bluuebunny/crossref_metadata_2025 huggingface dataset:
C1s-induced vascular permeability in C2-deficient guinea pigs.
Authors: Strang C J , Auerbach H S , Rosen F S
Published: December 2022

Normal guinea pigs that have been intradermally injected with C1s exhibit increased vascular permeability at the injection site. Guinea pigs that are genetically deficient in complement component C2 do not exhibit increased vascular permeability when given a similar injection. The C2-deficient guinea pigs respond normally to injections of bradykinin and kallikrein, suggesting that these animals can respond to kinins and have a normal kininogen pathway. When the C2-deficient guinea pigs are given guinea pig C2 before C1s injection, increased vascular permeability is observed. These results demonstrate a definite requirement for complement component C2 in the generation of C1s-induced vascular permeability.
Analysis of neoplasms induced by Cas-Br-M MuLV tumor extracts.
Authors: Holmes K L , Langdon W Y , Fredrickson T N , Coffman R L , Hoffman P M , Hartley J W , Morse H C
Published: December 2022

Cas-Br-M is an ecotropic murine leukemia virus isolated from wild mice that induces a wide spectrum of hematopoietic neoplasms, including T and B cell lymphomas, myelogenous leukemias, and erythroleukemias. The purpose of this study was to determine if the induction of neoplasms belonging to multiple lineages was due to the ecotropic virus itself or to the generation of cell lineage-specific recombinant viruses. The results demonstrate that in some instances (two of 12 tumor extracts tested), recombinant viruses can be recovered from primary Cas-Br-M-induced tumors that will induce lymphomas of single lineages in mice inoculated as newborns. One of these viruses is a recombinant mink cell focus-forming virus that induces T cell lymphomas, and the other is a replication-defective, fibroblast-transforming virus that induces early B lineage lymphomas in mice. Histologic and flow microfluorometric cell surface antigen analyses of primary and in vitro adapted tumors are presented in support of a modified scheme of hematopoietic cell development.
Do sugar residues contribute to the antigenic determinants responsible for protection and/or abolition of protection in Leishmania-infected BALB/c mice?
Authors: Gorczynski R M
Published: December 2022

Intravenous inoculation of irradiated virulent promastigotes of Leishmania mexicana, or intradermal inoculation of avirulent temperature-sensitive clones of the same parasite, can protect BALB/c mice from progressive disease when challenged with parental organisms. However, if animals are prechallenged s.c. with irradiated parental parasites before (or shortly after, i.e., within 10 days) any other immunization regime is used, the s.c. challenge effectively suppresses development of protective immunity. The role of N-linked sugars on promastigotes in providing the determinants responsible for protection and/or suppression of protection in these assays has been examined. Growth of parasite in 2 micrograms/ml tunicamycin has no effect on incorporation of [3H]leucine but decreases incorporation of [3H]mannose by some 40 to 50%. Such tunicamycin-treated avirulent clones or irradiated virulent organisms are now unable to induce a protective response. However, tunicamycin-treated parental parasites given s.c. could still suppress the protection seen when irradiated parasites were given i.v. as immunogen. Tunicamycin-treated avirulent organisms given s.c. subcutaneously unlike the untreated avirulent clones, now also caused suppression of protection. These data suggest that the determinants responsible for development of protective immunity to L. mexicana in BALB/c mice are dependent on N-linked glycoproteins for their expression, unlike the determinants responsible for suppression of that protection. By using swainsonine as an alternative inhibitor of N-linked glycoprotein synthesis, the data additionally suggest that no complex processing of N-linked sugars takes place to reveal the immunogenic determinants in this system.
B lymphocytes are required for the generation of T cells that mediate healing of cutaneous leishmaniasis.
Authors: Scott P , Natovitz P , Sher A
Published: December 2022

The role that B lymphocytes and/or antibodies play in the healing of Leishmania major infections in genetically resistant C3H/HeN mice was investigated by monitoring the course of infection in animals that had been B cell depleted by treatment from birth with anti-IgM sera (mu-suppressed). L. major infection of mu-suppressed C3H/HeN mice produced lesions that were significantly larger than those induced in control animals, and failed to heal. Moreover, vaccinated mu-suppressed mice also developed chronic nonhealing infections, although their lesions were initially smaller than those developed by nonvaccinated mu-suppressed controls. The enhanced susceptibility of mu-suppressed mice could be completely overcome by adoptive transfer of T lymphocytes from mice that had spontaneously healed their lesions, and to a lesser extent by T lymphocytes from normal animals. Anti-leishmanial antibody responses were completely absent in mu-suppressed mice, regardless of whether they were lymphocyte reconstituted, whereas delayed type hypersensitivity (DTH) to leishmanial antigens was present in normal and mu-suppressed animals. The ability of immune T cells to protect mu-suppressed mice without restoring humoral responsiveness clearly indicates that antibodies are not necessary for healing leishmanial infections. Instead, the observed effect of mu-suppression argues that B lymphocytes are required for the generation of an effector T cell population, apparently unrelated to DTH, which mediates the healing of cutaneous lesions. These results thus provide the first evidence for the B cell and/or Ig dependency of a T cell population that is critical for the development of immunity against a microbial agent.
The murine gamma-chain of the T cell receptor is closely linked to a spermatocyte specific histone gene and the beige coat color locus on chromosome 13.
Authors: Owen F L , Taylor B A , Zweidler A , Seidman J G
Published: December 2022

With the use of standard genetic techniques, we have mapped the T cell gamma-chain gene locus (Tcrg) to the proximal end of mouse chromosome 13. Close genetic linkage was demonstrated between a restriction fragment length polymorphism in the Tcrg locus and a locus (HlS) encoding a spermatocyte-specific histone electrophoretic variant (HlS) by using recombinant inbred strains. Because HlS is closely linked to the dominant visible marker extra toes (Xt), Tcrg must be within 5 cM of the beige (bg) locus. Mice homozygous for the recessive mutation beige (bg/bg) show diminished pigmentation, enlarged lysosomes in granulocytes, and a selective deficiency of natural killer cell function. The bg mutation in mice is an animal model for humans with Chediak-Higashi syndrome; we speculate that the human gamma-chain may be closely linked to the human mutation and may be a useful diagnostic tool. Seven restriction enzymes identify five haplotypes of the highly polymorphic gamma-chain in mice.
A genetically engineered murine/human chimeric antibody retains specificity for human tumor-associated antigen.
Authors: Sahagan B G , Dorai H , Saltzgaber-Muller J , Toneguzzo F , Guindon C A , Lilly S P , McDonald K W , Morrissey D V , Stone B A , Davis G L
Published: December 2022

Chimeric immunoglobulin genes were constructed by fusing murine variable region exons to human constant region exons. The ultimate goal was to produce an antibody capable of escaping surveillance by the human immune system while retaining the tumor specificity of a murine monoclonal. The murine variable regions were isolated from the functionally expressed kappa and gamma 1 immunoglobulin genes of the murine hybridoma cell line B6.2, the secreted monoclonal antibody of which reacts with a surface antigen from human breast, lung, and colon carcinomas. The kappa and gamma 1 chain fusion genes were co-introduced into non-antibody producing murine myeloma cells by electroporation. Transfectants that produced murine/human chimeric antibody were obtained at high frequency as indicated by immunoblots probed with an antisera specific for human immunoglobulin. Enzyme-linked immunoabsorbent assay analysis demonstrated that this chimeric antibody was secreted from the myeloma cells and retained the ability to bind selectively to membrane prepared from human tumor cells. The chimeric immunoglobulin was also shown by indirect fluorescence microscopy to bind to intact human carcinoma cells with specificity expected of B6.2. The ability of chimeric antibody to recognize human tumor-associated antigen makes feasible a novel approach to cancer immunotherapy.
Influence of immunoglobulin-dependent T cells on antibody class switching.
Authors: Teale J M , Janeway C A
Published: December 2022

Normal and B cell-deficient, carrier-primed mice were irradiated and were adoptively transferred with B cells to evaluate the role of putative Ig- and B cell-dependent T cells in anti-hapten antibody responses. The response was analyzed by using the splenic focus assay, which allowed us to examine the frequency of responding B cells and the production of multiple isotypes by single precursor B cells. This analysis revealed that both primary and secondary B cells were activated at higher frequency in the spleens of normal recipients, and production of isotypes other than IgM and IgG1 was enhanced in normal recipients as compared with anti-mu-treated recipients. Both changes could be restored to control levels by co-transfer of T cells from normal donors primed with an unrelated carrier, provided the free carrier was added to the assay culture. These results are consistent with a role for Ig or B cell-dependent helper T cells in the optimal activation and the resulting isotype expression of both primary and secondary B cells.
The role of the accessory cell in mitogen-stimulated human T cell gene expression.
Authors: Kern J A , Reed J C , Daniele R P , Nowell P C
Published: December 2022

The role of the accessory cell in optimizing T cell proliferative responses to mitogens is a well known but poorly understood phenomenon. To further dissect the function of the accessory cell in allowing T cell proliferation, we compared mitogen-induced c-myc, interleukin 2 (IL 2), and IL 2 receptor gene expression in peripheral blood mononuclear cells (PBMC) and in T cells rigorously depleted of accessory cells through differential adherence and anti-Dr (anti-class II major histocompatibility antigen) monoclonal antibody complement-directed cytotoxicity. In cultures stimulated with phytohemagglutinin (PHA), a mitogen that requires accessory cells to induce T cell proliferation, expression of all measured genes was accessory cell dependent, since accumulation of their mRNA in PBMC was greater than that in cultures depleted of accessory cells. These genes varied in their accessory cell dependence, with IL 2 expression most dependent, c-myc expression least dependent, and IL 2 receptor expression intermediate in dependency. Use of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or ionomycin, mitogens that stimulate T cell proliferation independent of accessory cells, induced equal levels of gene expression in PBMC and in T cells depleted of accessory cells. These results suggest that PHA-stimulated T cells are dependent on an accessory cell signal(s) for optimal expression of the genes for c-myc, IL 2, and IL 2 receptor, and for proliferation. In addition, this signal(s) appears to be delivered early in the course of T cell activation events, since it can be bypassed by mitogens that directly activate protein kinase C (TPA) or induce calcium translocation (ionomycin). In addition, these data provide further evidence that expression of the c-myc protooncogene is insufficient for T cell mitogenesis, since PHA-induced accumulation of c-myc mRNA was only partially accessory cell dependent, whereas proliferation was completely accessory-cell dependent.
Role of self carriers in the immune response and tolerance. X. A lymphoid dendritic-like tumor, P388AD.2, acts as a novel immunogenic carrier for hapten.
Authors: Cogswell J P , Phipps R P , Scott D W
Published: December 2022

Hapten-modified spleen cells, peritoneal exudate cells, and certain lymphoid tumors preferentially induce specific tolerance after i.v. administration. In contrast to these tolerogenic carrier cells, we found that a haptenated lymphoid dendritic-like tumor, P388AD.2, acts as a potent immunogen after i.v. injection. The immunogenicity of P388AD.2 was analyzed by measuring the specific augmentation of plaque-forming cell (PFC) responses when spleen cells from mice previously injected with haptenated tumor cells were challenged in vitro with thymus-independent antigens. Optimal immunization was found to be dependent on cell dose and hapten concentrations. Further studies indicated that P388AD.2 elicited a response which was T cell-dependent and which involved both the so-called Lyb-3,5,7- and Lyb-3,5,7+ B cell populations. Injection of haptenated tumor into different mouse strains suggested that H-2 compatibility was required to prime B cells in vivo, although significant augmentation could also be achieved in allogeneic C57B1/6J mice. The enhanced PFC responses elicited in H-2b mice could not be explained by allo-recognition of class I or II MHC determinants. In toto, these results suggest that P388AD.2 acts as a unique accessory cell for the presentation of hapten-modified self.
Immunodeficiency of aging: restorative effects of phorbol ester combined with calcium ionophore.
Authors: Miller R A
Published: December 2022

Current models for lectin-induced T cell proliferation suggest that activation of protein kinase C (PK-C) and elevation of cytoplasmic Ca2+ may both play important roles in the earliest phases of signal transduction. To learn more about the relative inability of T cells from old mice to proliferate in response to mitogenic stimuli, we attempted to stimulate T cells by the synergistic effects of a PK-C activator, phorbol myristate acetate (PMA), and the calcium ionophore ionomycin. T cells from young mice respond as well to optimal combinations of these agents as they do to the strong polyclonal activator Con A, but T cells from old mice respond much better to PMA plus ionomycin than they do to Con A. This result suggests that an inability to transduce the signal supplied by extracellular ligands into the intracellular signals represented by Ca2+ and PK-C activators may underlie the age-associated loss of T cell reactivity. We also found evidence for a second defect in old T cells related to their response to elevated intracellular Ca2+: old T cells, compared with young, required higher levels of ionomycin for maximal proliferation.
Alloantigen-specific suppressor T cells are not inhibited by cyclosporin A, but do require IL 2 for activation.
Authors: Bucy R P
Published: December 2022

Alloantigen-specific suppressor T cells are activated from normal murine spleen cells in mixed lymphocyte reactions (MLR). These T cells are radioresistant and suppress the activation of cytotoxic T lymphocytes (CTL) in second primary MLR cultures. This report demonstrates that cyclosporin A (CsA) blocks the activation of these suppressor cells at a dose of 1 microgram/ml. However, reconstitution of CsA blocked cultures with IL 2 restores the activation of the suppressor T cells, but fails to significantly restore the activation of CTL in these same cultures. This differential activation requirement was used to establish T cell lines that demonstrate enriched suppressor cell activity but depletion of CTL activity. These findings are discussed in terms of the mechanism of action of CsA in these distinct T cell subsets and the relevance to models of allograft unresponsiveness.
Slower processing, weaker beta 2-M association, and lower surface expression of H-2Ld are influenced by its amino terminus.
Authors: Beck J C , Hansen T H , Cullen S E , Lee D R
Published: December 2022

To determine why Ld antigens are expressed on the cell surface at levels three to four times lower than Dd or Kd antigens, pulse-chase experiments were used to compare their rates of biosynthesis and processing. Electrophoresis on sodium dodecyl sulfate gradient polyacrylamide gels resolved immunoprecipitates of each of these histocompatibility complex class I molecules into a slower and faster species. During the chase period, the faster migrating species appeared to be converted to the slower migrating species in a time-dependent manner. However, the conversion of Ld from the faster to the slower migrating species proceeded significantly more slowly than did the conversion of either Dd or Kd. Endoglycosidase H sensitivity and cell surface radiolabeling were used to determine the glycosylation state and cell location of each species of Ld and Dd. The results from these experiments, along with the pulse-chase studies and cytofluorometric analyses, suggest that Ld possesses a much slower rate of processing from a faster migrating, high mannose-bearing species to a slower migrating, complex oligosaccharide-bearing species found on the cell surface. Analysis of the beta 2-microglobulin (beta 2-m) association confirmed that Ld is associated with less beta 2-m than Dd. To localize the structures on class I molecules influencing their surface expression, rate of processing, and beta 2-m association, the Ddm1 molecule was analyzed. The Ddm1 molecule of the mutant B10.D2-H-2dm1 has previously been shown to be a chimeric Dd (amino-terminal)/Ld (carboxyl-terminal) polypeptide. The surface expression, processing and beta 2-m association of Ddm1 were found to be similar to Dd rather than Ld, suggesting that each of these phenomena are influenced by protein structure in the amino terminus.
E alpha u and E beta u chain association: where lies the anomaly?
Authors: Ayane M , Mengle-Gaw L , McDevitt H O , Benoist C , Mathis D
Published: December 2022

H-2u haplotype mice are unique among all E alpha+ strains because they do not provide in heterozygotes an E alpha chain that interacts with E betak,s,etc. sufficiently well to allow certain E-restricted immune responses. As a first step in understanding this peculiarity, we have sequenced E alpha u and E beta u cDNA and compared the derived amino acid sequences with those of previously analyzed alleles. Although no glaring structural abnormalities were found, we have identified some u-specific residues and suggest which are the most likely to provoke a pairing anomaly.
Nonspecific T suppressor factor (nsTsF) cascade in contact sensitivity: nsTsF-1 causes an Ly-1+2- I-A+ immune T cell to produce a second, genetically restricted, nsTsF-2.
Authors: Zembala M , Romano G C , Colizzi V , Little J A , Asherson G L
Published: December 2022

We studied the mode of action of the nonspecific T suppressor factor (nsTsF-1) made in the picryl (TNP) system when T acceptor cells armed with antigen-specific TsF are triggered by antigen in the context of I-J. This suppressor factor does not inhibit the passive transfer of contact sensitivity directly, as shown by its failure to inhibit passive transfer by immune cells deprived of I-A+ cells. Its immediate target is an immune, antigen-specific, Ly-1+2-, I-A+ T cell. This cell, which may be regarded as a T suppressor effector cell (Ts-eff-2), produces nsTsF-2 when exposed sequentially to nsTsF-1 and antigen. This nsTsF subsequently inhibits the passive transfer of contact sensitivity. The action of nsTsF-2 is MHC genetically restricted. As the nsTsF-2 bears I-A determinant(s), this raises the possibility that it may act by combining with the recognition site for I-A on the T cell that mediates contact sensitivity.
Effect of human syncytiotrophoblast plasma membrane-soluble extracts on in vitro mitogen-induced lymphocyte proliferation. A possible inhibition mechanism involving the transferrin receptor.
Authors: Khalfoun B , Degenne D , Crouzat-Reynes G , Bardos P
Published: December 2022

Transferrin (Tf) binding to lymphocytes and to syncytiotrophoblast plasma membranes (STPM) and their soluble extracts (STPM-SE) was examined, as well as the effect of the latter on mitogen-induced lymphoproliferation. Lymphocytes only express Tf receptors (Rtf) after mitogen (phytohemagglutinin or concanavalin A) stimulation, and the percentage of Tf bound by stimulated lymphocytes increased as a function of contact time with the mitogen, reaching a maximum at 72 hr. Studies of Tf binding to STPM-SE showed that the percentage of bound Tf increased proportionally to the protein concentration, but was additionally enhanced by a factor of three when STPM were pretreated with 3 M KCl. Scatchard analysis of Tf binding to lymphocytes cultured for 72 hr in the presence of mitogen, as well as to STPM and STPM-SE, revealed that this binding was specific and occurs via a single category of identical and independent receptors the numbers and affinity constants (Ka) of which have been determined. The results obtained by using STPM indicate that the Ka does not vary significantly from one preparation to another, but that the number of sites per milligram of protein increases by a factor of 10 when the STPM are pretreated with 3 M KCl (KCl-STPM). Finally, STPM-SE inhibited the mitogen-induced lymphoproliferative response whether or not they were treated with 3 M KCl. This inhibition was not due to lymphocytotoxicity, was dose dependent regardless of the preparation used, but was maximized with the KCl-STPM-SE fraction. The correlation between the inhibitory capacities of the soluble STPM extracts and the numbers of RTf sites present on their membranes leads to the hypothesis that the observed inhibition could involve the RTf. This effect may help in protecting the fetus from the maternal immune system at the time of the semi-allogenic fetal graft.
Monoclonal antibody (H107) inhibiting IgE binding to Fc epsilon R(+) human lymphocytes.
Authors: Noro N , Yoshioka A , Adachi M , Yasuda K , Masuda T , Yodoi J
Published: December 2022

A hybridoma-producing monoclonal antibody blocking the binding of human IgE to lymphocytes Fc receptor (Fc epsilon R) was established by the fusion of murine myeloma cells. P3X63.653.Ag8, with BALB/c spleen cells immunized with Fc epsilon R(+) human B lymphoblastoid cell line cells, RPMI1788. A clone of the hybridoma (H107) produced a monoclonal IgG2b antibody that inhibited the rosette formation of Fc epsilon R(+) human B lymphoblastoid cell line cells (RPMI1788, RPMI8866, CESS, Dakiki, and IM9) with fixed ox red blood cells (ORBC) conjugated with human IgE (IgE-ORBC). In contrast, the rosette formation with IgG-conjugated ORBC (IgG-ORBC) on Fc gamma R(+), Fc epsilon R(-) Daudi cells were not affected by the H107 antibodies. A close association of Fc epsilon R and the antigenic determinant recognized by H107 antibody was suggested by the following results. First, the bindings of 125I-labeled IgE (125I-IgE) or 125I-labeled H107 IgG2b antibody (125I-H107) to RPMI8866 cells were inhibited by cold human IgE and H107 IgG2b but not by other classes of human Ig (IgA and IgG), MPC11 IgG2b, or unrelated monoclonal antibodies. Second, H107 antibody reacted with Fc epsilon R(+) B cell lines but not with Fc epsilon R(-) B cell lines as determined by an indirect immunofluorescence. Third, Fc epsilon R(+) cells were depleted by the incubation in the dish coated with H107 antibody or IgE but not in the dish coated with unrelated antibodies. Finally, there was a correlation between the increase of Fc epsilon R(+) cells and that of H107(+) cells in the peripheral blood lymphocytes of the patients with atopic dermatitis. The surface antigens on Fc epsilon R(+) RPMI8866 cells recognized by H107 antibodies had the molecular size of 45,000 as determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.
Noncytolytic terminal complement complexes may serve as calcium gates to elicit leukotriene B4 generation in human polymorphonuclear leukocytes.
Authors: Seeger W , Suttorp N , Hellwig A , Bhakdi S
Published: December 2022

Complement effects on human polymorphonuclear leukocytes (PMN) have generally been ascribed to the anaphylatoxin C5a, which induces degranulation, superoxide anion generation, migration, and cell aggregation via interaction with membrane receptors. We here report that complement activation on the surface of antibody-sensitized human PMN provokes generation of the potent lipid mediator leukotriene B4 (LTB4) in strict dependence on complement component C8, but in the absence of detectable C9. The kinetics of LT generation are rapid, comparable with those observed after challenge with the calcium-ionophore A23187. LTB4 release is a distinct event that is dissociable from cytotoxicity as assessed by lactate dehydrogenase (LDH) release (dependent on C9) and from superoxide generation (independent of C8 and C9). It is dose dependent on extracellular calcium and is not observed in the absence of calcium. It is inhibited by substances interfering with calcium-calmodulin function (trifluoperazine and W7), but not by blockers of physiologic calcium channels (nimodipine, verapamil, and D 888). Addition of purified C8 to cells bearing C5b-7 induces a severalfold increase in their passive permeability to 45calcium. Sieving experiments with the use of marker molecules of different sizes collectively indicate the existence of small hydrophilic channels consisting exclusively or predominantly of C5b-8 complexes, which allow passive transmembrane flux of small molecules with Mr less than 200. Thus, noncytolytic terminal complement complexes may serve as a biological bypass gate for calcium in PMN membranes, triggering the arachidonic acid cascade with generation of LTB4 at doses well below the threshold required to invoke overt cell damage.
Concanavalin A induces Ca2+ mobilization, but only minimal inositol phospholipid breakdown in mouse B cells.
Authors: Bijsterbosch M K , Klaus G G
Published: December 2022

Concanavalin A (Con A) induces DNA synthesis in bulk cultures of murine T cells. In contrast, the lectin has been shown to stimulate purified B cells to leave G0, but not to proliferate. We demonstrate here that Con A induces comparable increases in intracellular Ca2+ levels in the two cell types. In B cells this appears to be due to Ca2+ influx. As expected, the lectin also provokes significant degradation of inositol phospholipids in T cells. However, it causes only minimal release of inositol phosphates in B cells. We therefore postulate that Con A may stimulate B cells by causing influx of Ca2+. In line with this, activation of B cells by the lectin is inhibited by cyclosporine, which preferentially affects stimulation of lymphocytes by Ca2+ mobilizing agents.
Amiloride inhibition of DNA synthesis and immunoglobulin production by activated human peripheral blood mononuclear cells is independent of sodium/hydrogen antiport.
Authors: Yamaguchi D T , Sakai R , Bahn L , Cragoe E J , Jordan S C
Published: December 2022

Activation of sodium/proton (Na+/H+) antiport activity has been shown to occur as an early event in mitogenesis. Because amiloride inhibits Na+/H+ antiport activity, it is hypothesized that mitogenesis may be inhibited by amiloride. In this work, we examined the effect of amiloride on DNA synthesis as measured by [3H]thymidine uptake and immunoglobulin (Ig) production as measured by an ELISA system in human peripheral blood mononuclear cells (PBM). Amiloride at 100 microM concentration inhibited irradiated Raji cell (*R)-activated and phytohemagglutinin-P (PHA-P)-stimulated DNA synthesis by 50 +/- 11% and 72 +/- 12%, respectively. IgG production was inhibited by 71% at 100 microM amiloride concentration in *R-activated PBM. This concentration of amiloride inhibited Na+/H+ antiport activity by 92%. Because amiloride is known to inhibit other pre-replicative cellular functions such as protein synthesis, we used an amiloride analogue, dimethylamiloride, which inhibited Na+/H+ antiport activity by 90% at a concentration of 1 microM without inhibition of PBM Ig or DNA synthesis. Furthermore, neither PHA-P nor *R-stimulated PBM demonstrated an intracellular alkalinization even after 6 hr of stimulation. Similarly, T cell-enriched or B cell-enriched populations did not show intracellular alkalinization after PHA-P or *R activation. Thus, it appears that Na+/H+ antiport activation is not an early event in PBM mitogenesis. The inhibition of mitogenesis by amiloride may be due to abrogation of premitotic events such as protein synthesis.
The mechanism of action of lymphokines. IX. The enzymatic basis of hydrogen peroxide production by lymphokine-activated macrophages.
Authors: Freund M , Pick E
Published: December 2022

The purpose of this study was to elucidate the biochemical basis of the enhanced hydrogen peroxide (H2O2) production by guinea pig peritoneal macrophages (MP) cultured in lymphokine (LK)-containing medium. The markedly augmented H2O2 generation by these cells, demonstrable by the horseradish peroxidase (HRP)-catalyzed oxidation of phenol red, is distinguished by its lack of dependence on a second stimulus. We demonstrate that H2O2 production is truly spontaneous and is not caused by a stimulant present among the H2O2 assay reagents. The principal candidate for such a role was HRP type II (a mixture of five isoenzymes) that was reported to be capable of eliciting an oxidative burst in MP. Four distinct HRP isoenzymes that were found incapable of provoking an oxidative response were nevertheless adequate for demonstrating H2O2 production by LK-activated MP. Blocking the MP receptor for mannose by the addition of mannan to the assay system resulted in enhanced detection of H2O2 by low concentrations of HRP type II and by three out of four HRP isoenzymes. Treatment of MP with LK-containing medium for 72 hr did not result in a significant change in the activity of cellular superoxide dismutase (SOD) compared with MP cultured for the same length of time in control medium. By using the specific inhibitor of copper, zinc-containing SOD, sodium diethyldithiocarbamate (DDC), and the universal SOD inhibitor, sodium nitroprusside, we found that the predominant enzyme in guinea pig peritoneal MP is probably manganese-containing SOD. Incubation of LK-activated MP with nitroprusside resulted in almost total inhibition of H2O2 production and a simultaneous switch to superoxide (O2-) liberation. Similar exposure to DDC had no effect. These data indicate that H2O2 produced by LK-activated MP is derived exclusively by enzymatic dismutation of O2- mediated by a manganese-containing SOD. The increase in spontaneous H2O2 production induced by LK is therefore secondary to augmented O2- production that occurs at a cellular location where O2- is accessible to SOD. The enzymatic basis of the enhanced oxygen radical production was investigated by determining the kinetic parameters of the O2- -forming NADPH oxidase of resting LK-treated MP in a cellfree system in which O-2 production was induced by sodium dodecyl sulfate. The Km for NADPH and the Vmax of the enzyme of LK-treated MP were not different from those of the enzyme of MP incubated in control medium. We conclude that LK treatment of MP does not modulate the NADPH oxidase itself but, most likely, a process related to activation of the enzyme.
Bone marrow-derived macrophage expression of endogenous and transfected class II MHC genes during differentiation in vitro.
Authors: Pullen J K , Schook L B
Published: December 2022

C57BL/6 (H-2b) mice fail to express I-E molecules on the surface of their cells and thus are unable to respond to I-E-restricted antigens such as GL phi and cytochrome c. Previous experiments in our laboratory have involved developing a system for studying differentiation of bone marrow cells into mature macrophage to gain a better understanding of class II MHC gene expression and function. In this study, we have used this system to transfect the E alpha d gene (cosmid 17.2) into C57BL/6 bone marrow cells and subsequently observed I-E expression on bone marrow-derived macrophages (BMDM) after differentiation in vitro. By using a modified calcium phosphate protocol, we found that the optimal period for transfection of the bone marrow cells was after 2 days of culture in vitro. By using the anti-I-E monoclonal antibody (Ia.7) derived from hybridoma 14-4-4, we detected the I-E molecule on the surface of transfected macrophages by a radiobinding assay and immunoprecipitation. BMDM expressed the I-E product maximally at 5 days of differentiation, and expression then declined. Furthermore, we have found that the expression of the I-E molecule on transfected macrophage was dependent upon exposure to interferon-gamma. Expression of I-E molecules was also detected by the generation of an allogeneic response. Transfected BMDM were compared with (CB6)F1 BMDM for their ability to stimulate C57BL/6 T cells and they were found to be equally effective. By using these initial findings, we hope to further optimize the conditions for insertion and expression of class II MHC genes in bone marrow cells.
Conversion of specific to nonspecific cytotoxic T lymphocytes.
Authors: Havele C , Bleackley R C , Paetkau V
Published: December 2022

The type of cytotoxic T lymphocyte (CTL) generated during a mixed leukocyte culture has been shown to depend on the purity of interleukin 2 (IL 2) used to stimulate growth. Antigen-specific CTL requiring both antigen and IL 2 to proliferate or express cytotoxicity (designated Type I in this work) are generated and maintained with highly purified IL 2. In the presence of IL 2 alone, they persist in a quiescent, noncytotoxic state, and on its removal they rapidly die. A second type of CTL (Type II), of nonspecific cytotoxicity, and dependent only on IL 2 for growth, is generated by exposure to an impure lymphokine preparation containing IL 2. Type I cells are T lymphocytes which are nongranular and nonadherent, whereas Type II cells are larger, adhere to plastic, and contain many granules. Cloned Type I cells are converted to Type II under three conditions: by low levels of IL 2 in the presence of a crude lymphokine preparation; by high levels of IL 2 in the presence of antigen; or by high levels of IL 2 in the presence of the phorbol diester PMA. Conversion results from an effect of the growth conditions on individual cells, and not from selection of a minor population.
Identification and characterization of a suppressor T cell hybridoma specifically inducible by L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT).
Authors: Sorensen C M , Pierce C W
Published: December 2022

In vitro activation of naive spleen cells from C57BL/10 mice with GAT and the monoclonal GAT-TsF1, 372B3.5, followed by fusion with BW5147 resulted in generation of a hybridoma that fails to produce GAT-TsF constitutively, but upon reexposure to GAT and 372B3.5 is induced to secrete GAT-TsF2. The induction is GAT specific and requires de novo RNA, protein synthesis, and DNA synthesis. Although both GAT and 372B3.5 are required for induction, they may be added sequentially, provided the GAT is added first. The GAT-TsF produced by the induced cell is antigen specific and composed of two polypeptide chains: one capable of binding antigen, the other bearing determinants encoded by the I-J region of the MHC. The utility of this inducible GAT-TsF2 cell line for molecular biology and other studies is discussed.
Suppressive effect of human natural killer cells on Epstein-Barr virus-induced immunoglobulin synthesis.
Authors: Kuwano K , Arai S , Munakata T , Tomita Y , Yoshitake Y , Kumagai K
Published: December 2022

The suppressive effect of human natural killer (NK) cells on Epstein-Barr virus (EBV)-induced immunoglobulin (Ig) synthesis by autologous B cells was investigated. By Percoll discontinuous density gradient centrifugation, low-density fractions enriched for NK cells were isolated from human peripheral blood lymphocytes. These NK-enriched fractions were added to purified autologous B cells in the presence of EBV, were cultivated for 8 days, and were examined for their suppressive effect on Ig synthesis by an enzyme-linked immunosorbent assay. The fractions markedly suppressed both IgM and IgG synthesis induced by EBV. It was possible to reduce the suppressive effect of NK-enriched cells by complement-dependent lysis of NK cells and Leu-11, but not by OKT3 monoclonal antibody, indicating that NK cells may be responsible for the suppression of Ig synthesis. Upon close examination of interferon (IFN) activity, it was revealed that the co-cultures of NK-enriched cells and EBV-infected B cells generated production of IFN-alpha, which might be produced by NK cells in response to EBV-stimulated B cells. Addition of anti-IFN-alpha but not anti-IFN-gamma serum almost completely abrogated the suppressive effect of NK-enriched cells on Ig synthesis, indicating that IFN-alpha produced are required for the NK cell-mediated suppression of Ig synthesis. However, addition of IFN-alpha into purified B cells showed no direct suppressive effect on EBV-induced Ig synthesis by B cells in the absence of NK cells. Nevertheless, NK cells when previously incubated with IFN-alpha and added to B cells showed a suppressor activity on Ig synthesis to a level higher than that of untreated NK controls. These results strongly suggest the possibility that NK cells display an interaction with EBV-infected B cells and produce IFN-alpha, which in turn activates NK cells. These activated NK cells suppress the Ig synthesis by B cells, which undergo transformation induced by EBV.
Structure-function relationships for the IL 2-receptor system. II. Localization of an IL 2 binding site on high and low affinity receptors.
Authors: Kuo L M , Rusk C M , Robb R J
Published: December 2022

The ligand-binding component of high and low affinity IL 2 receptors is a 55,000 m.w. glycoprotein termed Tac. Correlating the structure and function of this molecule should provide insight into the mechanism of IL 2-initiated signal transduction and the structural basis for high and low affinity receptor forms. As a first step in this process, various approaches were used to localize the IL 2 binding region of the Tac molecule. Antibodies prepared to synthetic fragments of Tac were tested for their ability to interfere with IL 2 binding and bioactivity. The results delineated segments in the C-terminal portion of the molecule which appeared to be distal to the ligand binding site. In a more direct approach, radioiodinated IL 2 was cross-linked to high and low affinity receptors, and the resulting complexes were subjected to mild tryptic digestion. Consistent with the antibody data, the IL 2 remained covalently associated with an N-terminal tryptic fragment which apparently consisted of residues 1-83 of the Tac protein. These results suggest that the N-terminal region of the Tac molecule contains important contact sites for ligand-receptor interaction.
Characterization of the human trophoblast-leukocyte antigenic molecules defined by a monoclonal antibody.
Authors: Stern P L , Beresford N , Thompson S , Johnson P M , Webb P D , Hole N
Published: December 2022

The murine monoclonal antibody H316 recognizes cell surface glycoproteins shared in expression by human trophoblast and peripheral blood leukocytes. It is also strongly expressed by many human tumor cell types, including choriocarcinoma and teratocarcinoma. The H316 antigenic determinant from these different cell types is carried on two wheat germ agglutinin-reactive glycoproteins of approximately 65,000 and 55,000 m.w. The antigenicity is not dependent on N-linked glycosylation. Biochemical fractionation procedures suggest that the molecules are related to each other, are not associated through intermolecular disulfide bonds, but do exhibit intramolecular disulfide interactions. These glycoproteins demonstrate m.w. heterogeneity between different cell lines and individual trophoblast membrane preparations. Such properties suggest that H316 molecules may be representative of TLX antigens (trophoblast-leukocyte common antigens), which have been suggested to influence maternofetal immunogenetic interactions.
Endogenous inhibitor of protein kinase C: association with human peripheral blood neutrophils but not with specific granule-deficient neutrophils or cytoplasts.
Authors: Balazovich K J , Smolen J E , Boxer L A
Published: December 2022

A Ca2+-activated and phospholipid-dependent protein kinase (PKC) has been described in several cell systems, including the human neutrophil. We found that less than 30 pmol 32P/10 min/10(6) cell equivalents of phorbol ester- or 1-oleoyl-2-acetylglycerol-stimulated PKC activity was obtained when neutrophil homogenates were used as an enzyme source. However, detergent-soluble and detergent-insoluble fractions prepared from the same homogenates, respectively, catalyzed 578 +/- 50 and 136 +/- 40 pmol 32P/10 min/10(6) cell equivalents. Recombining detergent-soluble and detergent-insoluble fractions resulted in the complete loss of activity. We therefore explored the possibility of an endogenous inhibitor of PKC in neutrophils. Homogenates from neutrophil cytoplasts, which lack the nuclei and intracellular granules of whole neutrophils, yielded 813 +/- 28 pmol 32P/10 min/10(6) cell equivalents. In addition, total neutrophil homogenates from a patient with a specific granule deficiency yielded high activities, namely 424 +/- 48 pmol 32P/10 min/10(6) cell equivalents. Specific granule-deficient neutrophils possessed translocated and activated PKC, and phosphoprotein patterns from these cells resembled those from activated normal neutrophils. Our results suggested the existence of an inhibitor of previously active PKC. That this inhibitor is primarily associated with neutrophil-specific granule membranes was suggested by our finding of high PKC activity associated with cell preparations or combinations of cell fractions that were free of specific granules, but not necessarily of other cellular organelles. Preliminary characterization of the endogenous inhibitor indicated that it was protease- and heat-sensitive, and did not exhibit either protease or phosphatase activity. We speculate that the inhibitor may play a physiologic role in regulating the activity of its target enzyme.
Regulation of the expression of multiple class II genes in murine B cells by B cell stimulatory factor-1 (BSF-1).
Authors: Noelle R J , Kuziel W A , Maliszewski C R , McAdams E , Vitetta E S , Tucker P W
Published: December 2022

We have studied the mechanisms governing the induction of class II major histocompatibility complex (MHC) antigens on murine B lymphocytes by B cell stimulatory factor-1 (BSF-1). BSF-1 induced a dramatic (10- to 15-fold) and selective increase in class II MHC antigen expression, as judged by flow cytometry. Analysis of radiolabeled membrane proteins on BSF-1-induced B cells also showed that the expression of class II MHC antigens was increased 10- to 15-fold. Biosynthetic labeling studies established that a selective increase in the translation of class II MHC molecules could be detected at 5 to 6 hr post-addition of BSF-1 to culture. After 16 hr of culture, when cell surface expression was induced 12- to 15-fold, biosynthesis rates of class II MHC antigens was induced fivefold to sixfold. The biosynthesis of the I region-associated invariant chain (Ii) was also enhanced. Actinomycin D abrogated the increased biosynthetic labeling of class II products, suggesting transcriptional regulation of expression. At 6 hr after addition of BSF-1, there was a twofold increase in the steady-stage level of class II mRNA, which was slightly increased at 16 hr. The BSF-1-induced increase in surface Ia antigen is dependent on new transcription and translation. However, posttranslational events that lead to a decrease in membrane antigen turnover rate or to an increase in protein stability probably also play a significant role in the hyperexpression of class II antigens on BSF-1-treated B lymphocytes.
Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin 2.
Authors: Rosenstein M , Ettinghausen S E , Rosenberg S A
Published: December 2022

Adoptive immunotherapy with lymphokine-activated killer cells and recombinant interleukin 2 (IL 2) can produce significant reduction of visceral metastases in tumor-bearing mice and, as shown recently, in humans with disseminated cancer. Because further dose escalations of IL 2 have been prevented by the development of a vascular leak syndrome (VLS) in both mice and humans, we investigated this VLS in mice undergoing the systemic administration of high-dose IL 2. A model for quantitating capillary permeability was used in which 125I-bovine serum albumin was injected i.v., and 2 hr later, tissues were counted in a gamma analyzer. A permeability index (PI) was calculated by dividing the mean counts per minute (cpm) of tissues from IL 2-treated mice by those from control animals. The injection of IL 2 produced increases in vascular permeability that were most pronounced in the thymus, spleen, lungs, liver, and kidneys (PI = 18.0, 10.0, 9.7, 6.7, and 6.3, respectively, on day 6). The development of the VLS was highly dependent on the number of days of IL 2 treatment (for example, the lungs contained 638, 1382, 3350, and 6187 cpm after 0, 1, 3, and 6 days of IL 2, respectively). Moreover, the degree of the VLS was directly related to the dose of IL 2 administered. Measurement of the wet and dry weights of lungs from IL 2-treated mice demonstrated that IL 2 produced a dramatic increase in their water weight (from 0.10 g at base line to 0.22 g after 200,000 U of IL 2 for 6 days). The injection of the IL 2 excipient failed to induce capillary leakage in tissues. Immunosuppression of mice by pretreatment irradiation (500 rad) or by injection of cyclophosphamide or by concurrent use of cortisone acetate markedly reduced or eliminated the development of the VLS. Similarly, the VLS was not observed in nude mice receiving IL 2. Thus, the administration of IL 2 produces a dose-limiting VLS that may be mediated, directly or indirectly, by host lymphoid elements.
Regulation of primary cytotoxic T lymphocyte responses generated during mixed leukocyte culture with H-2d identical Qa-1-disparate cells.
Authors: Huston D P , Tavana G , Rich R R , Gressens S E
Published: December 2022

Cytotoxic lymphocyte (CTL) responses are not usually generated during primary mixed leukocyte culture (MLC) with H-2 identical cells. Thus NZB mice are unusual in that their spleen cells do mount CTL responses during primary MLC with H-2d identical stimulator cells; the predominant target antigen for these NZB responses is Qa-1b. Considering the numerous immunoregulatory defects in NZB mice, we postulated that these NZB anti-Qa-1 primary CTL responses were due to an abnormality in T suppressor cell activity. Cellular interactions capable of suppressing NZB anti-Qa-1 primary CTL responses were investigated by using one-way and two-way MLC with spleen cells from NZB mice and other H-2d strains. Although H-2d identical one-way MLC with the use of NZB responders resulted in substantial CTL responses, only minimal CTL responses were detected from two-way MLC with the use of NZB spleen cells plus nonirradiated spleen cells from other H-2d mice. Thus the presence of non-NZB spleen cells in the two-way H-2d identical MLC prevented the generation of NZB CTL. Noncytotoxic mechanisms were implicated in the suppression of the NZB CTL responses during two-way MLC, because only minimal CTL activity was generated when NZB spleen cells were cultured with semiallogeneic, H-2d identical (e.g., NZB X BALB) F1 spleen cells. The observed suppression could be abrogated with as little as 100 rad gamma-irradiation to the non-NZB spleen cells. The phenotype of these highly radiosensitive spleen cells was Thy-1+, Lyt-1+, Lyt-2-, L3T4+. The functional presence of these cells in the spleens of semiallogeneic, H-2d identical F1 mice indicated that their deficiency in NZB mice was a recessive trait. These data suggest that NZB mice lack an L3T4+ cell present in the spleens of normal mice that is capable of suppressing primary anti-Qa-1 CTL responses. This model system should facilitate additional investigations of the cellular interactions and immunoregulatory mechanisms responsible for controlling primary CTL responses against non-H-2K/D class I alloantigens. The model may also provide insight into the immunoregulatory defects of autoimmune NZB mice.