNP-KLH) in complete Freund's adjuvant produced precipitating anti-DNP antibody with characteristics different from that produced by frogs immunized against DNP-Salmonella in saline.

The antibody from animals injected with DNP-KLH sedimented as a 7S protein on sucrose density gradients and gave precipitin curves similar to the precipitin curve of mammalian 7S-IgG except for the lack of 100% precipitability of the antigen over most of the frog curve.

DNP-Salmonella induced antibody which sedimented as a macroglobulin and was distinguished from the light antibody induced by DNP-KLH by radioimmunoelectrophoresis. The precipitin curve for this immunoglobulin and the persistence of high titers 1 year after a single injection suggest that the immunoglobulin is IgM.
Studies of Non-Migrating, Long-Lived Lymphocytes in Rats
Authors: Miller John J. , Shortman Ken , Byrt Pauline
Published: December 2022

Rat lymph node lymphocytes were labeled by hind footpad injections of tritiated thymidine in the absence of an immune stimulus or after priming with a polymerized Salmonella flagellin. Fifty or more days later the rats were killed and the lymph node cells were fractionated on continuous albumin density gradients. The proportions of lymphocytes of lighter density (< 1.07 g/cm3) were greater in primed than in unprimed nodes. The density distributions of the “long-lived” cells, i.e., those with persistent label, were identical to those of the total lymph node cell population. After challenge with either antigenically identical or different flagellins, the density distribution of the persistently labeled cells demonstrated relative loss of cells in the density range from 1.068 to 1.072 g/cm3 in comparison to the bulk of lymph node cells, but not in patterns distinct for the specificity of the antigens used for challenge. Comparison of the density distributions of long-lived lymphocytes with flagellinbinding cells and with cells transferring adoptive immune reactions revealed partial overlap with these cells from primed but not from unprimed lymph nodes.
Phylogeny of Immunoglobulin Structure and Function
Authors: Leslie Gerrie A. , Clem L. William
Published: December 2022

Three different sizes of immunoglobulins containing anti-dinitrophenyl activity were isolated from turtles immunized with dinitrophenyl bovine γ globulin. The sedimentation coefficients of the three immunoglobulins were ∼17S, 7.5S and 5.7S and their molecular weights were 850,000, 180,000 and 120,000, respectively. The 17S immunoglobulin was similar to IgM and was composed of five subunits each with a molecular weight of ∼170,000. The heavy and light chain molecular weights were 70,000 and 22,500, respectively. The 7.5S molecule had H chains of ∼67,500 and L chains. The 5.7S molecule had H chains of ∼35,000 and L chains. Partial reduction of the 5.7S molecule resulted in the formation of half molecules of ∼61,000. Antigenic analyses suggested that the 5.7S molecule may be a fragment of the 7.5S molecule. Neither the 7.5S H chains nor the 5.7S H chains cross-reacted with the 17S H chains. These findings are discussed in relationship with the phylogeny in immunoglobulin structure and function.
Inhibition by Anti-µ Chain Sera of the Cellular Transfer of Antibody and Immunoglobulin Synthesis in Mice
Authors: Herrod Henry G. , Warner Noel L.
Published: December 2022

Unprimed mouse spleen cells were incubated in vitro with various class specific rabbit anti-mouse immunoglobulin sera. Cells were washed and transferred into syngeneic or allotype congenic irradiated recipients. Syngeneic recipients were stimulated at the time of cell transfer with sheep erythrocytes (SRBC) and Brucella abortus. At 7 days their IgM response to SRBC and B. abortus was significantly depressed following pretreatment with anti-µ chain sera, but not with anti-γ chain sera. Congenic recipients were serially bled and levels of donor type IgG immunoglobulins were determined by allotype specific inhibition of precipitation assays. Marked suppression of the transfer of donor type IgG of all classes was obtained by pretreatment with anti-µ chain sera.
Immunogenicity of Soluble HL-A Alloantigens
Authors: Ferrone S. , Natali P. G. , Hunter A. , Terasaki P. I. , Reisfeld R. A.
Published: December 2022

In recent years extensive investigations have been directed toward determining the biologic and chemical profiles of soluble HL-A alloantigens isolated from a variety of tissue sources. During the course of such studies, the in vitro reactivity of these materials was established serologically by their ability to inhibit specifically cytotoxic alloantisera (1). The soluble HL-A antigens have been shown to have further immunologic significance as they can induce specifically blastic transformation of histoincompatible but not of isogeneic leukocytes in vitro (2). Chemical studies thus far suggest that HL-A antigens are essentially protein in nature and apparently consist of single polypeptide chains with a molecular weight of 31,000 (3). The data concerning the immunogenic properties of soluble HL-A alloantigens thus far are scanty and conflicting. Batchelor (4) reported that rabbits immunized with partially purified soluble HL-A substances produced only species-specific lymphocytotoxic antibodies.
The Pathogenesis of Aleutian Disease of Mink
Authors: Porter David D. , Larsen Austin E. , Porter Helen G.
Published: December 2022

Administration of an inactivated Aleutian disease virus vaccine to mink induced detectable antibody in one of 26 animals. When immunized mink were challenged with live virus, they developed more severe lesions than mink given a control vaccine. The immunized mink were also more susceptible to oral virus challenge than control mink. Passive administration of antiviral antibody at the peak of virus replication produced necrotizing acute inflammatory lesions, followed by mononuclear cell infiltrates.
Attempts to Reconstitute Immunoglobulin G from Heavy Chain and Halves of Light Chain
Authors: Karlsson F. A.
Published: December 2022

The noncovalent recombination of normal human immunoglobulin light chains and their halves with normal heavy chains and of a monoclonal light chain and its constant and variable halves with the autologous heavy chain has been studied. When the recombination mixtures were analyzed at an ionic strength close to the physiologic, intact light chains recombined readily with heavy chains, whereas light chain halves showed no tendency to recombine under these conditions. At higher ionic strength some binding of constant and variable fragments to heavy chains was observed. The results indicate that in an immunoglobulin molecule both the constant and the variable half of the light chain participate in the noncovalent bonding to heavy chain and demonstrate that a covalent bond between the halves is of great importance for recombination.
Functional Development of the Interacting Cells in the Immune Response
Authors: Fidler John M. , Chiscon Martha O. , Golub Edward S.
Published: December 2022

Young CBA spleen cells respond initially to sheep red blood cells (SRBC) in vitro at 15 days of age but the response is less than 10% of the adult response. The magnitude of the response reaches levels comparable to those in the adult at 7 weeks of age. Spleen cells of 1-week-old mice can reconstitute the anti-SRBC response of lethally x-irradiated adult mice but cannot respond to SRBC when placed in diffusion chambers implanted in the peritoneal cavity of sub-lethally irradiated adult syngeneic recipients. In an attempt to determine the cellular basis of the “immunologic immaturity,” reciprocal recombinations of adult and young macrophage-rich adherent (A) cells and lymphocyte-rich non-adherent (NA) cells were made. It was found that young A cells are functional, but young NA cells are not. The implications of these findings in terms of the mechanism of maturation and the acquisition of immunocompetence by the young mouse are discussed. It is suggested that the cause of incompetence in the young spleen cells is the presence of pluripotent stem cells but not of committed lymphoid precursors. This may be due to the lack of functional lymphocyte-directed-inducing-microenvironments which would induce stem cells to differentiate into committed lymphoid precursors.
Activity of Migration Inhibitory Factor in the Absence of Antigen
Authors: Yoshida Takeshi , Janeway Charles A. , Paul William E.
Published: December 2022

The possibility that the activity of migration inhibitory factor (MIF) requires the presence of antigen in the culture of nonimmune peritoneal exudate cells has been suggested by several authors. In order to study such antigen-dependent MIF, we stimulated lymph node cell cultures from guinea pigs immunized with various DNP-protein conjugates with the immunizing antigen in vitro. The antigen was removed from MIF-containing supernatants by affinity chromatography on anti-DNP-agarose bead columns. The effluent material retained full MIF activity despite the absence of antigen. Addition of antigen did not increase the activity of these antigen-free supernatants. Moreover, no MIF activity could be subsequently eluted from the anti-DNP-agarose bead column by treatment with dilute acid, suggesting that no MIF, as antigen-MIF complex, had been removed by the anti-DNP column. In addition, DNP-protein-agarose bead conjugates stimulated lymphocytes to produce active MIF, although no antigen could be detected in these MIF-containing supernatants. Addition of antigen did not increase the activity of the supernatant. Thus, in these systems, no evidence for antigen-dependent MIF has been obtained.
Antineuraminidase Antibody Response of Man to Influenza Virus Neuraminidase N2: Results Obtained with an Improved Hemagglutination Inhibition Technique and an Enzyme Inhibition Test
Authors: Hennessy Albert V. , Minuse Elva , Davenport Fred M.
Published: December 2022

Sets of sera obtained from subjects vaccinated or infected with influenza A2 strains from 1957 to 1968 were examined by an improved hemagglutination inhibition procedure for measuring antineuraminidase antibodies with recombinant viruses, and by a neuraminidase inhibition test with human serum Cohn fraction IV-4 as substrate. Recombinants prepared with viruses containing 1957 and 1968 antigenic variants of neuraminidase were employed. The improved HI procedure was found to be as reliable as the NI test for detecting antineuraminidase antibody and antineuraminidase antibody increase. Antineuraminidase antibody was generally not found on first exposure to A2 strains but antibody increase occurred with increasing frequency during subsequent years. The antineuraminidase antibody response of man is broad, making recognition of antigenic enzymatic variants difficult by use of human serum.
Macrophage-Bound Antigens
Authors: Seeger Robert C. , Oppenheim Joost J.
Published: December 2022

The effectiveness of macrophage-bound antigens (macrophage-antigen) and soluble antigens in inducing delayed hypersensitivity (DH) and serum antibodies in syngeneic guinea pigs were compared. Peritoneal macrophages from unimmunized guinea pigs were incubated in vitro with 125-I tetanus toxoid (tet), dinitrophenylated guinea pig albumin (GPA.DNP) or dinitrophenylated ovalbumin (OA.DNP) for 60 to 90 min at 37°C. After this incubation period the cells were washed, and the quantity of 125I antigen which had been taken up determined. Then either macrophage-antigen or soluble antigen was injected into the footpads of syngeneic nonimmune guinea pigs.

A single injection of macrophage-antigen induced DH demonstrable 7 or 21 days after immunization, whereas equal or higher doses of soluble antigen did not. Immunization with macrophage-antigen followed by secondary and tertiary immunizations with macrophage-antigen induced maximal DH and only minimal antibody titers. Less DH and higher titers of antibodies developed when primary immunization with macrophage-antigen was followed by secondary and tertiary immunizations with soluble antigen. Macrophage-antigen was more effective than an equal dose of soluble antigen in priming for the production of antibodies. High doses of soluble antigen with or without secondary immunization induced antibodies as effectively as macrophage-antigen boosted with a secondary immunization of soluble antigen. Our studies suggest that macrophage-antigen interaction with thymus dependent lymphocytes (T cells) leads to their activation. Such activated T cells mediate DH and also may facilitate the production of antibodies by thymus independent cells (B cells).
In Vitro Demonstration of Two Antigenically-Distinct Rabbit Lymphocyte Populations
Authors: Fanger Michael W. , Pelley Ronald P. , Reese Alice L.
Published: December 2022

Two populations of lymphocytes have been demonstrated in the rabbit by using a goat antiserum directed to rabbit thymus cells. When incubated with rabbit lymphocytes from peripheral blood the IgG fraction of this antiserum (ATG) completely suppressed lymphocyte transformation initiated by phytohemagglutinin (PHA) or concanavalin A (Con A) but did not affect stimulation by goat anti-rabbit Fab. Neither preimmune IgG nor ATG absorbed with thymus or lymph node cells was capable of affecting transformation. The method of purification of ATG and studies involving order of addition, the effect on human lymphocytes and the effect of the F(ab′)2 and Fab′ fragments of ATG indicate that the primary mechanism of action of ATG is not to prevent the binding to the lymphocyte of Con A or PHA. ATG but not preimmune IgG appeared to be lymphocytotoxic under conditions similar to those used for transformation experiments. The most straightforward interpretation of these data is that ATG in vitro selectively eliminates the population of lymphocytes susceptible to stimulation by Con A and PHA but does not affect lymphocytes carrying immunoglobulin determinants. Thus thymus-derived cells have on their surface receptors for ATG, PHA and Con A while the other lymphocyte population expresses receptors for immunoglobulin.
Inhibition of Antigen-Stimulated in Vitro Proliferation of Thymic Dependent Chicken Spleen Cells by Specific Antibody
Authors: Jacobs R. P. , Blaese R. M. , Oppenheim J. J.
Published: December 2022

The ability of passively administered antibody to inhibit active immune responses suggests that antibody may be an important element in normal immunoregulation. This report describes studies of in vitro lymphocyte transformation in which cells from agammaglobulinemia bursectomized-irradiated (Bx-X) and rabbit anti-chicken lymphocyte serum (ALS)-treated chickens were used to study the effects of specific antibody on the in vitro proliferative responses of thymus dependent (T) and bursal dependent (B) lymphocytes. Intact chickens immunized with keyhole limpet hemocyanin (KLH) and Brucella abortus (BA) developed positive in vitro proliferative responses to these antigens and these responses were specifically inhibited when the appropriate antibody was added to the cultures. Low level “natural” or background proliferative responses were observed in unimmunized chickens in response to Brucella, and these responses could not be inhibited by specific anti-Brucella (anti-BA) antibody.

Agammaglobulinemic (Bx-X) chickens immunized with both KLH and Brucella responded in vitro only to KLH. This response was also inhibitable by specific anti-KLH antibody, indicating that proliferative responses of T cells can be suppressed by specific antibody. Because spleen cells from immunized Bx-X birds failed to respond to Brucella, the in vitro proliferative response to this antigen is probably a B lymphocyte dependent process. This was investigated further by using spleens from immune ALS-treated chickens. These cells failed to respond to either antigen in vitro. When these ALS spleen cells were mixed with immune Bx-X spleen cells, the in vitro proliferative response to BA was reconstituted, demonstrating synergy between the two populations of cells. However, since both T and B cells are needed for the proliferative response to BA, we cannot determine which lymphoid cell was inhibited by anti-BA.
Reaction Mechanisms of Nascent C5̄6̄7̄ (Reactive Lysis)
Authors: Goldman John N. , Ruddy Shaun , Austen K. Frank
Published: December 2022

An activated complex of the fifth and sixth components of complement (C) was generated by treatment of certain human sera with either zymosan or the low molecular weight anticomplementary factor of cobra venom. The interaction of C with C7 and unsensitized erythrocytes was studied. The formation of EC from these reactants was temperature dependent and proceeded most favorably at pH 6.0, ionicity 0.065. The half-life of the hemolytic activity of nascent C was 5 hr at 0°C and less than 2 min at 30°C. When the average numbers of C sites per erythrocyte generated in the presence of optimal C7 were plotted as a function of C input, linear responses were obtained, indicating that a single competent C site is sufficient to prepare the cell for lysis by C8 and C9. Nascent formed by the interaction of C and C7 operates in the absence of antibody and extends the membrane-damaging capacity of the complement system to unsensitized cells.
The Response to SRBC by Nude Mice Injected with Lymphoid Cells Other Than Thymus Cells
Authors: Kindred Berenice , Weiler Eberhardt
Published: December 2022

Nude mice from a stock partially backcrossed to BALB/c were able to produce hemagglutinating antibody if they were injected with BALB/c-Igb spleen, lymph node, peritoneal or educated T cells. C57 spleen cells and, in one experiment, BALB/c-Igb bone marrow cells induced only a transitory responsiveness. Usually bone marrow cells had no apparent effect on the ability to produce hemagglutinating antibody, nor did BALB/c-Igb fetal cells from thymus, spleen or liver.

The allotype of the serum antibody was studied by facilitated hemolysis. In nude mice, regardless of the type of cell injected, the antibody was almost always of host type although donor type antibody predominated if the cells were injected into irradiated BALB/c.
Mitogenic Factors Produced by Lymphocyte Activation: Effect on T- and B-Cells
Authors: Vischer Thomas L.
Published: December 2022

During activation of lymphocytes in vitro by specific and nonspecific stimulants, various biologically active factors are produced which have been called “lymphokines” (1). Such lymphokines include factors cytotoxic for target cells, macrophage-migration inhibitor factor, a skin reactive factor, factors inducing blastogenesis, 3H-thymidine incorporation and mitosis, and others (2).

The mitogenic factors could be important in recruiting cells during immune reactions and in explaining the effect of adjuvants (3). Immune responses to many antigens require cooperation of thymus-dependent (T) and bone marrow-derived (B) lymphocytes (4). It is not clear yet whether such mitogenic factors activate T- or B-lymphocytes, or both of them.

In order to investigate the effect of mitogenic factors on T- and B-cells, we produced active culture supernatants in the following way: spleen cells from BALB/c mice immunized 8 weeks previously with keyhole limpet hemocyanin (KLH) were exposed to the antigen in serum-free medium for 2 days (5).
Differentiation of Type 1 and Type 2 Herpes Simplex Virus by in Vitro Stimulation of Immune Lymphocytes
Authors: Rosenberg Gary L. , Wohlenberg Charles , Nahmias André J. , Notkins Abner Louis
Published: December 2022

In a previous report we demonstrated that lymphocytes from rabbits immunized with herpes simplex virus (HSV) or vaccinia virus could be stimulated in vitro by the homologous but not heterologous virus (1). These observations indicated that the lymphocyte stimulation technique, believed to be an in vitro correlate of cell-mediated immunity (2), could distinguish between antigenically unrelated viruses. Recently it has been shown (3, 4) that HSV could be differentiated by serologic techniques into two closely related antigenic types (HSV-1 and HSV-2). The present study was initiated to determine whether the two types of HSV also could be distinguished by the in vitro lymphocyte stimulation technique.

Materials and Methods. The methods employed in the present study have been described in detail elsewhere (1). HSV-1 (strain VR3) and HSV-2 (strain MS) were prepared and assayed in primary rabbit kidney cells grown in the presence of 5% rabbit serum (3, 5).
Radioimmune Precipitation of Group a Arboviruses
Authors: Dalrymple J. M. , Teramoto A. Y. , Cardiff R. D. , Russell P. K.
Published: December 2022

A radioimmune precipitation (RIP) test was used to study selected group A arboviruses. Purified preparations of Sindbis (SIN), western equine encephalitis (WEE) and eastern equine encephalitis (EEE) virus were examined in homologous and heterologous reactions. RIP reactions were comparable to plaque reduction neutralization tests in both sensitivity and specificity. When SIN virus was precipitated by WEE virus antiserum, the RIP curve was biphasic. RIP inhibition of the homologous and biphasic heterologous reaction suggested three separate antibody populations of differing specificities. The results indicated that WEE virus antiserum contained: a) a high titered specific antibody, b) a broadly crossreactive antibody at intermediate antiserum dilutions and c) a low titered antibody which detected only the intra-group WEE-SIN virus complex.
Serologic Cross-Reactivity Between H-2 and HL-A Antigens
Authors: Götze Dietrich , Ferrone Soldano , Reisfeld Ralph A.
Published: December 2022

Soluble HL-A antigen extracted from cultured human lymphoid cells (RPMI 1788, HL-A 2, 10, 7, W14) by the 3M KCl method was injected into rabbits. The rabbit anti-HL-A sera contained antibodies against all the HL-A specificities present on the immunogen. Moreover, these same xenoantibodies reacted also with certain murine lymphocytes bearing distinct H-2 specificities in the direct complement dependent lymphocytotoxic test. Absorption studies confirmed this selected reactivity and led to the conclusion that mainly antigenic determinants of the D region of H-2d and the K region of H-2b are involved in this cross-reactivity. Rabbit anti-HL-A sera absorbed with cells of different mouse strains were tested against a large panel of human lymphocytes from unrelated donors for remaining cytotoxic activity. The results indicate that absorption with cells of the genotypes H-2b (KbDb) and H-2a (KkDd) remove antibodies against determinants of the first segregant series (cross-reacting with HL-A 2 and 10).

These data suggest a specific cross-reactivity between human and murine histocompatibility antigens and suggest a certain structural homology between these cell surface antigens and perhaps even a similar organization of the genetic fine structure of the HL-A and H-2 systems.
Tick-Bite Injury: Mediation by a Complement-Derived Chemotictic Chemotactic Factor
Authors: Berenberg J. L. , Ward P. A. , Sonenshine D. E.
Published: December 2022

The intensely destructive tick-bite injury is dependent on the presence of neutrophils. The present study examines the nature of the tick-host interaction and the attraction of neutrophils to the site of injury. Tick salivary gland extracts have no intrinsic chemotactic activity but can generate this activity from human or dog serum. Activity was also generated from human C5 but not from C3. Antibody to C5 eliminated the chemotactic activity produced in human serum. The chemotactic factor generated in human serum appeared to be a C5 cleavage product of low sedimentation velocity in sucrose gradient ultracentrifugation. Complement depletion of C5 in rats by cobra venom treatment greatly reduced the tissue necrosis and inflammation associated with the tick feeding lesion. These results suggested that this inflammatory lesion was related to the cleavage of host C5 by tick saliva to produce a chemoattractant for neutrophils. This non-immunologic model of tissue injury is analogous to the immunologic models of the Arthus reaction and acute nephrotoxic nephritis. In each case, the complement system provides the chemotactic factors for neutrophils, and the ensuing damage is neutrophil-dependent.
Radioimmunoassay Procedure for Rabies Binding Antibodies
Authors: Wiktor Tadeusz J. , Koprowski Hilary , Dixon Frank
Published: December 2022

Purified rabies virus was labeled with 125I, and preparations with specific activities of 4.4 to 12.7 × 107 cpm/mg of viral protein were obtained. The labeled virus was reacted with dilutions of rabies antibodies and the resulting virus-antibody complexes were precipitated with anti-IgG serum. The specific binding activity for each serum dilution could be calculated after subtraction of the background activity determined from samples of normal serum treated in the same manner. When sera from immunized humans and rabbits and a serum sample from a patient with a clinical rabies infection were evaluated by this procedure, the virus-binding ability of anti-rabies serum was shown to be similar to or higher than the values obtained by the neutralization test based on plaque reduction method. The radioimmunoassay procedure was also used to determine the proportion of rabies-specific γ globulins in a mixture of 131I-labeled γ globulins from immunized rabbits and 125I-labeled γ globulins from normal rabbits. Ten to twelve per cent of all γ globulins represented rabies-specific antibody in this serum sample.
The Clonal Nature of Antibody Formation
Authors: Bosma M. , Davis G.
Published: December 2022

Clonal antibody responses to the haptens, poly-d-alanine (poly-ala) or poly-O-acetyl-d-serine (poly-ser), were produced in lethally-irradiated mice after the injection of limiting numbers of mouse spleen cells and protein-hapten immunogens. Syngeneic F1-hybrid mice permitted the use of antibody allotypes as genetic markers. Allotype markers together with the aid of Poisson statistics enabled us to identify mice whose spleens contained antibody-forming progeny of only one or two hapten-specific precursor cells (antibody cell clones). The results obtained indicated that poly-ala or poly-ser haptens were able to stimulate only about one in 108 spleen cells to form antibody cell clones of a given allotype; that regardless of antibody specificity or allotype, the relative size of most antibody cell clones was limited to 212 to 214 cell divisions; and that most antibody cell clones which shared specificity for the same hapten could be inhibited differentially with hapten analogues.
The Clonal Nature of Antibody Formation
Authors: Bosma M. , Davis G. , Bosma G.
Published: December 2022

Antibody cell clones of (C57BL/6 C3H/HeJ) F1 mice directed against the haptens, poly-O-acetyl-d-serine (poly-ser) and poly-d-alanine (poly-ala), were evaluated with the aid of a cell transfer system and a modified plaque assay. When antibody responses to poly-ser or poly-ala were initiated by a few precursor cells, unequal associations of a given antibody specificity with one of two parental antibody allotypes became apparent: namely, poly-ser specific clones of C3H/HeJ allotype were more frequent than those of C57BL/6 allotype, whereas poly-ala specific clones of C57BL/6 allotype were more frequent than those of C3H/HeJ allotype. The degree of these unequal associations could be accentuated when both poly-ser and poly-ala specific clones were induced with proteins bearing random copolymers of O-acetyl-d-serine and d-alanine (ser-ala). In this instance the proportion of clones which were of C57BL/6 allotype was greater than that obtained with either homopolymer hapten. We interpreted these results as consistent with the following: 1) that closely-linked genes encoded for the two distinct portions of mouse immunoglobulin heavy-chain that determine allotype (constant region) and combining site specificity (variable region); and 2) that more of the immunoglobulin structural genes of the C3H/HeJ allelic chromosomal region could encode for antibodies to the poly-ser hapten than those of the C57BL/6 allelic chromosomal region, while the converse seemed true for antibodies specific to the poly-ala hapten.
Characterization of Three Feline Paraproteins
Authors: Kehoe J. Michael , Hurvitz Arthur I. , Capra J. Donald
Published: December 2022

Serum protein electrophoretic studies on three cats, two with lymphosarcoma and one with multiple myeloma, revealed M components in the γ region. These proteins were isolated by zone electrophoresis and were characterized immunologically, physically and chemically. Antisera to two feline isolates demonstrated individual antigenic specificity. All three proteins were classified as γG lambda immunoglobulins on the basis of cross-reactivities with human antisera. Cross-reactions were also documented between each of the feline proteins and previously studied canine light chains and intact human γG proteins. By analytical ultracentrifugation these feline immunoglobulins had Svedberg constants of approximately 6.8S. Molecular weight determinations of the intact γG proteins by SDS acrylamide gel analysis indicated an average m.w. of 154,000. The amino terminal amino acid residue of one of the feline heavy chains was glutamic acid and that of another aspartic acid. Thus these chains could be partially sequenced by an automatic protein sequencer. Comparison of the results with sequences of human VHIII heavy chains showed a striking sequence homology between the two species.
Antigen-Induced Inhibition of Experimental Allergic Encephalomyelitis
Authors: Swanborg Robert H.
Published: December 2022

Encephalitogenic basic protein (BP) was isolated from bovine brain. Portions of BP preparations were rendered non-encephalitogenic for guinea pigs by modification of the single tryptophan residue with 2-hydroxy-5-nitrobenzyl bromide. This modified BP (benzyl-BP) was employed for antigen-induced inhibition of experimental allergic encephalomyelitis (EAE). In some experiments, non-encephalitogenic diazonium derivatives of BP were also employed for inhibition. The results show that non-encephalitogenic modified BP preparations are capable of suppressing EAE when administered to previously sensitized guinea pigs. On the other hand, if the animals were treated before sensitization, EAE was almost completely prevented. Moreover, the modified preparations inhibited the disease as effectively as unmodified BP.
Radioimmunoassay of Mammalian Type C Viral Proteins
Authors: Scolnick Edward M. , Parks Wade P. , Livingston David M.
Published: December 2022

A radioimmune precipitation assay (RIPA) for the major internal polypeptide of mammalian type C viruses is described. Gel chromatography and isoelectric focusing resulted in a polypeptide of approximately 30,000 daltons which migrated as a single band in three different dissociating systems as analyzed by polyacrylamide gel electrophoresis. This VP3(gs) protein from murine and feline type C viruses has species specific antigenic reactivities which were not altered by chemical iodination. 125I-labeled VP3(gs) was a sensitive probe for antibodies. By employing limiting concentrations of antibody in RIPA, unlabeled murine or feline VP3(gs) antigen could be quantitated by competition with labeled polypeptide. This double antibody VP3(gs) antigen assay was at least 500 times more sensitive than complement-fixation or immunodiffusion and was specific for type C viruses of the homologous species.
Characterization of Migration Inhibitory Factor (MIF) from Guinea Pig Lymphocytes Stimulated with Concanavalin A
Authors: Remold Heinz G. , David Roberta A. , David John R.
Published: December 2022

A migration inhibitory factor (MIF) obtained from cultures of guinea pig lymphocytes stimulated with the mitogen concanavalin A (MIFCon A) was characterized and compared to MIF produced by stimulation with the o-chlorobenzoylbovine γ globulin antigen (MIFOCB-BGG). MIFCon A behaves like MIFOCB-BGG on acrylamide gel electrophoresis, in its buoyant density on isopycnic centrifugation, and in its susceptibility to neuraminidase and chymotrypsin. On the other hand, MIFCon A showed greater heterogeneity than MIFOCB-BGG when chromatographed on Sephadex G-100, and Con A stimulated the production of more MIF than antigen. The increase in heterogeneity and activity of MIFCon A over MIFOCB-BGG would be expected if Con A affects more cells than antigen. These experiments suggest that the triggering of different kinds of receptors results in the production of similar mediators, probably through a common pathway.
The Alternate Complement Pathway in Cell Damage: Antibody-Mediated Cytolysis of Erythrocytes and Nucleated Cells
Authors: May Joseph E. , Green Ira , Frank Michael M.
Published: December 2022

Components of the alternate complement pathway present in C4 deficient guinea pig serum have been studied for their ability to lyse cells sensitized with cytotoxic antibody. Five different cell types, including erythrocytes, nucleated cells and tumor cells, were examined. Cytotoxic antibodies utilized were obtained from guinea pig, mouse, burro and rabbit sources. The specific assay systems included sheep erythrocytes-rabbit anti-Forssman antibody; TNP-coated sheep erythrocytes-rabbit anti-DNP antibody; guinea pig thymocytes-burro anti-thymus cell antibody; mouse thymocytes-mouse anti-ϑ antibody; guinea pig leukemia cells (L2C)-guinea pig anti-histocompatibility antibody. In all cases the alternate complement pathway mediated lysis inefficiently or not at all. The data indicate that in most situations the sequential participation of C1, C4 and C2 is requisite for the efficient lysis of cells sensitized with anti-membrane antibodies.
Regulation of Antibody Response in Vitro
Authors: Kishimoto Tadamitsu , Ishizaka Kimishige
Published: December 2022

Rabbits were immunized by intraperitoneal injections of DNP-coupled ascaris antigen (DNP-Asc) precipitated with aluminum hydroxide gel, and the anamnestic antibody response of the mesenteric lymph node cells was studied in vitro. The cells were stimulated with homologous antigen, cultured in the absence of antigen for 6 days, and the IgM, IgG and IgE anti-DNP antibodies in the culture fluids were measured. In this system, formation of the anti-hapten antibodies was suppressed when the primed cells were stimulated with antigen in the presence of either ε-DNP lysine or ascaris antigen. At a given concentration of either the hapten or carrier, a similar extent of suppression was observed for the formation of the three immunoglobulins. The results suggest that both hapten-specific memory cells and carrier-specific helper cells are involved in the formation of anti-hapten antibodies of the three immunoglobulin classes.

The primed lymph node cells gave maximal antibody response when they were stimulated with a certain concentration of DNP-Asc. The optimal concentration of the antigen for maximal response was found to depend upon the interval between the priming of donors and stimulation of primed cells. When the same cell suspension was employed, however, the optimal concentration of antigen was the same for the formation of IgG, IgM and IgE antibodies.

The DNP-Asc-primed lymph node cells formed both IgM and IgG anti-hapten antibodies upon stimulation with DNP-bovine γG globulin (DNP-BGG), the amounts of the antibodies formed in the culture were only 10 to 20% of that formed by stimulation with DNP-Asc. The optimal concentration of the antigens for maximal antibody formation, and the ratio of IgM/IgG antibodies in the culture fluids was comparable whether DNP-Asc or DNP-BGG was used for stimulation. The results indicated that the optimal concentration of antigen and the distribution of anti-hapten antibodies between IgG and IgM classes is decided by the population of hapten-specific memory cells rather than carrier-specific helper cells. This idea was supported by the experiments in which rabbits primed with hapten primary-carrier conjugates received a supplemental immunization with a secondary carrier. The supplemental immunization enhanced the in vitro antibody response of their lymphocytes to the secondary carrier conjugates but did not influence the distribution of anti-hapten antibody between IgM and IgG.

IgE antibody, however, was not formed in vitro when DNP-Asc-primed lymph node cells were stimulated by DNP-BGG, even when the donors had received supplemental immunization with BGG. The results indicated theat BGG-specific helper cells, which enhance both IgG and IgM antibody responses, did not collaborate with hapten-specific memory cells for IgE antibody formation.
The Correlation Between the Proliferative and the Cytotoxic Responses of Mouse Lymphocytes to Allogeneic Cells in Vitro
Authors: Wagner Hermann
Published: December 2022

The relationship between the antigen recognition phase (mixed lymphocyte culture) and the cytotoxic effector phase of a mouse allograft reaction in vitro has been studied. In addition, the responsiveness of thymus-derived (T) cell depleted and bone-marrow derived (B) cell enriched lymphocyte populations to alloantigens was investigated. Using CBA mouse spleen cells, there was a separation in time (48 hr) between the maximal proliferative response (3H-thymidine uptake) and the peak generation of cytotoxic lymphocytes as measured by a 51Cr release assay. Quantitation of the cytotoxic response obtained by using spleen cells of neonatal thymectomized (NnTx) mice or adult thymectomized lethally irradiated and bone marrow-protected (ATxBM) mice demonstrated that the cytotoxic allograft response may be useful as a functional test for residual mature T cells within a lymphocyte population.

Spleen cells of congenitally athymic (“nude”) mice did not contain precursor cells of cytotoxic lymphocytes. However, they proliferated in the “one way” mixed lymphocyte culture as did spleen cells of NnTx or ATxBM mice. Evidence is given that the proliferation of nude spleen cells was due to dividing lymphocytes and not to contaminating erythroid or myeloid elements. It is concluded that in the mouse both B and T cells are capable of proliferation in vitro due to alloantigens. Whereas T cells differentiate into cytotoxic effector cells, B lymphocytes respond independently of helper cells (T lymphocytes) and the mitotic response may be part of a humoral response to alloantigens in vitro.
Serologic Identity of the b4 Allotypic Determinants Present on Homogeneous Rabbit Light Chains with Different N-Terminal Amino Acid Sequences
Authors: Kindt T. J. , Seide R. K. , Lackland H. , Thunberg A. L.
Published: December 2022

Homogeneous rabbit light chains with marked differences in their N-terminal sequences possess identical b4 allotypic markers. Homogeneous antibodies isolated from b4 rabbits absorbed anti-b4 antisera with the same efficiency as a pool containing multiple b4 light chains. These results indicate that the molecular determinant(s) of the b4 allotype is not related to the sequence variations at the N-terminus of the light chains. The same b4 allotypic marker is present on homogeneous light chains even though they have marked differences in electrophoretic mobility and amino acid composition. These data suggest that the b4 determinant is in the constant region or in a constant part of the variable region of the rabbit κ light chain.
Antigenic Competition and Nonspecific Immunity after a Rickettsial Infection in Mice: Restoration of Antibacterial Immunity by Phenyl-Imidothiazole Treatment
Authors: Renoux Gérard , Renoux Micheline
Published: December 2022

A prior Coxiella burneti infection renders mice nonspecifically resistant to a Brucella abortus challenge but depresses the specific activities of either dead vaccine (in incomplete adjuvant) or of live Brucella vaccine and inhibits Brucella antibody formation that usually follows the use of live vaccine. Thus, a first antigenic stimulus interferes with responses to a second unrelated antigen to cover both nonspecific immunity and antigenic competition. Phenylimidothiazole treatment 2 days after anti-Brucella vaccinations restores most of the specific activities of these vaccines which were reduced by C. burneti priming, but leaves as impaired as they were Brucella antibodies titrated through three serologic tests. This finding suggests that phenylimidothiazole stimulates above all cell-mediated immunity.
Purification of Human Lymphotoxin
Authors: Russell Stephen W. , Rosenau Werner , Goldberg Melvin L. , Kunitomi Gene
Published: December 2022

This paper describes the first preparation of highly purified lymphotoxin, a soluble cytotoxin produced by lymphocytes. Lymphotoxin was produced in vitro by phytohemagglutinin-stimulation of human lymphocytes and was then purified greater than 2000-fold by a sequence of column chromatography (Sephadex G-150 followed by DEAE-cellulose) and preparatory acrylamide gel electrophoresis. Ten per cent of the cytotoxic activity was recovered in the final product. The results of the purification steps were evaluated by a new quantitative assay for cytotoxic activity.
Immunoglobulin Class of Influenza Antibodies Investigated by Radioimmunoassay (RIA)
Authors: Daugharty Harry , Davis Marianne L. , Kaye Harold S.
Published: December 2022

Influenza antibodies were quantitated by radioimmunoassay (RIA) binding of immunoglobulin (Ig) class-specific antibodies labeled with 125-iodine as indicators. Indicator binding values by RIA were tested by chi-square for association with hemagglutination inhibition (HI) and complement fixation (CF) titers. IgG-specific indicators for RIA had binding values which appeared to be more closely associated with HI titers than with CF titers. IgM-specific indicators appeared to be more closely related to CF titers than with HI titers. RIA indicators with combined Ig heavy chain specificity proved to be more sensitive and had a higher chi-square value when tested against both HI and CF test results than did either IgG- or IgM-specific indicators. RIA with Ig class-specific indicators has made HI and CF tests more useful in ascertaining the phase of the immune response to viral antigens. Class-specific indicators exhibited a high degree of accuracy for detecting 7S and 19S viral antibodies fractionated by sucrose density gradient ultracentrifugation. Variations in the strain of viral antigen in the RIA test revealed a predominant 7S (IgG) class of antibodies against an influenza virus which was prevalent 7 to 8 years previously. However, a predominant 19S (IgM) class of antibodies was evident when a more recent influenza strain was used for the assay. Precisely distinguishing the major Ig class of viral antibody by RIA should be helpful in determining how recently a viral infection occurred or how antigenically variable viral strains are.
Proliferation by Bone Marrow-Derived Lymphocytes in Response to Antigenic Stimulation in Vitro
Authors: Elfenbein Gerald J. , Shevach Ethan M. , Green Ira
Published: December 2022

The extent of tritiated thymidine (3H-THY) incorporation into bone marrow derived cells (B cells) in cultures of sensitized guinea pig lymph node cells was determined by the simultaneous identification by radioautography of grained cells and cells bearing the receptor for antigen antibody complement complexes found on B cells. By this criterion 14.2 to 27.3% of the cells incorporating 3H-THY in these cultures are B cells whether or not antigen was present. Furthermore, in the cultures to which antigen was added there is an increase in the per cent of proliferating B cells.