bluuebunny/crossref_metadata_2025 huggingface dataset:
Myeloma Cell Immunoglobulin Expression during in vivo Growth in Diffusion Chambers: Evidence for Repetitive Cycles of Differentiation
Authors: Rohrer James W. , Vasa Kathy , Lynch Richard G.
Published: December 2022

The DNP-binding BALB/c plasmacytoma MOPC-315 undergoes repetitive cycles of differentiation of immunoglobulin expression when serially passaged at 10-day intervals in normal syngenic mice. Within 24 hr of implantation of MOPC-315 cells in i.p. diffusion chambers there is a preferential loss of cells which: a) secrete IgA paraprotein, b) have large amounts of cell surface membrane localized IgA paraprotein, and c) morphologically have a plasmacytic appearance. In addition, myeloma stem cells capable of forming macroscopic splenic colonies following i.v. injection not only survive implantation preferentially, but they also increase in number sooner than any other measured myeloma cell subset. Following the stem cell increase there is a period of progressive increase in the number of myeloma cells with cell surface membrane localized immunoglobulin, and this is followed by an increase in the number of myeloma cells which secrete the homogeneous immunoglobulin. These observations demonstrate that marked changes in the frequency and absolute numbers of myeloma cell subsets occur following implantation in normal syngenic hosts. It is also clear that the changes observed during the initial 7 to 10 days of growth would not have been detected without the DNP-binding property of MOPC-315 used in combination with diffusion chambers which permit sequential recovery of all the surviving cells and their progeny.

We interpret our data as indicating linear maturation of the surviving MOPC-315 cells and their progeny, and raise the possibility that this variation in immunoglobulin expression may have fundamental implications for the idiotype-specific suppression of myeloma cell grafts achieved by immunization with purified homologous myeloma immunoglobulin.
Augmentation of Proliferation of Human Mixed Lymphocyte Culture by Human α-Fetoprotein
Authors: Charpentier Bernard , Guttmann Ronald D. , Shuster Joseph , Gold Phil
Published: December 2022

We have studied the effects of purified human α1-fetoprotein (AFP) of hepatoma origin on the kinetics of one-way lymphocyte proliferation in a mixed human lymphocyte culture system. Human serum albumin was employed in parallel control studies. Although AFP, over a wide range of concentrations from 5 ng/ml to 2 mg/ml, had no mitogenic activity on a single lymphocyte population, significant augmentation of the proliferative response was seen at the three different times of harvesting in the system of one-way allogeneic stimulation. When the addition of the AFP to this system was delayed to 24 hr after culture initiation, the AFP was still effective in producing augmented proliferation. However, no effect on the normal mixed lymphocyte reaction was seen upon addition of the AFP at either 48 or 72 hr after initiation of the cultures. In completely parallel control studies, human serum albumin used at the same concentrations as the AFP produced no modifications of the normal mixed lymphocyte culture response when compared to control cultures incubated in media without added serum proteins. Thus, the preparation of highly purified human AFP studied had no immunosuppressive activity, but actually showed a stimulatory activity on mixed lymphocyte proliferation when employed at relatively high concentrations.
Development of a Hemolytic Plaque Assay for Glutamic Acid, Lysine-Containing Polypeptides: Demonstration that Nonresponder Mice Produce Antibodies to These Peptides When Conjugated to an Immunogenic Carrier
Authors: Cheung Nai Kong V. , Dorf Martin E. , Benacerraf Baruj
Published: December 2022

Fowl γ-globulin, when chemically conjugated to GLØ or GL, functions as a T-dependent immunogenic carrier and stimulates anti-GLØ and anti-GL antibody production in nonresponder mice. The conjugation procedure utilizes the Schiff base reaction. The anti-GL and anti-GLØ responses were detected by hemagglutination and hemolytic plaque assays by using GL-coated erythrocytes. The coupling of GL to erythrocytes utilizes a novel procedure in which a palmitoyl derivative of GL is adsorbed onto red blood cells. The optimal conditions for preparing the palmitoyl derivative and for coupling to SRBC are presented. With the hemolytic plaque assay, we have verified that GLØ responder animals make both IgM and IgG responses, whereas nonresponder mice fail to make either IgM or IgG plaque-forming cells.
Humoral Immunostimulation
Authors: Shearer William T. , Crouch Judith A.
Published: December 2022

Uptake of phosphate (32P) in L cells was modulated by reaction with anti-L cell antibody. A biphasic response was noted with high, cytotoxic concentrations inhibiting 32P uptake and low, cytostimulatory concentrations stimulating 32P uptake. Stimulation of 32P uptake was dependent upon multivalent binding as immune IgG and F(ab′)2 were effective, but univalent Fab′ was ineffective in enhancing 32P uptake into cells. Antibody stimulation of 32P uptake appeared to be an energy independent process and to take place by activation of 32P membrane transport with an increased Vmax (19.3 pmoles/min to 25.3 pmoles/min), but the same Km (0.22 mM). Isolation and measurement of cellular (primarily membrane) phospholipids demonstrated a dramatic increase of (2-fold) specific radioactivity in phosphatidylinositol. Early turnover of phosphatidylinositol may be an important signal for tumor cells to grow at an enhanced rate when exposed to cytostimulatory concentrations of antibody.
Immunosuppression in Man: Suppression by Macrophages Can be Mediated by Interactions with Regulatory T Cells
Authors: Stobo John D.
Published: December 2022

Previous data from this laboratory indicated that profound defects of in vivo and in vitro T cell function noted in some patients with fungal infection reflected the presence of immunosuppressive forces. Investigation of the cellular interactions required for this immunosuppression to become manifest revealed the following. Patients' macrophages liberated, in vitro, a soluble material (SM)3 capable of inhibiting the blastogenic response of normal T cells to mitogens and antigens. However, not all T cells were equally susceptible to its suppressive effect. Incubation of normal PBMC in culture fluid for 7 days yielded a population of cultured T cells whose reactivity could not be inhibited by SM. Binding studies indicated that this “resistance” did not reflect any gross alterations in the ability of cultured T cells to interact with SM. Rather, it reflected the functional deletion of a subpopulation of T cells required for SM to mediate its suppressive effect. This was demonstrated by the finding that in the presence of low density, fresh, autologous T cells, SM was capable of suppressing the reactivity of cultured T cells. Similar interactions between SM and low density T cells appeared to be required for SM to inhibit T-dependent responses among fresh noncultured PBMC. Thus, removal of low density T from fresh normal PBMC yielded high density T whose reactivity was not suppressed by SM. These studies are interpreted as indicating that immunosuppression in the patients studied does not reflect direct interactions between suppressive macrophages and responding T cells. Instead, it represents an indirect interaction whereby macrophages may generate or potentiate active suppressive T cells.
Immune Ascites in the Guinea Pig: Specificity of Cells and Antibody in an Induced Peritoneal Exudate
Authors: Yaron Arieh , Dunham Edward K. , Stashenko Philip P. , Campos-Neto Antonio , Levine Herbert , Schlossman Stuart F.
Published: December 2022

Strain 2 guinea pigs, immunized with Lys10-Lys(Dnp) in CFA and repeatedly injected intraperitoneally with adjuvant, developed ascites. The fluid was harvested over 8 months in total amounts up to 2 liters per animal and contained substantial amounts of cells and antibody which reacted with the immunizing antigen and related peptides. The antibody was of the IgG and IgA classes and showed restricted heterogeneity. Among synthetic Dnp-oligopeptides, both the cells, studied by antigen-stimulated thymidine incorporation, and the purified antibody, studied by fluorescence quenching, demonstrated the same specificity for the immunizing antigen as has previously been noted in lymph node cells and in serum antibody. The technique offers a means for studying more cells and more antibody than has previously been possible from individual guinea pigs.
Presidential Address to the American Association of Immunologists, Delivered in Chicago, Illinois, April 6, 1977
Authors: Mayer Manfred M.
Published: December 2022

During the past several years this talk has been an occasion for historical, philosophical, or pedagogic presentations. I want to return to the earlier tradition and address my remarks to a scientific subject that is currently of great interest to me, namely, the similarities between humoral and cellular immunity.

For a century, immunology has been divided into two worlds, one humoral, the other cellular. I believe there are clear indications that this era is now drawing to a close as a consequence of studies of the various mediators that are operative in the trans-antibody domain of humoral and cellular immunity. It is my impression that we are witnessing one of those exciting developments in scientific research in which a series of diverse facts gives rise to hypothetical concepts that are eventually developed into the satisfying pattern of a theory.
Induction of Plasma Cell Differentiation of Human Fetal Lymphocytes: Evidence for Functional Immaturity of T and B Cells
Authors: Hayward A. R. , Lawton A. R.
Published: December 2022

Resembling the in vitro antibody response of the newborn, cultures of cord blood lymphocytes stimulated with pokeweed mitogen (PWM) generated fewer plasma cells (PC) than comparable adult lymphocyte cultures and the response was almost exclusively of the IgM class. We investigated the cellular basis of this difference by preparing mixed cultures of newborn T or B lymphocytes with adult B or T cells. Substitution of adult for newborn T cells enhanced the response of newborn B cells, particularly of the IgG and IgA classes. The response of second trimester fetal spleen cells was also increased by adult T cells, although no IgA PC appeared. Conversely, adult B cells generated fewer PC particularly of the IgG and IgA classes when cultured with newborn T cells. The relatively poor IgA and IgG responses of newborn cells seems partially but not entirely due to deficiency of T cell helper function. Suppressor activity of newborn T cells was investigated by adding excess unrelated newborn or adult T cells to adult T + B cells: adult T cells improved the response whereas newborn T cells were variably suppressive. The results indicate that newborn T cells, although capable of helper function, are balanced toward suppression.
20α-Hydroxysteroid Dehydrogenase: A T Lymphocyte-Associated Enzyme
Authors: Weinstein Yacob
Published: December 2022

20α-Hydroxysteroid dehydrogenase (20α-SDH), an enzyme which reduces progesterone to 20α-dihydroprogesterone, was found to be associated with T lymphocytes. 20αSDH activity was present in spleen cells bearing θ antigen, spleen cells nonadherent to nylon wool (T lymphocyte-enriched population), and in thymocytes. Almost no enzymatic activity was found in bone marrow cells from normal mice and in spleen cells from neonatally thymectomized or athymic nude mice. T cell mitogens (PHA and Con A), but not the B cell mitogen LPS, induced high levels of enzymatic activity 48 hr after addition to spleen cell cultures.

The level of 20αSDH activity in lymphocytes was age dependent. At the age of 4 weeks 20αSDH activity in thymocytes, spleen cells, and lymph node lymphocytes was 3 to 5 times higher than at 8 and 16 weeks. Progesterone (5.0 × 10-7 M) was found to inhibit thymocyte proliferation after exposure to mitogens, but not 20α-dihydroprogesterone (10-6 M). 20α SDH may protect the embryonic thymocytes against high concentrations of progesterone.
Physicochemical Characterization of a Vascular Permeability Factor Produced by Con A-Stimulated Human Lymphocytes
Authors: Sobel Alain T. , Branellec Anne I. , Blanc Christian J. , Lagrue Gilbert A.
Published: December 2022

Stimulation of human lymphocytes with Con A resulted in the liberation of a soluble lymphokine that increased the permeability of guinea pig skin capillaries. This factor, termed vascular permeability factor (VPF), was characterized by physicochemical methods. Upon gel filtration chromatography on Sephadex G-100, it eluted in a narrow peak with an apparent molecular weight of 12,000 daltons. The sedimentation rate of VPF, estimated by sucrose density gradient ultracentrifugation, was 1.8 S. Polyacrylamide gel electrophoresis showed the biologic activity to migrate in the β region; the factor displayed a pI of 6.4 upon isoelectric focusing. These characteristics allow VPF to be distinguished from other human lymphokines.
Purification of C3b Inactivator and Demonstration of Its Two Polypeptide Chain Structure
Authors: Fearon Douglas T.
Published: December 2022

C3b inactivator (C3bINA) was isolated from plasma by sequential ammonium sulfate gradient solubilization, anion exchange chromatography, gel filtration, and repeat ammonium sulfate gradient solubilization. The final product was pure as assessed by alkaline disc gel electrophoresis, isoelectric focusing, and SDS polyacrylamide gel electrophoresis, and elicited a monospecific rabbit antiserum. The normal serum concentration of C3bINA was found to be 53 ± 9 µg/ml (mean ± S.D.). Heterogeneity of purified C3bINA was apparent on alkaline disc gel electrophoresis and isoelectric focusing, but not with SDS polyacrylamide gel electrophoresis and thus is attributed to forms of C3bINA that differ in charge rather than in size. SDS polyacrylamide gel electrophoresis of unreduced, alkylated C3bINA yielded a single stained band with an apparent m.w. of 93,000, whereas the reduced protein demonstrated two bands of 55,000 and 42,000 m.w., thereby establishing a composition of two disulfidelinked polypeptide chains for C3bINA.
Antigenic Requirements for the Generation of Secondary Cytotoxicity
Authors: Grillot-Courvalin Catherine , Alter Barbara J. , Bach Fritz H.
Published: December 2022

The in vitro role of products of the H-2 complex in restimulation of secondary proliferative and cytotoxic responses has been studied. In this paper we have studied in particular the role of CD antigens, the target antigens for cytotoxic T lymphocytes. We have used UV-treated cells that express their CD antigens but no LD. Like others, we demonstrate that CD antigens alone are able to generate a secondary cytotoxic response and furthermore that these CD antigens must be the same as those used for primary sensitization. However, in contrast to the results of others, in some cases UV-treated cells were unable to restimulate such a response even when appropriate controls showed that the CD antigens on UV-treated cells were functional. Furthermore, in these cases the addition of an LD stimulus in the presence of UV-treated cells results in a secondary cytotoxic response that is significantly greater than that elicited by LD restimulation alone. Such potentiation is not observed if the CD antigens used for restimulation are different from those used for the primary sensitization. In addition, a non-H-2 differences(s), presumably the Mls locus, appears to be able to generate a secondary response. The implications of these results are discussed in terms of “memory” T lymphocytes in cell-mediated immunity.
Characterization of Suppressive Immunoglobulin-Binding Factor (IBF)
Authors: Neauport-Sautes Catherine , Fridman Wolf H.
Published: December 2022

L-5178-Y thymoma cells were used to produce radioactive immunoglobulin-binding factor (IBF). For this purpose, the cells were internally labeled by incubation with radioactive amino acids and/or fucose. The supernatants contained radioactive material that bound to IgG-sensitized erythrocytes and suppressed the in vitro antibody response to sheep red blood cells. Upon filtration on Sephadex G-200 both the IgG-binding activity and the suppressive activity eluted at peaks of 140,000 and above 300,000 d. However, on SDS polyacrylamide gels, after precipitation with antigen-IgG-antibody complexes, IBF was found in a single peak of 80,000 d. This molecule could be dissociated in the presence of mercaptoethanol into a major unit of 40,000 d and a minor unit of 20,000 d.

These data suggest that IBF is a molecule of 80,000 d, which contains chains of 40,000 d and probably 20,000 d linked by disulfide bridges. In cell supernatants, however, the factor exists in polymeric forms of 140,000 d and more than 300,000 d.
Antibody-Dependent, Complement-Mediated Homologous and Cross-Cytolysis of Togavirus-Infected Cells
Authors: King Bernadette , Wust Carl J. , Brown Arthur
Published: December 2022

The appearance of viral induced antigen(s) on Sindbis and Semliki Forest (SF) virus-infected chick embryo cells is detected with homologous mouse anti-viral antibody-dependent, complement-mediated cytolysis in a 51Cr release assay. Such antigen(s) is detected within 3 to 4 hr after infection whereas viral cytopathologic effects do not occur until 10 to 12 hr. In addition, significant heterologous cytolysis (anti-Sindbis on SF virus-infected cells, or, anti-SFV on Sindbis-infected cells) could be observed with serum samples tested. These sera were assayed for neutralizing and hemagglutination-inhibition activities. Heterologous cytolysis cannot be correlated with hemagglutination-inhibition antibody titers nor is cross-neutralizing antibody detected. The possibility is discussed that another previously undefined antigen on infected cell membranes may be involved in heterologous cytolysis.
Select Growth of Human T Lymphocytes in Single Phase Semisolid Culture
Authors: Shen Jasmin , Wilson Floyd , Shifrine Moshe , Gershwin M. Eric
Published: December 2022

Selective growth and clonal proliferation of human T lymphocytes can be achieved by using a single-phase semisolid methylcellulose system without the requirement of preincubation with lectins. Significant proliferation, however, depends upon the continued presence of Con A or PHA, but not pokeweed mitogen or lipopolysaccharide within the methylcellulose. This procedure eliminates nonspecific agglutination by lectins and allows for direct visualization of colonies and their specific removal and subsequent cloning in liquid phase. Optimal growth and production of colonies greater than 40-cell size require 3 to 9 days. Individual cells can be identified on the basis of E rosette formation and absence of surface immunoglobulin or ability to phagocytize latex particles. Moreover, proliferation is inhibited by anti-thymocyte but not anti-B cell sera and can be demonstrated with peripheral blood T and MOLT-4 cells, but not with B or Raji cells. Finally, colony formation is not enhanced by the presence of 2-mercaptoethanol. The clonal proliferation of human T lymphocytes has wide application in the study of both antigen recognition and lymphocyte alterations in specific diseases.
Plasma Cells and Epithelial Immunoglobulins in the Mouse Mammary Gland during Pregnancy and Lactation
Authors: Weisz-Carrington Paul , Roux M. Estela , Lamm Michael E.
Published: December 2022

Mammary glands of virgin, pregnant, lactating, and post-weaning CAF1 mice were studied by immunofluorescence for epithelial immunoglobulins and stromal plasma cells. In normal virgins, both females and males, only occasional plasma cells and but scanty intraepithelial immunoglobulins are present. During pregnancy and the early days of lactation, an increase in the number of plasma cells occurs, and by 1 week of lactation, there is a marked in crease in the number, and most are synthesizing IgA. This increment parallels the development and proliferation of the glandular epithelium, in anatomical relation to which the plasma cells are observed. The intraepithelial content of IgA is also maximal when the glandular epithelium is most developed. These findings are consistent with a local production of the IgA in milk. Weaning, or deliberate interruption of suckling for more than 10 days results in a sharp decrease in the number of IgA plasma cells and an involution of the epithelium, whose content of immunoglobulin also declines. The changes in the secretory IgA system which occur locally during lactation are thought to be hormone-dependent.
In Vitro Induction of Tumor Specific Immunity
Authors: Burton Robert C. , Chism Stanley E. , Warner Noel L.
Published: December 2022

The 51Cr release cytotoxicity assay was used to evaluate whether self-reacting clones of T cells develop in vitro when normal spleen cells are cultured with irradiated syngeneic cells. This phenomenon has previously been described as autosensitization and has been thought to be due to reactivity against self-determinants. It was shown, however, in this present study that the autosensitization phenomenon differs qualitatively and quantitatively from specific immune induction by oncofetal antigens and tumor-associated antigens. A variety of tumor cell lines of different H-2 types are lysed by autosensitized cells, and the lysis appears to be nonspecific and not restricted to identity at the major histocompatibility complex (MHC). Induction of autosensitization follows the same kinetic pattern, and lysis is semilogarithmically related to effector cell to target ratio as in other systems. Analysis of the specificity of the phenomenon by competitive inhibition with tumor cells or congenic derived spleen cells did not, however, indicate any reactivity for self-MHC determinants, but rather showed that a wide range of culture tumor cells were all capable of inhibiting lysis. Since it was further shown that the level of autosensitization was markedly dependent on the batch of fetal calf serum, it is provisionally concluded that this phenomenon does not represent specific T cell activation to self-determinants, but rather reactivity against minor antigens in the system, possibly a cell-bound fetal calf serum component.
Immunosuppressive Properties of a Peptic Fragment of BSA
Authors: Muckerheide Annette , Pesce Amadeo J. , Michael J. Gabriel
Published: December 2022

The immunogenic properties of a peptic fragment of BSA were investigated. BSA was subjected to limited proteolysis by pepsin and the resulting fragments were separated on DEAE cellulose. The fragment under consideration, Fraction Ia (m.w. 8000 to 10,000), did not precipitate with anti-BSA serum but did inhibit the binding of specific antibody to labeled BSA, indicating the presence of determinants found on the native antigen. BDF1 mice immunized with Fraction Ia in A1(OH)3 gel or in complete Freund's adjuvant produced no significant antibody response as measured by passive cutaneous anaphylaxis (PCA) or by a modified Farr assay. The fragment elicited a PCA reaction in mouse skin sensitized with anti-BSA serum. Treatment of mice with single doses of Fraction Ia at various time intervals before immunization with BSA resulted in significant suppression of the formation of anti-BSA antibody. The conditions of suppression of the IgE response by the peptic fragment were studied in greater detail. Evidence is presented that such suppression can be attributed to the presence of specific T suppressor cells in our system.
Enhancement of C5̄6̄-Initiated Lysis by Cell-Bound C3 Fragments: Evidence for a Mechanism Independent of the Prior Binding of C5̄6̄ to C3b
Authors: Yamamoto Ken-Ichi , Lint Thomas F. , Gewurz Henry
Published: December 2022

Cell-bound C3b can reversibly bind C, activated complex of the fifth (C5) and sixth (C6) components of complement, and in this way potentiate C-initiated lysis by favoring the formation of C at the cell surface. We report here another way in which cell-bound C3 fragments can enhance C-initiated lysis, which involves C generated in the fluid phase rather than at the cell surface. Evidence for the involvement of fluid phase was obtained by use of dextran sulfate, which is known to inhibit the hemolysis of E mediated by fluid phase . Dextran sulfate strongly inhibited the formation of sites on cells bearing C4b and C3b (EAC4b3b) as well as on unmodified E when and C7 were added simultaneously to the cells. By contrast, dextran sulfate had virtually no effect on the reaction sequence involving the prior binding of C to C3b and subsequent formation of at the cell surface. Treatment of EAC4b3b with either anti-C3 Fab′ fragments or the C3b inactivator reduced but did not eliminate the enhancement of hemolysis, raising the possibilities that a C3 fragment(s) other than C3b also can enhance C-initiated lysis and/or that the enhancement is indirect without a requirement for an interaction between and the cell-bound C3 fragment itself.
Antibodies to Bacterial and Tumor-Derived Antigens in Sera from Normal Guinea Pigs
Authors: Brunda Michael J. , Minden Percy
Published: December 2022

Antibodies that react with radiolabeled antigens derived from guinea pig line-10 tumor cells and Mycobacterium bovis (BCG) were detected in sera from normal tumor-free strain-2 guinea pigs (NGPS). Binding by NGPS to the two antigens was inhibited by extracts of either line-10 cells or BCG. Binding by NGPS to the line-10 antigen was inhibited by a number of other bacterial extracts. NGPS was tested after absorption with a variety of cells including line-10, line-1, normal guinea pig spleen, normal adult and fetal liver cells. Results indicated that some of the antibodies in NGPS were directed to line-10-specific determinants. The specific stimulating antigen for these antibodies was not identified but because of the antigenic relationship between BCG, line-10 cells and other bacteria, antibodies to line-10-associated antigens might have been induced by exposure to environmental microorganisms.
Initiation of Bone Resorption by the Classical and Alternative C Pathways and Its Mediation by Prostaglandins
Authors: Sandberg Ann L. , Raisz Lawrence G. , Goodson J. Max , Simmons Hollis A. , Mergenhagen Stephan E.
Published: December 2022

Activation of C by immunoglobulins reactive with cell surface antigens stimulated synthesis of prostaglandin E and resultant release of 45Ca from organ cultures of fetal rat bones labeled in utero. Absorption of the C source (rabbit serum) with rat spleen cells or dilution of C abolished this activity which was, however, restored by addition of immunoglobulin preparations from sera of rabbits immunized with either rat erythrocytes or sonicated fetal rat bones. The active reconstitution factors were present in the 50% (NH4)2SO4 cut of the antisera and were eluted from Sephadex G-200 in both the 19S and 7S fractions and from DEAE cellulose in the IgG-containing fraction. The C-dependent resorption of bone by the F(ab′)2 fragments of the IgG antibody implicated a role for the alternative C pathway in this event. Complete inhibition of this biologic effect by indomethacin and detection of enhanced levels of prostaglandin E in the media of cultures containing antibody and C demonstrated a role for prostaglandins as mediators in the destruction of bone initiated by immune activation of C.
Echinococcus Granulosus: Identification of Subunits of the Major Antigens
Authors: Piantelli Mauro , Pozzuoli Roberto , Arru Efisio , Musiani Piero
Published: December 2022

This paper describes a procedure for obtaining the two “major” Echinococcus granulosus antigens (antigens 4 and 5) from sheep hydatid fluid. Purified antigen 4 (m.w. 400,000 and over) and antigen 5 (m.w. 150,000) were separated in order to identify molecular subunits of each. SDS 12.5% polyacrylamide gel electrophoresis of antigen 4 gave a single band for which a m.w. of 67,000 was calculated. When 5% 2-mercaptoethanol was added to the antigen 4 preparation, SDS 12.5% polyacrylamide gel electrophoresis gave two bands (m.w. 47,000 and 20,000). SDS 12.5% polyacrylamide electrophoresis of antigen 5, with or without 2-mercaptoethanol, gave at least three bands with m.w. ranging from 10,500 to 20,000. Low m.w. subunits of both antigens were eluted in a single peak from a preparatory SDS 7.5% acrylamide gel electrophoresis; these subunits were found to be immunogenic and antigenic.
Evidence for Cyclic GMP and Calcium Mediation of Lymphocyte Activation by Mitogens
Authors: Coffey Ronald G. , Hadden Elba M. , Hadden John W.
Published: December 2022

The results of 47 experiments indicate that the lectin mitogens, PHA and Con A, increase cyclic GMP (cGMP) levels in human peripheral blood lymphocytes and their incubation medium 2- to 9-fold within 10 min of stimulation. At concentrations supraoptimal for the induction of proliferation, Con A, but not PHA, induces progressive increases in cAMP and lesser increases in cGMP. Succinylated Con A (S-Con A) does not exhibit inhibition of proliferation up to concentrations of 250 µg/ml and correspondingly does not produce the cAMP increases observed with native Con A. PHA and both forms of Con A induce modest increases in cGMP in the absence of calcium and no increases in the presence of EGTA. After preexposure to Con A in the absence of calcium, the addition of calcium produces increases in cGMP. The data are consistent with the interpretation that mitogen-induced increases in lymphocyte cGMP are totally Ca2+-dependent, yet only in part dependent on extra-cellular Ca2+. The interpretation is confirmed with a divalent cation ionophore A23187, which was shown to induce lymphocyte proliferation in close association with Ca2+-dependent increases in cGMP and at higher concentrations to inhibit the mitogenic effects in association with Mg2+-dependent increases in cAMP. Dose-response curves for each agent showed that this capacity to increase cGMP levels was correlated with the optimal mitogenic concentration as determined by assay of DNA synthesis. The data are consistent with the postulate that cGMP participates in the positive aspects of mitogen action and that cAMP participates in the inhibitory aspects. It is concluded that coparticipation of cGMP and Ca2+ as essential mediators of the mitogenic signal is a sound hypothesis.
Characterization of Antigen-Binding Receptors in Vitro
Authors: Benfari Mary Jane F. , Cooperband Sidney R. , Moolten Frederick L.
Published: December 2022

We examined the kinetics and affinity of antigen binding in lymphoid populations in mice after immunization. There is increased binding capacity in lymphoid cells from animals that have undergone primary immunization. This increase would seem to be related to increased numbers of antigen-binding cells (rosette-forming cells). The serum antibody titers rise after the increasing binding capacity and numbers of BSA rosette-forming cells have increased. There is an increased amount of antigen bound per antigen-binding cell at certain times after immunization with two peaks in this capacity being demonstrable—one occurring at 4 days after immunization and the second occurring approximately 12 days after immunization and persisting for prolonged periods after that. With time, after immunization two separable peaks of increased antigen-binding cells become apparent, one very early (before Day 4) and one later (after Day 20 to 30). The affinity constants for antigen-binding cells have been measured and found to be high, and to increase with time after immunization. It appears that the heterogeneity of the affinity constants for antigen-binding cells is high early in immunity and becomes more homogeneous with time after immunization.
Activation of T and B Lymphocytes in Vitro
Authors: Eshhar Zelig , Armerding Dieter , Waks Tova , Katz David H.
Published: December 2022

The biologic and metabolic conditions required for optimal production of a nonantigen-specific biologically active factor termed allogeneic effect factor (AEF) have been explored in greater depth. AEF is produced in mixed lymphocyte cultures (MLC) of in vivo alloantigen-activated T cells (ATC) and the respective target cells. In these studies, we describe conditions for production of AEF in serum-free media supplemented with appropriate concentrations of 2-mercaptoethanol, Ficoll, or gelatin. Analysis of the cellular components required for AEF production revealed that AEF is not produced by macrophages nor are macrophages required for optimal production of AEF in vitro. The production of AEF requires the participation of T cells of the Ly-1+, Ly-2, 3- phenotype in the effector cell population and target cells of appropriate specificity differences determined by H-2 haplotype; Mls locus differences are neither essential nor effective in inducing AEF production in this secondary MLC system.

Investigation of the possible contribution of target cell products to the active components of AEF involved the use of glutaraldehyde-fixed normal spleen cells of lipopolysaccharide-induced blasts as stimulators in the MLC. Although it was found that such fixed cells could serve as effective stimulators of AEF production, the finding that 7 to 10% of radiolabeled surface membrane proteins were shed from target cells despite glutaraldehyde fixation precluded a definitive conclusion on the role of target cell products in AEF activity. Finally, metabolic inhibitors were used to explore the metabolic processes required of the participating cells; such studies demonstrated that neither proliferation nor DNA synthesis is required for generation of active AEF, whereas protein synthesis, glycolysis, and electron transfer are required.
Lytic Activity of C5̄‑9̄ Complexes for Erythrocytes from the Species Other Than Sheep: C9 Rather Than C8-Dependent Variation in Lytic Activity
Authors: Yamamoto Ken-Ichi
Published: December 2022

With the reactive lysis system, a form of hemolysis mediated solely by the late-acting complement components (C, C7, C8, and C9), guinea pig C9 (C9gp) was found to be very inefficient in inducing the lysis of guinea pig and mouse erythrocytes bearing human C. By contrast, C9gp could efficiently induce the lysis of sheep and goat erythrocytes bearing human C. By contrast, C9gp could efficiently induce the lysis of sheep and goat erythrocytes bearing human C5-8. Human C9 was efficient in the lysis of erythrocytes from the species mentioned above. Further study showed: 1) the observed inefficiency in the lysis of guinea pig erythrocytes was not due to incompatibility between human C7 or C8 and C9gp; 2) C9gp could efficiently bind to guinea pig erythrocytes bearing human C but was inactive in the subsequent lytic process. The present finding emphasizes a role for C9 in complement-mediated membrane damage which may not be a simple effector function of C8 action.
Cytotoxic Activity of Human Lymphocytes: Quantitative Analysis of T Cell and K Cell Cytotoxicity, Revealing Enzyme-Like Kinetics
Authors: Zeijlemaker Wim P. , van Oers Rien H. J. , de Goede Ruud E. Y. , Schellekens Peter Th.A.
Published: December 2022

The mechanism of action of human cytotoxic lymphocytes was studied in two systems: T cell cytotoxicity in cell-mediated lympholysis (CML) and K cell cytotoxicity in antibody-dependent lymphocytotoxicity (ADL). The mechanisms resemble that of an enzyme, if the effector cells are regarded as enzyme and the target cells as substrate. On the basis of a kinetic analysis it was shown that the experimental data are compatible with a simple rate equation, which allows the definition of three constants: p, representing the maximal killing rate of the effector cell suspension (at target →∞); q, representing the maximal lysis of the target cells (at effector →∞); K which represents the Michaelis constant (i.e, the target cell number giving ½ Vmax), when extrapolated to effector = 0. The constant K may be taken to reflect the affinity between effector and target cells.

Furthermore, for the ADL system the influence of the effector cell concentration on Km was due not to the addition of inert cells but rather to the presence of cells that can bind but not kill. Finally, it was shown that in the ADL a K cell can recycle, i.e., kill more than one target cell.

On the basis of these results, experimental conditions are proposed for optimal measurement of cellular cytotoxicity.
The Demonstration of Subtype (D or Y)-Specific Determinants on the Surface of the Presumed Hepatitis B Virus
Authors: Hess G. , Shih J. W-K. , Kaplan P. M. , Gerin J. L.
Published: December 2022

Dane particles isolated from the sera of HBsAg/ad and HBsAg/ay carriers were reacted with monospecific antibodies to the d and y subtype-specific determinants of HBsAg/. Dane particles from HBsAg/ad sera expressed the d determinant on their surfaces and those from HBsAg/ay sera contained the y specificity. Both complete (DNA-P and HBcAg) and defective (HBcAg alone) Dane particles expressed the subtype-specific determinants.
Effect of Cyclophosphamide on Interstitial Nephritis and Tubule Cell Proliferation in NZB/NZW Mice
Authors: Hurd Eric R.
Published: December 2022

The effect of cyclophosphamide on renal interstitial mononuclear cell infiltration and renal tubule cell (TC) proliferation has been examined in (NZB × NZW)F1 hybrid (B/W) and control mice. TC proliferation was measured by tritiated thymidine (3HTdr) injection, autoradiographic examination of kidney sections, and enumeration of labeled tubular cells. Cyclophosphamide administered 2 months before sacrifice virtually obliterated interstitial infiltration and significantly decreased tubule cell proliferation at 3, 5, 7, 9, and 12 months of age.
Hydrocortisone Affects Tumor Growth by Eliminating Precursors of Suppressor Cells
Authors: Schechter Bilha , Feldman Michael
Published: December 2022

Suppressor lymphocytes responsible for the suppression of anti-tumor immune response were previously demonstrated in spleens of C57BL mice carrying the syngeneic Lewis lung carcinoma (3LL). Based upon earlier observations on the inactivating effect of hydrocortisone (HC) on precursors of suppressor lymphocytes, we have now studied the effect of the steroid on the anti-tumor cellular immune response and on tumor development in 3LL-bearing mice. Mice were given a single injection of HC before, together with, or after the inoculation of 3LL tumor cells. Tumor development and mouse mortality were delayed when HC was administered after tumor inoculation. The highest delay was observed when the steroid was injected to the mice 3 to 5 days after the tumor cells. HC-resistant spleen cells (15 to 30% of the total number of cells) obtained from such tumor-bearing mice (TBM) 2 or 3 days after HC treatment enhanced tumor development significantly more than cells from untreated TBM, when transferred together with tumor cells into syngeneic recipients. However, this enhancing activity rapidly disappeared upon spleen regeneration. Spleen cells obtained 7 to 14 days after HC injection manifested a protective activity against tumor growth. These results suggest that HC affects the balance between lymphocyte subpopulations which participate in either the rejection or the enhancement of the tumor. HC-resistant spleen cells recovered from the spleens of TBM shortly after HC treatment seem to be enriched for preformed mature suppressor cells, but are deficient for their precursors. This deficiency, which is transient due to thymus and spleen regeneration, is later on expressed in an increased cytotoxic response against the tumor.
The Expression of Ia Antigens on Immunocompetent Cells in the Guinea Pig
Authors: Yamashita Uki , Shevach Ethan M.
Published: December 2022

Only 15 to 25% of purified oil-induced guinea pig macrophages could be lysed by treatment with anti-Ia serum and C. Those cells remaining alive after treatment were not damaged and were metabolically active since they readily phagocytized latex beads. However, the “Ia-negative” macrophages were markedly deficient in their ability to present protein antigens to immune T lymphocytes and to function as stimulator cells when mixed with allogeneic T cells in the mixed leukocyte reaction. It thus appears that Ia antigens are expressed on a subpopulation of macrophages and that this subpopulation plays a critical role in the activation of T cell proliferation to soluble protein antigens and to alloantigens.
Cleavage of Human C5 by Trypsin: Characterization of the Digestion Products by Gel Electrophoresis
Authors: Minta Joe O. , Man Doreen P.
Published: December 2022

Human C5 is composed of two nonidentical polypeptide chains, α and β (m.w. 130,000 and 80,000, respectively) linked together by disulfide bonds and noncovalent forces.

Cleavage of C5 by trypsin was restricted to the α-chain and resulted in the generation of major fragments with increased anodic mobilities. Limited digestion of C5 by trypsin (substrate to enzyme ratio 10:1 w/w at 37°C for 1 min) resulted in the release of a small terminal α-chain peptide (α1, m.w. 15,000) probably analogous to C5a, from a large fragment, C5b (m.w. 195,000) composed of an intact β-chain disulfide linked to an α-chain that has a lower m.w. (α′ 115,000). Further digestion (37°C, 5 min) resulted in cleavage of the α-chain at multiple sites with the production of three peptides from the α′-chain (α2I, 23,500; α2II 15,700 and α2III 10,200) and a residual fragment, C5c (m.w. 144,000). The α1 and α2 peptides are not covalently linked to the β-chain nor to one another. The C5c fragment on the other hand is composed of small peptides of the α′c chain (α3 14,000; α4I 9,000; α4II 11,000; α5 23,000 to 30,000) which are linked to the β-chain and also probably to one another by covalent bonds. Secondary cleavage occurred upon prolonged digestion with trypsin (37°C, 20 min), and this resulted in the progressive erosion of the α′c peptides and the conversion of C5c to smaller C5c-like species.
Demonstration of Activation of B Lymphocytes in New Zealand Black Mice at Birth by an Immunoradiometric Assay for Murine IgM
Authors: Moutsopoulos Haralampos M. , Boehm-Truitt Marilyn , Kassan Stuart S. , Chused Thomas M.
Published: December 2022

We have developed a highly specific and sensitive “two-site” immunoradiometric assay for murine IgM in which antigen bound to immobilized antibody reacts with affinity-purified radiolabeled antibody. We utilized the sensitivity of this assay to study the rate of IgM secretion in short-term cultures by spleen cells from the autoimmune strains, New Zealand Black (NZB) and New Zealand Black by New Zealand White F1 hybrid (BW), and from normal (C57BL/6, DBA/2, NZW) mice. The temperature dependence of IgM secretion in short-term cultures, its pentameric structure, and the similar viability of NZB and normal strain spleen cells indicate that active IgM synthesis is being measured. We observed that the splenic B lymphocytes of NZB and BW mice, in contrast to normal strains, produce IgM in vitro at birth. By 6 to 8 weeks of age NZB and BW spleen cells produce 40 times more IgM than spleen cells from normal strains of mice. The IgM produced in vitro by splenic lymphocytes from NZB and BW mice is not absorbed by syngeneic or allogeneic thymocytes or erythrocytes.
Reconstitution of C1 in Native, Proenzyme from and Its Use in a Quantitative C1 Activation Test
Authors: Cooper Neil R. , Ziccardi Robert J.
Published: December 2022

C1 was reconstituted in macromolecular proenzyme, nonactivated form by incubation of highly purified C1q, C1r, and 125I-C1s together in the presence of calcium. C1 reformed in this manner had an equimolar ratio of C1 subcomponents, as is found in serum, and sedimented in sucrose density gradients with the 16S rate characteristic of C1 in serum. Reconstituted C1 was activatable as shown by cleavage of the 87,000 dalton polypeptide chain of C1s into disulfide linked subunits of 59,000 and 28,000 daltons, respectively, after incubation with aggregated IgG. The extent of activation may be quantitated. Reformed activatable proenzyme C1 can be used to quantitatively assess the C1-activating properties of various substances in addition to its use in the analysis of the C1 activation process.
The Requirement of Viable Thymocytes for Species-Specific Attachment to and Release from Macrophages
Authors: Lopez Luis R. , Vatter Albert E. , Talmage David W.
Published: December 2022

The role of the thymocyte in its species-specific binding to macrophages has been explored. Although formalin treatment of macrophages resulted in loss of binding to thymocytes, formalin treatment of thymocytes did not have this effect. However, two differences between living and formalin-treated thymocytes were noted. Formalin-treated thymocytes bound to macrophages of any species whereas the binding of living thymocytes was species specific. Living thymocytes attained maximum binding in approximately 1 hr and then the fraction bound gradually diminished. Formalin-treated thymocytes remained bound to the macrophage and appeared to be phagocytized. Released thymocytes did not bind to fresh macrophages, but released macrophages bound to fresh thymocytes. The results suggest that the binding of thymocytes to macrophages results in maturation of thymocytes.
Spontaneous Cytotoxicity of Human Peripheral Blood Mononuclear Cells Toward Red Blood Cell Targets
Authors: Muchmore Andrew V. , Decker Jean M. , Blaese R. Michael
Published: December 2022

In a previous paper we demonstrated that human peripheral blood mononuclear cells become strikingly cytotoxic toward a wide variety of red blood cell targets after 7 days of in vitro culture. The cell responsible for cytotoxicity does not rosette with SRBC and demonstrates both surface adherence and phagocytic properties. In this paper we wish to show that development of spontaneous cytotoxicity is due to a time-dependent loss of suppressor cell function. Fresh autologous lymphocytes, when added to cultured cells, abrogate the subsequent expression of spontaneous cytotoxicity toward RBC targets. The suppressor cell is radioresistant; requires 24 hr to suppress optimally; is inactivated by heating at 56°C for 15 min, and is enriched in the non-T interface after SRBC rosette depletion over a discontinuous Ficoll-Hypaque gradient. Furthermore, the addition of a cell-free sonicate of fresh lymphocytes is capable of inhibiting spontaneous cytotoxicity toward RBC targets. However, if mononuclear cells are allowed to incubate in tissue culture medium for 7 days they are no longer suppressive after sonication. These data suggest that fresh mononuclear cells exert a potent negative regulatory influence on monocyte killing. Our culture conditions by removing this negative influence have produced a new model of spontaneous nonspecific killing by monocytes.
Genetic Control of Immune Response to the H-2Dk Private Specificity, H-2.32
Authors: Shinohara Nobukata , Ricks John , Hansen Ted H. , Sachs David H.
Published: December 2022

A variety of congenic resistant strains of mice sharing the B10 background have been examined for humoral responsiveness to the H-2D region private specificity H-2.32. All of these strains were found to produce little or no anti-H-2.32 antibody, confirming previous indications that the B10 background is defective with respect to this response (1). However, both strain B10 (H-2b) and strain B10.D2 (H-2d) did produce reasonable titers of antibodies reactive with public H-2D region specificities of H-2k. In the case of B10.D2, this response could be shown to be due to a response to the D region product itself rather than a cross-reaction with public specificities shared in both K and D regions. By using sera to public and private H-2Dk specificities sequentially to precipitate radiolabeled solubilized H-2 antigen preparations, both private and public specificities were shown to be present on the same molecules. Thus, the failure of B10 background mice to respond to the private specificity H-2.32 appears to be peculiar to this specificity and not to all specificities on the H-2Dk molecule. Analysis of (AKR.M × B10.AKM)F1 × B10.AKM backcross mice revealed that the magnitude of the antibody response to H-2d antigens did not correlate with that of the anti-H-2.32 response, indicating the specific nature of the anti-H-2.32 low responsiveness of B10 congenic mice.