bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Staphylococcal Enterotoxins on the Primary in Vitro Immune Response
Authors: Smith Bennett G. , Johnson Howard M.
Published: December 2022

Staphylococcal enterotoxins, like several plant lectins, have been shown to stimulate mitogenic activity and lymphokine production in lymphocytes. The effect of enterotoxins A and B on the primary in vitro plaque-forming cell (PFC) response of mouse (C57BL/6) spleen cells to sheep red blood cells (SRBC) was examined. PFC responses in 4-or 5-day cultures were inhibited τ; 90% by 0.1 µg enterotoxin A and by 3- to 6-µg enterotoxin B when the toxins were added to the cultures either at the time of SRBC addition or 24 hr before. Both enterotoxins A and B were shown to be T lymphocyte mitogens. Kinetic data indicated that the enterotoxins (10 µg) inhibited the PFC response as early as day 3, but were relatively more inhibitory at days 4 and 5. The PFC response was inhibited when enterotoxins were added to cultures at the same time as antigen or a day later; it was enhanced when added at day 2; and it was unaffected when added at day 3 of 4-day cultures. Further, the presence of enterotoxin A during the first 24 hr of culture and subsequent removal was still as effective in inhibiting the PFC response as when it was present throughout the culture period. The PFC inhibitory properties of enterotoxins appear, then, to affect some early event(s) in the in vitro immune response.

α-Methyl-D-mannopyranoside blocked the PFC inhibitory effect of concanavalin A (con A), but it had no effect on enterotoxin A. The two mitogens appear, then, to react with different receptors on the lymphocytes or to differ in the dynamics of their reactivity.

The effects of staphylococcal enterotoxins on the PFC response of spleen cells were remarkably similar to those reported for lectins such as con A. Enterotoxins are structurally simpler than con A and should therefore be quite useful in studying various biologic activities of lymphocytes.
Ia Alloantigen(s) Detected on Thymocytes by Use of a Fluorescence-Activated Cell Sorter
Authors: Fathman C. G. , Cone J. L. , Sharrow S. O. , Tyrer H. , Sachs D. H.
Published: December 2022

With the increased sensitivity of detection of fluorescence made possible by a fluorescence-activated cell sorter (FACS), Ia antigen(s) have been demonstrated, although in very small amounts, on murine thymocytes by using an indirect immunofluorescent technique. Absorption studies indicate that the Ia antigens detected on thymocytes are also present on spleen B cells, however, they do not rule out the possibility that certain Ia antigens may exist only on B cells. These studies suggest that the surface distribution and/or the decreased density of Ia antigens on thymocytes relative to peripheral B cells may preclude their demonstration by more conventional assays. Although these studies demonstrate Ia antigens on less than 50% of thymocytes, the percentage may in fact be much higher since current methods do not allow an upper limit to be assigned.
Suppressor Cell Control of Unresponsiveness to Experimental Allergic Encephalomyelitis
Authors: Swierkosz James E. , Swanborg Robert H.
Published: December 2022

Lymph node cells (LNC) from Lewis rats rendered unresponsive to experimental allergic encephalomyelitis (EAE) by pretreatment with myelin basic protein markedly suppressed clinical (but not histologic) EAE in normal recipients later challenged with an encephalitogenic emulsion. Unresponsiveness was immunologically specific, and required viable LNC; serum transfer was ineffective. These findings suggest that suppressor cells exert control over this autoimmune disease.
Sera and the in Vitro Induction of Immune Responses
Authors: Shiigi Stanley M. , Mishell Robert I.
Published: December 2022

More than 200 samples of pooled fetal bovine sera (FBS) were tested to determine their capacity to support primary immune responses by cultured mouse spleen cells. Less than 10% of the FBS samples were fully supportive; the majority of the samples were moderately to very deficient. Several lines of evidence suggest that bacterial contamination during the processing of sera plays a major role in determining which sera are supportive. Serum samples known to have been temporarily contaminated during processing were strongly supportive. Samples of FBS which were likely never to have been contaminated were deficient. Bacteria from a specific lot of supportive serum converted a very deficient serum into one which was supportive. Several mechanisms by which bacteria could affect sera are discussed.
Intestinal Uptake of Macromolecules
Authors: Walker W. Allan , Wu Margaret , Isselbacher Kurt J. , Bloch Kurt J.
Published: December 2022

The mechanism(s) whereby immunization interferes with the intestinal absorption of macromolecules has been examined. After repeated intraperitoneal injection of protein antigens emulsified in incomplete Freund's adjuvant into rats, everted gut sacs from these animals and controls were exposed to radiolabeled antigen in vitro. There was a significant decrease in absorption of antigen by gut sacs from immunized rats as compared to control rats. On further analysis, it was found that, in contrast to controls, gut sacs from immunized rats showed an increased initial adsorption of labeled antigen. Furthermore, gut sacs from immunized rats showed enhanced breakdown of labeled antigen on prolonged incubation. Following incubation, gut sacs were rinsed and the amount of radioactivity eluted was determined. More radioactivity was present in the first rinse fluid of gut sacs from immunized rats than in rinse fluid from controls. On density gradient ultracentrifugation of the former fluids, radioactivity was localized in the middle or bottom of the gradient, whereas radioactivity in the latter fluids was located at the top of the gradient. The rapidly sedimenting labeled antigen was co-precipitated in the reaction of rabbit anti-rat IgG1 and IgG1 protein, suggesting the presence of antigen-IgG1 antibody complexes in the rinse fluid.

These findings suggest that in vitro, antigen becomes rapidly associated with antibodies present on the surface of the gut. Formation of antigen-antibody complexes seems to prevent binding of antigen to and subsequent pinocytosis by the intestinal epithelial cells. Antigen-antibody complexes retained in the mucous coat of the gut may be degraded by local proteases. The findings in this study suggest an additional role for antibodies present on mucosal surfaces, i.e., protection against absorption of ingested proteins which have escaped normal digestion in the intestinal lumen.
The Basis for Conflicting Results Obtained in Studies on the Plaque-Forming Cell Response to Type III Pneumococcal Polysaccharide
Authors: Baker Phillip J. , Prescott Benjamin
Published: December 2022

Treatment with several preparations of anti-lymphocyte or anti-thymocyte serum (ALS or ATS)1 has been shown to cause a significant increase (10- to 20-fold) in the magnitude of the plaque-forming cell (PFC) and serum antibody response to Type III pneumococcal polysaccharide (SSS-III) in different strains of mice (1–4); such enhancement is evident with respect to both PFC/106 nucleated spleen cells and PFC/spleen, indicating that it is not due simply to splenomegaly (3). These observations have been confirmed using preparations of ALS and SSS-III, made independently by different methods (5). In all of these studies, sheep erythrocytes, sensitized with purified SSS-III by the CrCl3 coupling method (6), were used as indicator cells for the detection of PFC making antibody specific for SSS-III. Despite these reproducible findings and their independent confirmation, some investigators have reported that treatment with ALS produces either no change, or a significant reduction in the number of PFC found after immunization with this antigen (7–9); here, sheep erythrocytes, sensitized with uncharacterized filtrates from broth cultures of pneumococci (10), were used as indicator cells in assays for PFC.
Immunochemical Properties of Streptococcal M Protein Purified by Isoelectric Focusing
Authors: Cunningham Madeleine , Beachey Edwin H.
Published: December 2022

Electrofucusing of an alkaline extract of type 24 streptococcal M protein yielded an antigenic fraction that was type specific and apparently homogeneous. The haptenic nature of this fraction was suggested by its inability to precipitate type-specific antiserum or to induce opsonic antibodies in rabbits, despite its ability to strongly inhibit opsonization of homologous-type streptococci by M antibody. The fraction migrated as a single band upon electrophoresis in sodium dodecyl sulfate (SDS) acrylamide gel. The mobility of the protein band was consistent with a molecular weight of 36,500 daltons. In some experiments using larger quantities of protein, a second faint protein band with an average molecular weight of 70,000 was observed, suggesting the presence of dimers of the 36,500-dalton protein. Amino acid analysis showed the predominant amino acid to be glycine followed by aspartic acid and glutamic acid.

Moreover, this M protein fraction was free of non-type-specific immunotoxic properties in guinea pigs and in man. Although apparently not immunogenic, this nontoxic fraction may provide a useful tool to study the relationship of the type-specific protective moiety to potentially harmful “impurities” in M protein vaccines.
Aleutian Disease of Mink: The Antibody Response of Sapphire and Pastel Mink to Aleutian Disease Virus
Authors: Bloom Marshall E. , Race Richard E. , Hadlow William J. , Chesebro Bruce
Published: December 2022

The specific antiviral antibody response of sapphire and pastel mink to Pullman strain of ADV has been examined. Sapphire mink inoculated with from 300,000-3 LD50 developed high levels of specific antibody and AD. Pastel mink inoculated with parallel doses of ADV also produced antibody but did not develop AD. The low incidence of AD in pastel mink inoculated with Pullman strain of ADV is probably related to factors other than antiviral antibody.
In Vitro Human Reactivity to Staphylococcal Phage Lysate
Authors: Dean J. H. , Silva J. S. , McCoy J. L. , Chan S. P. , Baker J. J. , Leonard C. , Herberman R. B.
Published: December 2022

The cell-mediated reactivity of normal individuals to staphylococcal phage lysate (SPL) were tested in vitro in the lymphocyte stimulation (LS) and leukocyte migration inhibition (LMI) assays. There were 95% positive responses in LS (stimulation ratio ≥ 3 with p < 0.01) and 67% positive responses in LMI (migration index ≤ 0.80). Enriched subpopulations of T and B lymphocytes were prepared with rosette formation and density gradient centrifugation. SPL stimulated lymphoproliferative responses in both T and B cell subpopulations whereas phytohemagglutinin (PHA) stimulated only the T cell subpopulation. Cord blood leukocytes were tested in the LS assay and 41% gave positive responses to SPL, 81% to PHA, and 17% with SLO. SPL appears to be a useful reagent for the in vitro study of cell-mediated reactivity, and may provide somewhat different information from that obtained with other mitogens or antigens.
Interaction of C3b, B, and D̄ in the Alternative Pathway of Complement Activation
Authors: Nicholson Anne , Brade Volker , Schorlemmer Hans-Ulrich , Burger Reinhard , Bitter-Suermann Dieter , Hadding Ulrich
Published: December 2022

The interaction of ZXd2, an insoluble intermediate of the alternative pathway on zymosan (Z5), with factor B and the enzyme D̄ proceeds in a two-step reaction: 1) B binds in the presence of Mg++ to ZXd2 to form the intermediate ZXd2B, 2) B bound to ZXd2 is subsequently activated enzymatically by D̄ to yield the complex ZXd2B̄ which cleaves C3. Evidence was obtained that C3b, which is present on ZXd2, is required for ZXd2B̄ formation. Studies of the functional role of C3b for ZXd2B̄ formation revealed that C3b is involved in the first reaction step, i.e., binding of B to ZXd2 to yield ZXd2B. Formation of ZXd2B is inhibited by pretreatment of ZXd2 with either anti-C3 Fab or with C3b-INA. Low ionic strength of about 2 mS was found to favor the interaction of the C3b with B. Mg++ concentrations from 1 to 31 mM as well as variation of pH in the range from 6.2 to 8.5 did not greatly influence the reaction of B with ZXd2. For the enzymatic activation of B only C3b on ZXd2 and factor D̄ are required. This is concluded from the finding that fluid phase C3b is sufficient for the activation of B in the presence of D̄. This does not exclude the fact that other proteins present on ZXd2 may help to stabilize the intermediate ZXd2B or the enzymatically active complex ZXd2B̄, or both of them.
Comparison of the Binding Affinities of Rabbit IgG Fractions to the Rabbit Fetal Yolk Sac Membrane: Use of 22Na to Facilitate Quantitation of 125I-IgG Binding
Authors: Tsay David D. , Schlamowitz Max
Published: December 2022

Rabbit IgG was purified, Fr-1-(G-200)2, and then separated by DEAE-cellulose column chromatography into three major fractions, Fr-I, -II, and -III. Binding affinities of the 125I-labeled IgG and its fractions to the rabbit fetal yolk sac membrane were studied. At a concentration of 2 mg/ml, rabbit IgG is bound to the extent of 9 µg/cm2 membrane, whereas the values for fractions Fr-I, -II, and -III are 13, 7, and 5 µg/cm2, respectively. The maximal amount of IgG bound appears to be approximately the same, i.e., 23 µg of IgG/cm2 membrane, for Fr-1-(G-200)2 and its three fractions. In contrast, bovine IgG does not bind to the membrane over the range of concentration tested. Binding constants for Fr-1-(G-200)2, fraction Fr-I, -II, and -III are estimated to be 5.4, 8.6, 4.0, and 2.0 × 104 M-1, respectively. The binding affinities of IgG fractions to the yolk sac membrane correlate with the chemical and physicochemical properties of the fractions.

Also detailed in the text is the use of 22Na to facilitate quantitation of specific binding of the 125I-IgG to the membrane under equilibrium conditions.
IgM Antibody-Dependent Cell-Mediated Cytotoxicity in the Moloney Sarcoma Virus System: the Involvement of T and B Lymphocytes as Effector Cells
Authors: Lamon E. W. , Whitten H. D. , Skurzak H. M. , Andersson B. , Lidin B.
Published: December 2022

Antibody-dependent cell-mediated cytotoxicity in the Moloney sarcoma virus (MSV) system was analyzed with respect to the subpopulations of effector cells involved in tumor target cell destruction when IgM was used as the sensitizing antibody. With unfractionated aera from animals that had undergone regression of primary MSV tumors it was found that macrophages did not contribute to the cytotoxicity induced by normal spleen cells that were syngeneic to the target cells. The IgM fraction of MSV regressor sera was found to induce cytotoxicity against the target cells by immunoadsorbent column-fractionated normal spleen cells, which were either depleted of T cells or B cells, according to the specificity of the columns. Immune IgM was also found to potentiate the activity of MSV regressor spleen cells that had been similarly fractionated. Furthermore, IgM antibody was found to induce cytotoxicity by normal spleen cells which had been depleted of either T or B cells by the appropriate antiserum (anti-T or anti-Ig) in the presence of complement and subsequent recovery of the viable cells by trypsinization, filtration, and washing. However, spleen cells treated with both anti-T and anti-Ig sera simultaneously in the presence of complement and subsequent recovery of viable cells, were not induced to be cytotoxic against the IgM-coated tumor target cells. Further support of the finding that IgM antibody could induce cytotoxicity by T cells was provided by an experiment showing the induction with IgM of cytotoxicity against the target cells by normal thymocytes.
The Interdomain Disulfide Bond of a Homogeneous Rabbit Pneumococcal Antibody Light Chain
Authors: Strosberg A. Donny , Margolies Michael N. , Haber Edgar
Published: December 2022

Rabbit light chain 3315, prepared from a homogeneous antipneumococcal antibody, was subjected to hydrolysis by pepsin without prior reduction and alkylation of the intrachain disulfide bonds. Gel filtration of the hydrolysate on Sephadex G-10, G-15, and G-25 and ion exchange chromatography on SP-Sephadex yielded several disulfide bridge peptides. These were fully reduced and alkylated and sequenced by Edman degradation. The peptides were located in the light chain sequence determined in independent studies from our laboratory.

The half-cystine residues in this κB rabbit chain are located at positions 23, 80, 88, 134, 171, 194, and 214. The extra disulfide bridge extends between residues 80 and 171, thus joining the variable and constant domains. This is consistent with x-ray diffraction crystallographic studies showing that the corresponding residues in human light chains are separated by a distance compatible with disulfide bond formation.
The Effect of Alloantisera on Antigen-Induced T Cell Proliferation
Authors: Ruhl HartmÜt , Shevach Ethan M.
Published: December 2022

In the guinea pig, alloantisera raised by cross-immunization of strain 2 and strain 13 animals are capable of specifically inhibiting the in vitro proliferative response of (2 × 13)F1 T lymphocytes to those antigens the response to which is controlled by Ir genes linked to the genes controlling the alloantigens against which the serum is directed. However, in similar studies performed in the two parental strains, the responses to antigens not known to be under unigenic control were also markedly inhibited by the appropriate alloantisera. We have extended our studies of this “nonspecific” inhibitory effect of alloantisera on T cell proliferation and have demonstrated that the proliferative response of strain 2 and strain 13 T cells to a large number of antigens is markedly inhibited by anti-2 and anti-13 sera, respectively. Antisera to the B alloantigen, the product of a linked but distinct histocompatibility locus, which is present in both strain 2 and strain 13 animals, also produced a marked inhibition of T-lymphocyte proliferation. A number of possible explanations for the generalized inhibitory effect of alloantisera on T cell proliferation are discussed.
Response of Mouse Splenic Lymphocytes to Timothy Pollen Antigens in a Microculture System
Authors: Fairchild Sally S. , Malley Arthur
Published: December 2022

Spleen cells from LAF1 mice were stimulated in a microculture system with T and B cell mitogens or antigens of timothy pollen. Only cells from mice immunized with crude timothy pollen extract (WST) or a major antigen of timothy pollen conjugated to Ascaris (antigen B-Ascaris) responded to timothy antigens in vitro. Optimum responses were obtained at 120 to 144 hr of culture with 5 to 10 μg WST per culture and ranged from three to 10 times greater than cell background. No correlations could be found between the optimum antigen concentration or the maximum response and the immune status of the spleen cell donor. Response could be inhibited by a dialyzáble fraction of timothy pollen, antigen D, which is a monovalent form of a major antigen of timothy pollen.
Histamine Release from Human Leukocytes: Relationships Between Cyclic Nucleotide, Calcium, and Antigen Concentrations
Authors: Gillespie Elizabeth , Lichtenstein Lawrence M.
Published: December 2022

The antigen-induced IgE-mediated release of histamine from human basophils has previously been shown to require calcium, to be inhibited by agents which raise cyclic AMP levels and by high antigen levels, and to be unaffected by cyclic GMP. The interrelationship between these phenomena has been studied. The major findings are: 1) in the region of antigen-excess inhibition dibutyryl cyclic AMP potentiates release; 2) antigen-excess inhibition is seen at lower antigen concentrations when the calcium concentration is reduced from 0.6 to 0.1 mM; and 3) cyclic GMP modestly potentiates release when the calcium concentration is 0.1 mM.
Ionophore A-23187 Induced Histamine Release from Rat Mast Cells and Rat Basophil Leukemia (RBL-1) Cells
Authors: Siraganian Reuben P. , Kulczycki Anthony , Mendoza Guillermo , Metzger Henry
Published: December 2022

Ionophore A-23187 releases histamine from normal mast cells apparently by promoting Ca++ influx (Foreman et al, Nature 245: 249, 1973). In our hands at concentrations of τ; 0.2 μg/ml release occurs in 1 to 2 min, is blocked by metabolic inhibitors, and is unaccompanied by cytotoxicity (trypan-blue uptake, lactic dehydrogenase (LDH) release). At higher doses (0.5 μg/ml) histamine release is followed by significant cytotoxicity, but again Ca++ is required. In parallel studies, we examined cultured rat basophilic leukemia (RBL-1) cells. These cells, which apparently have normal surface receptors for IgE, contained ∼700 ng histamine/106 cells but did not release histamine when IgE-mediated release was looked for. They do not respond to doses of ionophore which would be expected to give noncytotoxic histamine release. At higher doses histamine release is preceded by progressive LDH release: LDH release is 75% complete at 5 min whereas 10 min are required for 75% maximal histamine release. This reaction requires Ca++ and is temperature dependent but is not inhibited by metabolic poisons (2-deoxyglucose, dinitrophenol, CN-). These studies suggest that either Ca++ does not enter into these cells normally or that one or more mechanisms which are ordinarily triggered by the changes in Ca++ flow are unresponsive in the RBL-1 cells. These studies also underline the importance of ruling out cytotoxicity in ionophore-induced phenomena.
Immune and Antibody Responses to an Isolated Capsid Protein of Foot-and-Mouth Disease Virus
Authors: Bachrach Howard L. , Moore Douglas M. , McKercher Peter D. , Polatnick Jerome
Published: December 2022

The purified capsid proteins VP1, VP2, and VP3 of foot-and-mouth disease virus type A12 strain 119 emulsified with incomplete Freund's adjuvant were studied in swine and guinea pigs. Swine inoculated on days 0, 28, and 60 with 100-μg doses of VP3 were protected by day 82 against exposure to infected swine. Serums from animals inoculated with VP3 contained viral precipitating and neutralizing antibodies, but such serums recognized fewer viral antigenic determinants than did antiviral serums. Capsid proteins VP1 and VP2 did not produce detectable antiviral antibody in guinea pigs, and antiviral antibody responses in swine to a mixture of VP1, VP2, and VP3 were lower than the responses to VP3 alone. However, when swine were inoculated with VP1, VP2, and VP3 separately at different body sites, no interference with the response to VP3 was observed. Vaccine containing VP3 isolated from acetylethylenimine-treated virus appeared less protective for swine than vaccine containing VP3 from nontreated virus. Trypsinized virus, which contains the cleaved peptides VP3a and VP3b rather than intact VP3, produced approximately the same levels of antiviral antibody responses in guinea pigs as did virus. Conversely, an isolated mixture of VP3a and VP3b did not produce detectable antiviral antibody responses in guinea pigs. The VP3a-VP3b mixture did, however, sensitize guinea pigs to elicit such responses following reinoculation with a marginally effective dose of trypsinized virus.
Chemotaxis Under Agarose: A New and Simple Method for Measuring Chemotaxis and Spontaneous Migration of Human Polymorphonuclear Leukocytes and Monocytes
Authors: Nelson Robert D. , Quie Paul G. , Simmons Richard L.
Published: December 2022

A variety of methods have been devised for the study of spontaneous and directed cell migration. Among these, the membrane filter method introduced by Boyden in 1962, with its more recent modifications, has become the technique of choice for studies of leukocyte migration in vitro. This method, however, cannot be applied without alteration to studies of chemotaxis and spontaneous migration of cells of different types. We describe in this report a new and simple method for studying human leukocyte chemotaxis, in vitro, which is based upon migration of cells under agarose gel. This method has application to both polymorphonuclear leukocytes and monocytes, permits measurement of both chemotaxis and spontaneous migration, requires fewer cells per test, and is rapid, simple, reproducible, and inexpensive to set up.
Cyclic GMP and Lectin-Induced Lymphocyte Activation
Authors: Wedner H. James , Dankner Rand , Parker Charles W.
Published: December 2022

Purified human peripheral lymphocytes incubated with the mitogenic plant lectins phytohemagglutinin and concanavalin A were examined for alterations in intracellular cGMP and cAMP under a variety of experimental conditions and using multiple techniques for the isolation and purification of cGMP and cAMP before assay of the cyclic nucleotides by radioimmunoassay. In contrast to work reported by others, we have been unable to demonstrate consistent increases in cGMP under any of the experimental conditions used and with any of the various purification schemes. In these same experiments exogenous cGMP added to the lymphocytes could be measured, and the immunoreactive material was destroyed by cyclic nucleotide phosphodiesterase, indicating that our inability to measure increases in cGMP was not caused by our inability to measure cGMP.

Under identical experimental conditions, small but consistent and statistically significant increases in cAMP were noted. In addition, other parameters of lymphocyte activation, 45Ca uptake (an early parameter), and incorporation of 3H thymidine into DNA were unimpaired. These data call to question the concept of cGMP as the second messenger in lectin-stimulated human peripheral lymphocytes.
Enumerating Immunoglobulin-Secreting Cells Among Peripheral Human Lymphocytes. A Hemolytic Plaque Assay for A B Cell Function
Authors: Eby William C. , Chong Christine A. , Dray Sheldon , Molinaro Giuseppe A.
Published: December 2022

Cells bearing immunoglobulin (Ig) can be enumerated by current methods, but cells secreting Ig (with unknown antibody specificity) can not. By using a reverse plaque-forming cell assay, we counted the Ig-secreting cells in the blood of nonimmunized individuals. Isolated human lymphocytes were plated in gels together with erythrocytes coated with purified antibody to human Ig. After treatment with anti-Ig antibody and complement, the Ig-secreting cells formed hemolytic plaques. In 10 normal individuals, 1.2 ± 0.4% of the plated mononuclear cells formed plaques; in an individual tested on 7 different days during 3 months, 1.1 ± 0.2% of the cells formed plaques. From the number of mononuclear cells in the blood, we calculated 22 ± 8 Ig-secreting cells per mm3 of blood. Enumerating Ig-secreting cells is a new direct assay for a B cell function and need not correlate with quantitating serum Ig. Thus, this assay should be useful for studying Ig-secretion in normal and altered states.
Detection of Immune Complexes in Unheated Sera by a Modified 125I-C1q Binding Test
Authors: Zubler Rudolf H. , Lange Ghislaine , Lambert Paul H. , Miescher Peter A.
Published: December 2022

The 125I-C1q binding test was modified in order to allow for the detection of immune complexes in native unheated human serum. Indeed, heat-inactivation (56°, 30 min) was found to reduce the C1q-binding activity of immune complexes mixed with native serum. This effect was not observed when EDTA was added to the native serum before mixing with immune complexes. The modified 125I-C1q binding test was performed in two steps: first, the tested native serum sample was incubated for 30 min at 37°C with 0.13 M EDTA in order to prevent the integration of 125I-C1q into the intrinsic C1qrs complex, second, 125I-C1q and polyethylene glycol (final concentration 2.5%) were added to this mixture, and further incubated for 1 hr at 4°C. Under these conditions, free C1q remained soluble whereas C1q bound to macromolecular complexes was precipitated. The competitive effect of intrinsic C1q and the interference of other substances such as DNA or bacterial LPS were very limited. The modified C1q binding test was applied to the clinical investigation of 44 patients with systemic lupus erythematosus; and increased C1q binding activity (C1q-BA) was observed in 91% of the samples. The level of C1q-BA was found to be significantly correlated to the DNA-binding capacity and to the decrease of the level of some complement components.
Mechanism of Immunologic Resistance to Herpes Simplex Virus 1 (HSV-1) Infection
Authors: Rager-Zisman Bracha , Allison Anthony C.
Published: December 2022

Susceptibility of adult mice to i.p. infection with HSV-1 was greatly increased by administration of a single dose of cyclophosphamide. Mortality of cyclophosphamide-treated virus-infected mice was associated with increased virus replication and pathologic changes in brain and liver. The development of a fatal infection in immunosuppressed mice could be curtailed after transfer of specifically immune spleen cells. Passively transferred antibody had no such effect. Protective activity of spleen cells was significantly reduced after pretreatment with anti-θ serum. Significant protection was also achieved when normal spleen cells plus immune serum were administered simultaneously. Our results indicate that protection against this virus infection is predominantly T cell dependent, and suggests that antibody-dependent cell-mediated protection may also be operative in vivo.
Immunoglobulin Biosynthesis by the MOPC 173 Mouse Myeloma Tumor and a Variant Spleen Clone
Authors: Baumal Reuben , Scharff Matthew D.
Published: December 2022

The IgG2a producing MOPC 173 tumor synthesizes heavy (H) and light (L) chains and assembles them into H2L2 utilizing heavy chain dimers (H2) and H2L as the major precursors. Although the tumor cells secrete only H2L2, they synthesize excess L chains which appear to be degraded and are not secreted. A L chain-producing variant arising spontaneously from the MOPC 173 tumor also failed to secrete any L chains. MOPC 173 tumor cells were cloned in the spleens of normal BALB/c mice and 1 of 23 spleen clones appeared to differ from the parent tumor in demonstrating a) a transient block in the assembly of H2L2 at the H2 level, leading to delayed formation and secretion of H2L2, b) appreciable amounts of non-covalently bonded H2L, c) an abnormal intracellular immunoglobulin species, H4, d) production of equimolar amounts of H and L chains. The patterns of immunoglobulin synthesis and assembly demonstrable in the above studies were also observed when tumor cells were studied in site. Tryptic peptide mapping of the H and L chains produced by the MOPC 173 tumor and the variant spleen clone failed to demonstrate any differences in the H chains, but there were definite chemical differences in the L chains. These studies indicate that variant myeloma cells producing structurally altered immunoglobulins may continually be arising in myeloma tumors.
Complement Activation in Semi-Solid Medium: Insolubilization of Properdin and the Third Component of Complement (C3) in Agar Gels
Authors: Ziegler John B. , Watson Lillian , Goodkofsky Ira , Alper Chester A. , Lepow Irwin H.
Published: December 2022

Although the role of properdin in the alternative pathway of complement activation remains unclear, evidence has recently been obtained for the formation of complexes between properdin and other components, including C3. In this study such complexes have apparently been directly visualized. When normal human serum and properdin were allowed to diffuse toward each other in agar gel for 16 hr, a line of precipitation could be seen when stained with Coomassie brilliant blue. The reaction occurred at pH 8.6 in 0.05 M Veronal buffer at room temperature but not under physiologic conditions of pH or tonicity. Like the alternative pathway, the reaction was Me++ dependent, occurred with C2- or C5-deficient or hypogammaglobulinemic serum, and did not occur with aged, 52°C-inactivated, C3b inactivator-deficient, or C3-deficient serum. 125I-labeled C3 and properdin but not Factor B were incorporated in the precipitate. Eleven sera containing the C3 nephritic factor failed to produce a precipitate with properdin, but a line of precipitation occurred between seven of these sera and normal serum. This line showed identity with the line occurring between properdin and normal serum. The phenomenon appears to result from formation of insoluble complexes between proteins of the alternative pathway and agar.
In Vitro Studies of the Genetically Determined Unresponsiveness to Thymus-Independent Antigens in CBA/N Mice
Authors: Cohen Philip L. , Scher Irwin , Mosier Donald E.
Published: December 2022

The X-chromosome-linked B lymphocyte defect of CBA/N mice has been studied in vitro by comparing the ability of (CBA/N × DBA/2)F1 (X-/X- × X+/Y) male (X-/Y) and female (X-/X+) spleen cells to respond to the thymus-independent antigen DNP (or TNP)-AECM-Ficoll. (CBA/N × DBA/2)F1 male spleen cells failed to generate significant in vitro anti-TNP antibody responses to DNP- or TNP-AECM-Ficoll, in contrast to spleen cells from F1 female (X-/X+) mice which responded normally to these T-independent antigens. Spleen cells from male F1 mice responded almost as well as F1 female cells to the thymus-dependent antigen, TNP-sheep red blood cells (TNP-SRBC) in vitro. Adding F1 male cells to F1 female cells failed to reduce the response of the latter to DNP-AECM-Ficoll, suggesting that the inability of F1 male cells to respond was not due to active suppression. The response of F1 male spleen cells to TNP-SRBC was not impaired by adding high concentrations of TNP-AECM-Ficoll indicating that the mechanism of unresponsiveness was not tolerance induction in all TNP-specific precursors. Lymphocytes from F1 male mice were capable of forming anti-TNP antibody after stimulation with lipopolysaccharide (LPS) in high concentrations; DNP-AECM-Ficoll had no effect no this polyclonal response.

B lymphocytes from mice bearing only the X-chromosome of the CBA/N strain thus display a profound defect in B cell activation. This functional defect may represent either an inability of the defective B cells to be activated by thymus-independent antigens or the absence of a sub-class of B cells which respond to thymus-independent antigens.
Immunosuppressive Factor(s) Extracted from Lymphoid Cells of Nonresponder Mice Primed with L-Glutamic Acid60-L-Alanine30-L-Tyrosine10 (GAT)
Authors: Kapp Judith A. , Pierce Carl W. , De La Croix Fern , Benacerraf Baruj
Published: December 2022

The synthetic terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) not only fails to elicit a GAT-specific antibody response in nonresponder mice, but also prior injection of GAT specifically decreases the ability of nonresponder mice to develop a GAT-specific antibody response to a subsequent challenge with GAT-MBSA. This inhibition is mediated by GAT-specific suppressor T cells. Further, a suppressive factor can be extracted from lymphoid cells of GAT-primed nonresponder mice that inhibits the development of primary GAT-specific antibody responses to GAT-MBSA and to GAT-PRBC by normal syngeneic mice. The suppressive activity is dose-dependent and absorbed by GAT-Sepharose, but not by BSA-Sepharose. The suppressive activity elutes from a G-100 Sephadex column in the same fraction as ovalbumin, suggesting its m.w. is approximately 45,000 daltons.
In Vitro Response of Subpopulations of Human Lymphocytes
Authors: Gausset Philippe , Delespesse Guy , Hubert Claude , Kennes Bernard , Govaerts André
Published: December 2022

A significant and constant increase in DNA synthesis was observed in human lymphocytes cultured in the presence of purified anti-immunoglobulin antibodies specific for human IgG, IgA, and IgM. This has been found in cultures of lymphocytes isolated from blood, tonsils, spleen, and lymph nodes. The optimal culture conditions for blood and tonsil lymphocytes were determined. As a rule 6-day cultures containing 2 × 106 cells/ml and 100 µg/ml of antibody yielded the highest 3H-thymidine uptake.

Purified T cell cultures could not be stimulated, whereas a low response could be observed in most of the purified B cell cultures. Optimal culture conditions were the same for the B and total tonsil lymphocytes. However, when the purified B cells were totally depleted of T cells, no response was observed. A T and B cell synergy has been demonstrated by supplementing B cell cultures with purified T cells, whether treated or not with mitomycin. These experiments indicated a permissive and potentiating effect of T cells on the B cell response. Cultures containing mitomycin-treated B cells and purified T cells (mB + T) could be stimulated by a-Ig, thus indicating a T cell proliferation. In keeping with this finding was the observation of an increased response of total lymphocytes supplemented with T cells but not with B cells. Adherent cells are necessary for an optimal response to a-Ig; they enhanced the B cell proliferation observed in (Tm + B) cultures and suppressed the response of T cells in (T + Bm) cultures.
Properties of Antigen-Specific Suppressive T Cell Factor in the Regulation of Antibody Response of the Mouse
Authors: Taniguchi Masaru , Hayakawa Kyoko , Tada Tomio
Published: December 2022

An antigen-specific suppressive T cell factor, which was extracted from carrier-primed T cells, was further characterized in an in vitro secondary antibody response. The factor was capable of suppressing secondary IgG antibody response of primed spleen cells when it was added to the culture together with relevant antigen. The suppressive T cell factor was not released from primed T cells by a short-term culture with antigen, but was kept bound to the membrane of the residual cultured cells, only the physical disruption of which can release the T cell factor. The target of the suppressive T cell factor was determined as being the helper T cell, since the factor did not exert any effect in the absence of the helper T cell with identical specificity to that of the factor. The suppressive activity was completely absorbed with alloantisera specific for products of the I region of H-2 complex, although various anti-immunoglobulin antisera failed to do so. Close analysis of the specificity of alloantisera capable of absorbing the suppressor molecule indicated that the suppressive T cell factor may, in fact, be an I region gene product probably coded for by genes in I-A and/or I-B (including I-E) subregions.
Evidence for More Than One Ia Antigenic Specificity on Molecules Determined by the I-A Subregion of the Mouse Major Histocompatibility Complex
Authors: Cullen Susan E. , David Chella S. , Cone James L. , Sachs David H.
Published: December 2022

Ia antigenic specificities determined by the I-A subregion of the mouse major histocompatibility complex have been examined in strain B10.D2 (H-2d), C57BL/10 (H-2b), and in a (C57BL/6xDBA/2) hybrid (BDF1; H-2b/d). Detergent solubilized, 3H-leucine-labeled antigen preparations were mixed with appropriate alloantisera and precipitation was induced either by addition of goat anti-mouse γ-globulin or by addition of protein A-bearing Staphylococci. Sequential precipitation analysis showed that in strain B10.D2, Ia specificities 8 and 11 were co-precipitable, and that in strain C57BL/10, Ia specificities 8 and 9 were co-precipitable. In contrast, precipitation of specificities 9 and 11 from a BDF1 antigen preparation showed that these two Ia specificities were on separate molecules. The genetic implications of these data are discussed.
Artifactual Plaque Formation in Vitro and in Vivo Due to Passive Transfer of Specific Antibody
Authors: Muchmore A. V. , Koski I. , Dooley N. , Blaese R. M.
Published: December 2022

The localized hemolysis in gel (LHG) assay for antibody-secreting cells was used to evaluate antibody production in heterologous erythrocyte-immunized cultures of human peripheral blood lymphocytes supplemented with 10% human plasma. Antigen-specific plaques were detected in such cultures and the number of plaques varied with different lymphocyte donors and were inhibited by heat killing the cultures before assay. However, plaque number varied directly with the number of immunizing erythrocytes added to the cultures and inversely with the number of lymphocytes added. Maximum plaque production occurred within 10 min of initiation of the cultures and occurred in the absence of any lymphoid cells. Absorption of the plasma used as culture supplement with the immunizing erythrocyte resulted in complete abrogation of subsequent specific plaque formation. Mice previously passively immunized with high titered anti-erythrocyte antibody had large numbers of plaques detected 30 min after i.v. immunization with the appropriate erythrocyte. These plaques detected by LHG are the result of carry-over of aggregates of antibody-coated erythrocytes and subsequent release of this antibody in the LHG assay and are not the result of active antibody synthesis. This “pseudoplaque” production may lead to the false interpretation of plaque formation as indicating active synthesis of antibody in vivo and in vitro where such experiments are carried out in immune animals or with antibody containing serum culture supplements.
Cell-Mediated Immune Response in Vitro
Authors: Hirano Toshio , Nordin Albert A.
Published: December 2022

During the in vitro development of cytotoxic lymphocytes (CL), suppressor cells also develop. Spleen cells or lymph node cells harvested from mixed lymphocyte cultures (MLC) on day 2 (day-2 MLC) and added to a fresh MLC suppressed the development of CL. This suppressive effect was sensitive to treatment with anti-θ and C. The suppressive effect of day-2 MLC was not due to cytotoxic effects nor to altered kinetics of the development of CL. Thymidine hot pulse experiments showed that cell proliferation is necessary for the development of both suppressor cells and CL. Although CL develop from hydrocortisone-treated spleen cells, day-2 MLC of hydrocortisone-treated spleen cells did not suppress the development of CL. These studies suggest that suppressor cells and CL are derived from different T cell subpopulations.
Release of Xenotropic Type C RNA Virus in Response to Lipopolysaccharide: Activity of Lipid-A Portion upon B Lymphocytes
Authors: Phillips S. Michael , Stephenson John R. , Greenberger Joel S. , Lane Particia E. , Aaronson Stuart A.
Published: December 2022

The present studies demonstrate that lipopolysaccharide (LPS) causes the release of endogenous xenotropic type-C RNA virus from BALB/c spleen cells. The evidence suggests that virus release is stimulated by the lipid-A portion of LPS and primarily involves an action of LPS on B lymphocytes. LPS had little or no effect on virus release by T lymphocytes, macrophages, or fibroblasts. These results indicate that the differentiated state of the cell plays an important role in the regulation of endogenous virus release.
Cell Membrane IgD: Demonstration of IgD on Human Lymphocytes by Enzyme-Catalyzed Iodination and Comparison with Cell Surface Ig of Mouse, Guinea Pig, and Rabbit
Authors: Finkelman Fred D. , van Boxel John A. , Asofsky Richard , Paul William E.
Published: December 2022

A substantial fraction of human cord blood and peripheral blood lymphocytes have recently been shown to bear IgD. Although IgD has not been identified in mice, it has been suggested that it is also a major surface immunoglobulin of murine lymphocytes. Thus, lactoperoxidase-catalyzed iodination of surface immunoglobulin of mouse spleen and lymph node cells reveals the existence of an Ig H chain differing from µ, γ, and α-chain both antigenically and by mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This new H chain class has been previously proposed to be the mouse homologue of δ-chain.

In this paper, we analyzed human, mouse, guinea pig, and rabbit lymphoid cell membrane Ig by lactoperoxidase-catalyzed iodination, extraction with non-ionic detergent, precipitation with a variety of specific anti-Ig sera, and electrophoresis of dissolved reduced precipitates on sodium dodecyl sulfate-polyacrylamide gels. Our studies confirm the previous reports of a new mouse cell membrane H chain with a mobility more rapid than that of µ-chain. However, we fail to detect a molecule with this electrophoretic mobility on the surface of guinea pig or rabbit lymph node and spleen cells. Moreover, neither anti-κ nor anti-δ antibody precipitates a molecule with an H chain of this mobility from labeled extracts of human cord blood or peripheral blood lymphocytes. Cell surface δ was identified on both human cord blood and peripheral blood lymphocytes, but it proved to have mobility similar to human and mouse µ-chain. This result indicates either that mouse δ-chain has an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels which differs appreciably from that of human membrane δ-chain or that the newly described mouse H chain is not the homologue of human δ-chain.
Reply to the Letter of Dr. G. W. Warr
Authors: Baker Phillip J.
Published: December 2022

I wish to make two points in reply to the letter of Dr. G. W. Warr. First, the work of Warr et al. (1) hardly provides support for the view that ALS-induced enhancement of the antibody response to Type III pneumococcal polysaccharide (SSS-III) is demonstrable by using indicator erythrocytes sensitized with filtrates from cultures of pneumococci. In this paper, data are presented to indicate that treatment with ALS results in about a 3-fold increase in number of PFC detected. However, this is also accompanied by about a 3-fold increase in spleen weight and the spleen cell count. This is not ALS-induced enhancement, as described in our previous studies (reviewed in 2); rather, the increased numbers of PFC found, which may not be specific for SSS-III in the first place, is due entirely to splenomegaly. Under such circumstances, the failure of thymocytes to reverse this effect only serves to show that thymocytes are unable to abrogate this splenomegaly.
Separation of Effector Cells Mediating Antibody-Dependent Cellular Cytotoxicity (ADC) to Erythrocyte Targets from Those Mediating ADC to Tumor Targets
Authors: Pollack Sylvia B. , Nelson Karen , Grausz J. D.
Published: December 2022

Murine spleen cells mediate antibody-dependent cellular cytotoxicity (ADC) both to erythrocyte targets in a 51Cr release assay and to syngeneic tumor targets in a micro-cytotoxicity assay. The effector cells active in the two ADC assays can be separated by passage of the spleen cells through columns of Sephadex G-10 at 37°C. Cells mediating ADC to sarcoma cells did not adhere to the G-10 and were recovered in the column effluent. These nonadherent cells were not cytotoxic to antibody-coated chicken red blood cells. Spleen cells which mediated ADC in a 51Cr release assay to the red cell targets adhered to G-10. Adherent effector cells could subsequently be recovered from the columns by elution with 5 × 10-4 M EDTA.
Genetic Control of the Immune Response to Nuclease
Authors: Fathman C. Garrison , Sachs David H.
Published: December 2022

The humoral response of mice to staphylococcal nuclease has previously been shown to be controlled genetically by H-2-linked Ir gene(s). In order to examine the possible contributions of variable region immunoglobulin genes to this genetic control, we have developed a system for the detection of idiotypic determinants on anti-nuclease immunoglobulin molecules. Antisera to nuclease were raised in two high responder strains, A/J and SJL. The corresponding antibodies were purified by affinity chromotography on Sepharose-nuclease columns, and were used to immunize groups of Lewis rats. An assay system was developed to assess the inhibition of antibody-mediated inactivation of nuclease activity by the rat antisera thus produced. Despite the presence of many species-specific anti-mouse immunoglobulin antibodies in these sera, inhibition of antibody-mediated enzyme inactivation was found to be specific for anti-nuclease antibodies of the immunizing strain. The inhibition could not be removed by extensive absorption with normal serum proteins from the antibody-producing strain, and was shown to require antibodies directed toward binding sites of the anti-nuclease antibodies. This inhibition thus defines idiotypic determinants of anti-nuclease antibodies.