bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of newborn liver cells as a murine model for cord blood cell transplantation.
Authors: Davenport C , Kumar V , Bennett M
Published: December 2022

There is current interest in using umbilical cord blood cells as a substitute for bone marrow cells (BMC) in human transplantation. However, T cell-depleted BMC are more susceptible to rejection. Because CBC lack mature T cells, mouse newborn liver cells (NLC) were used as a model to investigate the role of T cells in engraftment. BALB/c BMC, C.B-17 scid/scid (SCID) BMC, and BALB/c NLC were transplanted into lethally irradiated BALB/c and (B6 x DBA/2) F1 (B6D2F1) hosts. Splenic 125IUdR incorporation 5 days later assessed engraftment. BALB/c BMC, NLC, and SCID BMC grew well in BALB/c hosts, but only BALB/c BMC grew in B6D2F1 hosts. This suggests that T cells are necessary for engraftment of H-2d stem cells in the B6D2F1 host. Addition of BALB/c thymocytes to SCID BMC or NLC allowed engraftment in the F1 hosts. It appears that NK cells mediated the resistance because their depletion by mAb increased engraftment of SCID BMC or NLC. Anti-NK1.1 mAb and -asialo GM1 serum eliminate all NK cells, although anti-5E6 mAb eliminate a subpopulation of NK cells that responds to Hh-1d (determinant 2+) stem cells. Treatment of F1 hosts with any of these mAb prevented rejection of SCID BMC or BALB/c NLC. Also, activation of NK cells by poly I:C caused rejection of donor cells in the F1 that could not be overcome by the presence of thymocytes. This effect of the poly I:C could also be reversed by mAb depletion of host NK cells. Thus, engraftment of stem cells is influenced by the presence of donor T cells and the activation level of host NK cells.
Preferential expansion and activation of V beta 5+ CD4+ T cells in murine acquired immunodeficiency syndrome.
Authors: Selvey L A , Morse H C , Granger L G , Hodes R J
Published: December 2022

Infection of B6 mice with LP-BM5 MuLV results in a syndrome characterized by progressive and profound immunodeficiency, termed murine acquired immunodeficiency syndrome (MAIDS). In this report we show that preferential expansion and activation of V beta 5+ CD4+ spleen cells occurs early in the course of disease, which then progresses to more widespread polyclonal activation of CD4+ T cells. V beta 5 expansion occurs only on infection with MAIDS-associated MuLV and is not observed after infection with other MuLV. The possible role of V beta 5 activation and expansion as a necessary early step in the pathogenesis of MAIDS is discussed.
Insulin and IGF-1 increase mitogenesis and glucose metabolism in the multiple myeloma cell line, RPMI 8226.
Authors: Freund G G , Kulas D T , Mooney R A
Published: December 2022

Insulin receptors and insulin-like growth factor-1 (IGF-1) receptors are present in circulating human B lymphocytes (B cells) and certain B cell malignancies, but no function has been attributed to either receptor. We report a human myeloma cell line, RPMI 8226, that exhibits insulin and IGF-1-dependent receptor and substrate tyrosine phosphorylation as well as hormone-responsive cellular metabolism. Competitive hormone-binding analysis revealed that the cell line expressed approximately 4 x 10(3) high affinity insulin binding sites and 1.1 x 10(4) high affinity IGF-1 binding sites per cell. The Kd of the insulin-binding sites for insulin was 0.32 nM. The Kd of high affinity IGF-1 binding sites for IGF-1 was 0.89 nM. Insulin receptor autophosphorylation was maximum at 200 nM as was tyrosine phosphorylation of the 180-kDa cytosolic receptor substrate. Insulin-dependent activation of phosphatidylinositol 3-kinase paralleled receptor phosphorylation. In contrast, IGF-1 produced its maximum effects at 200 nM for receptor phosphorylation and 20 nM for substrate phosphorylation and PI 3-kinase activation. In growth synchronized cells, IGF-1 and insulin at 200 nM increased DNA synthesis by 122 +/- 18% and 101 +/- 27%, respectively. IGF-1 increased DNA synthesis 88 +/- 21% at 2 nM and the effect of insulin at 2 nM was 34 +/- 12%. Flux through the glycolytic pathway was also increased by insulin and IGF-1. At 200 and 2 nM, insulin increased production of lactate by 33 +/- 9% and 19 +/- 11%, respectively. IGF-1 increased lactate production 47 +/- 3% and 23 +/- 3% at identical hormone concentrations. Finally, in two additional myeloma cell lines, U266 (human) and Ag8.653 (mouse), insulin and IGF-1 increased tyrosine phosphorylation of receptor beta-subunit (95 kDa), the prominent 180-kDa substrate (pp185), and several other substrates. Thus, functional insulin and IGF-1 receptors are present in myeloma cell lines. Through these receptors, insulin and IGF-1 regulate mitogenesis and glucose metabolism, and may be important in potentiating plasma cell malignancy.
Urtica dioica agglutinin. A superantigenic lectin from stinging nettle rhizome.
Authors: Galelli A , Truffa-Bachi P
Published: December 2022

Urtica dioica agglutinin (UDA) is an unusual plant lectin that differs from all other known plant lectins with respect to its molecular structure and its extremely low specific agglutination activity. We recently reported that this small lectin (8.5 kDa) is a T cell mitogen distinguishable from classical T cell lectin mitogens by its ability to discriminate a particular population of CD4+ and CD8+ T cells as well as its capacity to induce an original pattern of T cell activation and cytokine production. The mechanism by which UDA activates T cells was investigated and compared with the conventional T cell mitogen Con A and the known superantigen staphylococcal enterotoxin B. Our data show that T cell proliferation induced by UDA is strictly dependent on AC expressing MHC class II molecules but is not MHC restricted. This proliferation can be partially inhibited by anti-I-A or anti-I-E mAb and completely blocked by a mAb recognizing monomorphic determinants on the Ia molecule. UDA indeed binds to specific carbohydrate structures present on class II molecules. UDA-induced T cell stimulation is dependent on TCR recognition of the unprocessed intact molecule in association with various Ia molecules. T cell response to UDA is clonally expressed and correlates with particular TCR V beta gene families usage. This stimulation leads to a sixfold enrichment of V beta 8.3+ T cells within 3 days. Therefore, UDA appears to use the same molecular mechanism as structurally unrelated bacterial or retroviral superantigens and we propose that this lectin is a superantigen. UDA, which is not a pathogenicity factor, could provide a useful probe for the analysis of T cell activation by superantigens.
Early signals in alloantigen induced B-cell proliferation. Comparison between B-cell triggering by intact allogeneic cells and solubilized alloantigen.
Authors: Genaro A M , Boscá L
Published: December 2022

Stimulation of B cells from BALB/c with allogeneic lymphocytes from C57BL/6 mice resulted in a slight increase in cytosolic Ca2+ but in the absence of proliferative response. Immunization of BALB/c mice with C57BL/6 total lymphocytes resulted in an enhancement of cytosolic Ca2+ and of B cell proliferation. Phosphatidylinositol specific phospholipase C was activated immediately after allogeneic stimulation as deduced by the concomitant rise in inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Translocation of protein kinase C from the cytosol toward the membranes paralleled the elevation in cytosolic free Ca2+. Activation of BALB/c B cells with solubilized alloantigen from the plasma membrane of C57BL/6 lymphocytes produced qualitatively the same early responses as the treatment with allogeneic cells, although quantitatively more intense. Concerning protein kinase C, an important degradation was observed in these conditions. Soluble alloantigen failed to promote B cell proliferation, being observed when cells were costimulated with a low concentrations (2 ng/ml) of phorbol 12,13-dibutyrate before alloantigen addition. Analysis of the molecular weight of the active fraction of the solubilized alloantigen revealed the presence of a 51 kDa protein that mimicked all properties of the alloantigen preparation. This molecule was also recognized in Western blot by an anticlass I mAb and by the sera of immunized animals. A putative MHC class I antigen is proposed as the nature of the active molecule, and its interaction with specific membrane Ig on the B cell is analyzed. Although the results fit with a cellular response mediated through membrane Ig, the involvement of other B cell surface molecules interacting with the alloantigens cannot be disregarded.
Actin polymerization and pseudopod reorganization accompany anti-CD3-induced growth arrest in Jurkat T cells.
Authors: Parsey M V , Lewis G K
Published: December 2022

T cell activation via CD3/Ti linked pathways results in the polymerization and reorganization of actin. However, little is known about the morphology and temporal appearance of filamentous actin (F-actin) after activation. Similarly, little is known about the relationship between F-actin and changes in cell shape or other parameters of activation, such as the appearance of proteins newly phosphorylated on tyrosine, that occur after stimulation via the CD3/Ti complex. Accordingly, we have characterized changes in cell shape and F-actin morphology occurring in the Jurkat T cell leukemia attached to the surface of culture vessels by immobilized anti-CD3 antibodies (OKT3, UCHT-1, SPV-T3b). These antibodies induced activation within 30 min as measured by increased protein tyrosine kinase activity and conversion of the proto-oncogene product, lck, from 56 kDa to 60 kDa (p56lck conversion), and after 12 to 96 h as measured by growth arrest and, in some experiments, IL-2 production. Activation was not seen when cells were attached to the substrates using antibodies directed to other cell surface proteins including CD71 (transferrin receptor), CD7, and CD11a (LFA-1), demonstrating the specificity of activation for immobilized anti-CD3 antibodies. Temporal changes in cell shape and F-actin morphology were characterized in Jurkat cells attached by immobilized anti-CD3 antibodies (stimulatory antibodies) and compared with the patterns obtained obtained in Jurkat cells attached by antibodies specific for the other markers (nonstimulatory antibodies). In these experiments, Jurkat cells were incubated with antibody-coated substrates for 1 to 30 min at 37 degrees C and actin rearrangements were visualized on fixed, detergent-permeabilized cells using rhodamine-conjugated phalloidin. Analysis of cell shape and F-actin morphology during the first 30 min of activation revealed a unique pattern that was observed only when cells were stimulated with anti-CD3 antibodies. Jurkat cells attached by either stimulatory or nonstimulatory antibodies reorganized their actin similarly after the first minute of culture, as characterized by the formation of small, F-actin rich pseudopods at the sites of attachment. After 5 min of culture in cells attached by stimulatory antibodies, the actin was polymerized into a dense collar rimming the inner edge of the cell. From 15 to 60 min, this collar was replaced by numerous F-actin rich, branched pseudopods. These branched pseudopods were larger and had longer microfilament bundles than their earlier counterparts. By contrast, in cells attached by nonstimulatory antibodies, the initial configuration was maintained for at least 60 min, except that a decrease in microfilament bundle length was noted.(ABSTRACT TRUNCATED AT 400 WORDS)
IL-12 induces the production of IFN-gamma by neonatal human CD4 T cells.
Authors: Wu C Y , Demeure C , Kiniwa M , Gately M , Delespesse G
Published: December 2022

A major difference between "naive" and "memory" or "effector" Th cells is the spectrum of cytokines that they are capable of producing. After stimulation naive cells produce only IL-2, whereas memory cells produce several cytokines including IFN-gamma and IL-4. Using umbilical cord blood-derived CD4 T cells as a source of naive T cells, we first report that these cells are capable of producing large amounts of IFN-gamma when cultured with low concentrations of IL-12. The response is time- and dose-dependent, and it is observed at the protein and mRNA levels. IL-12 also induces neonatal CD4 T cells to produce lymphotoxin but not IL-2, TNF-alpha, or IL-4. The production of IFN-gamma by IL-12-stimulated neonatal T cells is associated with a small but significant T cell activation evidenced by DNA synthesis and by the expression of the activation markers CD25, CD71, and HLA-DR; moreover, it is inhibited by hydrocortisone, cyclosporin A, and transforming growth factor-beta. The response to IL-12 is enhanced and is much more rapid when CD4 T cells are cultured in the presence of accessory cells or of exogenous IL-1, IL-2, or TNF-alpha. Using a three-step culture system, we next show that IL-12 induces the maturation of resting naive CD4 T cells into cells producing both IL-2 and IFN-gamma but not IL-4 upon stimulation with PMA and ionomycin. Endogenously produced IFN-gamma plays a role in this IL-12-induced T cell maturation, as shown by the inhibitory effect of neutralizing IFN-gamma antibodies. Finally, we show that IL-12 supports the production of IFN-gamma during primary stimulation of neonatal T cells via the CD3/TCR complex by means of either immobilized anti-CD3 mAb or superantigen-coated (Staphylococcus enterotoxin B) fixed L cell transfectants expressing HLA-DR. It is suggested that IL-12 is involved in the selection of Th1 type immune responses.
Chimeric antibodies with anti-dextran-derived complementarity-determining regions and anti-p-azophenylarsonate-derived framework regions.
Authors: Kao C Y , Sharon J
Published: December 2022

The framework regions of antibodies fold into a conserved beta-sheet structure that acts as scaffolding for the antigen-contacting complementarity-determining regions (CDR). To test the structural equivalence of the frameworks between two antibodies with widely different combining sites, we created chimeric H and L chains by grafting the CDR of an alpha(1-->6)dextran specific antibody onto the framework of a p-azophenylarsonate (Ars) specific antibody through oligonucleotide-directed mutagenesis of the anti-Ars variable region genes. Antibodies consisting of various chain combinations of the chimeric, anti-dextran, and anti-Ars derived H and L chains were generated in transfectomas and tested for binding to dextran and Ars. Of the newly created chimeric and/or hybrid antibodies, an antibody with the chimeric H chain and the anti-dextran L chain bound to dextran with the same association constant as the parental anti-dextran antibody, and like the anti-dextran antibody was shown by immunochemical mapping to have a site complementary to six glucose residues. None of the other new variable region combinations, including the all-chimeric combination, showed binding to either dextran or Ars. These results indicate that the H chain but not the L chain anti-dextran and anti-Ars frameworks are functionally equivalent. Attempts to confer dextran binding on the H and L chain chimeric antibody, by mutagenizing selected framework residues, were unsuccessful. This study demonstrates the important role of the frameworks in the precise alignment of the CDR for Ag binding.
Transforming growth factor-beta suppresses activation and proliferation of microglia in vitro.
Authors: Suzumura A , Sawada M , Yamamoto H , Marunouchi T
Published: December 2022

Transforming growth factor-beta (TGF-beta) has been shown to be produced in the central nervous system (CNS). The functions of TGF-beta, however, remain to be elucidated. We investigated the effects of TGF-beta on the proliferation, activation, and cytokine production of isolated microglia in vitro. TGF-beta dose-dependently suppressed the acid phosphatase activity of and formation of superoxide anion by LPS activated microglia. It also suppressed the IFN-gamma-induced expression of class II MHC Ag- and the production of cytokines, IL-1, IL-6, and TNF-alpha, by these cells. TGF-beta also inhibited the proliferation of microglia that was induced by either GM-CSF or M-CSF. Because various immunoregulatory cytokines are produced in the CNS that serve as autocrine or paracrine mediators it is suggested that TGF-beta could be a negative regulator in the CNS cytokine network. It may play a role in the development of various disease processes in the CNS by inhibiting the function of microglia in inflammation or in immunoregulation.
Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8 beta, and the b domain of C9.
Authors: Tschopp J , Chonn A , Hertig S , French L E
Published: December 2022

Clusterin is a heterodimeric multifunctional protein expressed in a variety of tissues and cells. It forms high density lipid complexes in plasma and participates in the control of the lytic activity of the late complement complex (TCC, C5b-9). Together with vitronectin, clusterin binds to the nascent amphiphilic C5b-9 complex, rendering it water soluble and lytically inactive. To define the interactions that underlie the complement-inhibitory function of clusterin, we have examined the binding interactions between [125I]clusterin and the isolated components of the complex, C5b-6, C7, C8, and C9 and vitronectin. By using ligand blotting in the presence of Tween, specific binding of the labeled clusterin with C7, the beta-subunit of C8 and C9 was detected. Binding to C9 was competed by polymerized C9, but not by C8, C7, C6, and CD59, suggesting that the conformational change occurring during the hydrophilic-amphiphilic transition of C9 exposes the interaction site for clusterin. When thrombin-treated C9 was analyzed, clusterin was found to recognize the C9b fragment containing the hydrophobic membrane interaction segment. Both subunits of clusterin interact with C9 and are similarly potent in inhibiting C5b-9-mediated hemolysis and Zn+(+)-induced C9 polymerization. These results show that clusterin exerts its inhibitory effect by interacting with a structural motif common to C7, C8 alpha, and C9b.
Molecular analysis of IgE H-chain transcripts expressed in vivo by peripheral blood lymphocytes from normal and atopic individuals.
Authors: Efremov D G , Batista F D , Burrone O R
Published: December 2022

The low levels of IgE produced by PBMC from normal individuals has so far prevented an analysis of their IgE H chain repertoire. Using a nested polymerase chain reaction approach, we were able to detect epsilon transcripts in all normal and allergic individuals we investigated. We further cloned epsilon CDR3/FW4 regions from two normal and two atopic individuals with low serum IgE levels. Sequence analysis of 104 clones identified 26 different epsilon CDR3/FW4 regions and an additional number of clonally related transcripts in the two atopic individuals. Preferential usage of DH genes from the DXP family (33%) and of the JH4b gene (35%) were observed, similar to reported findings for the IgM-producing peripheral blood B cell subset. Using CDR3 specific oligonucleotides, we detected the CDR3/FW4 regions of a particular set of clonally related epsilon transcripts in mu and gamma 4 transcripts of the same individual. This finding demonstrates the in vivo production of IgE together with the two other Ig isotypes (IgM and IgG4) by the progeny of a common B cell precursor, and suggests a possible mechanism for regulating the allergic response. The clonally related epsilon transcripts were found to be only of the secreted form. We give also evidence that the IgE-producing B cells undergo somatic mutation because a number of identical mutations were observed in the FW4 regions of epsilon and mu clonally related transcripts. Some of these mutations were shared with other transcripts from the same and other individuals, supporting the existence of sequence-specific hot spots for the somatic hyper-mutation machinery in the JH gene segments.
Preferential V beta usage by cytotoxic T cells cross-reactive between two epitopes of HIV-1 gp160 and degenerate in class I MHC restriction.
Authors: Shirai M , Vacchio M S , Hodes R J , Berzofsky J A
Published: December 2022

The T cell response to HIV-1 gp160 is among the most thoroughly studied immune responses to HIV-1 products. In our previous work, the MHC class I molecule Dd as well as H-2u, p, and q, were found to present P18 and HP53, two determinants of HIV-1 gp160, to CD8+ CTL in mice. We have studied the TCR V beta chain expression in CTL lines, either cross-reactive for these two peptides or specific for P18 alone, in these four different MHC haplotypes. The usage of V beta in T cells showing cross-reaction between these two peptides was remarkably conserved (primarily V beta 8 family, with some use of V beta 14) despite the extensive TCR V beta diversity of the non-cross-reactive CTL, which did not use V beta 8 or 14. This correlation of V beta usage with fine specificity was consistent in H-2d, u, and p (p < 0.01), but not in H-2q. The correlation of V beta use with peptide fine specificity independent of MHC restriction was unexpected. The strong predominance of V beta 8 family TCR was all the more surprising in view of the finding that mice bearing a genomic deletion of V beta 8 can still produce T cells with the cross-reactive phenotype, implying that other V beta chains can produce this specificity. We therefore asked whether the complexes of P18 with H-2d, p, and u are recognized as identical, and observed the surprising result that H-2d, p, and u cells mutually cross-present the peptides P18 and HP53 to allogeneic CTL lines and individual clones of each of the other haplotypes, whereas none of these cross-present to H-2q CTL, nor do H-2q targets present to CTL of the other haplotypes. This degeneracy of MHC restriction is novel for class I molecules. Moreover, the observed restriction in V beta usage occurs only in the unique set of CTL that exhibit both peptide-cross-reactive fine specificity and MHC allogeneic cross-presentation. The observation that a strain of mice in which the V beta 8 family is genomically deleted can still make CTL of this phenotype using another V beta demonstrates the plasticity of the class I MHC-restricted repertoire when the dominating receptor is not available.
Cytotoxic T cell responses to minor H-43 alloantigens in H-43a and H-43b mice. Distinctive MHC class I restriction specificity and clonal inactivation are inherent properties of the H-43 system.
Authors: Ishikawa H , Hayakawa J , Shiohara T
Published: December 2022

Our previous studies demonstrated that CTL responses to the newly identified minor H-43a and H-43b alloantigens induced in H-43b and H-43a responder mice, respectively, utilized the same H-2Kb (Kb) MHC class I restriction element, whereas many allelic class I products were not utilized. Also, a single i.v. injection of H-43b recipient mice with spleen cells (SC) from H-43 congenic mice induced specific and lasting CTL tolerance to H-43a rather than priming. We have produced a H-43b C3H.AU mouse strain (H-2p), which we regard as H-43 congenic to the C3H.NB (H-2p, H-43a) strain. Using this strain we demonstrate that Kp and/or Dp alleles can serve as class 1 restricting elements in the CTL responses to H-43a and H-43b alloantigens. Injection of H-43a recipient mice with antigenic H-43b SC from the paired H-43 congenic abrogated the anti-H-43b CTL response, irrespective of MHC class I specificity, i.e., in the contexts of Kb and of Kp and/or Dp elements. A corresponding result was obtained upon injection of H-43b recipient mice with congenic H-43a SC. The tolerance-inducing capacity of H-43a congenic SC was so strong that injection of these cells into H-43b responders that had previously received immunogenic H-43a SC completely blocked the previously activated Kb-restricted memory CTL activity specific for the H-43a alloantigen.
Pattern of potassium channel expression in proliferating B lymphocytes depends upon the mode of activation.
Authors: Partiseti M , Korn H , Choquet D
Published: December 2022

Ionic channel expression is highly regulated during mitogenesis. But it is not clear whether these regulations only follow intrinsic programs during the course of the cell cycle or if they also depend upon the external factors used to promote cell activation. B lymphocytes express two classes of potassium channels and can be stimulated to enter the cell cycle by distinct pathways. Thus, we have analyzed, with the patch-clamp technique, if the expression of channels varies when the cells are activated by different signals that lead to cell proliferation. We found that stimulation through Ag receptors increases the expression of calcium- and voltage-activated potassium channels, whereas a bacterial mitogen, LPS, only enhances the expression of the latter. Moreover, channel expression can still be modified in proliferating cells because stimulation of LPS-activated cells through Ag receptors induces rapid expression of calcium-activated channels. The use of inhibitors of mRNA synthesis revealed that this process depends upon gene transcription. Thus, differential induction of the expression of potassium channels is not only linked to the entry into the cell cycle but depends also on pathways of stimulation.
Expression of the Bcl-2 protein in murine and human thymocytes and in peripheral T lymphocytes.
Authors: Veis D J , Sentman C L , Bach E A , Korsmeyer S J
Published: December 2022

Bcl-2, a proto-oncogene that can block apoptosis, was found to be expressed throughout the thymic medulla, but in only scattered cells in the thymic cortex. In order to determine the precise distribution of Bcl-2 protein during thymocyte development, we utilized mAb specific for either mouse or human Bcl-2. Thymocyte subpopulations were assessed using three-color flow cytometry and a saponin-permeabilization method. Staining of adult mouse and human thymocytes was comparable, with 20 to 35% of cells expressing Bcl-2. Bcl-2 was expressed in nearly all CD4+ and CD8+, and CD3hi cells, but in only 5 to 10% of CD4+8+ cells. The CD4-8- population was more variable, with 25 to 40% of human cells and 65 to 80% of murine cells expressing Bcl-2. In sorted adult murine CD4-8- cells, the very immature Pgp-1+/IL-2R alpha- subset had a high percentage of Bcl-2+ cells. Bcl-2 expression was also examined during murine fetal development. At fetal day 15.5 to 16.5, 60 to 70% of total thymocytes expressed Bcl-2. By fetal day 17.5, overall Bcl-2 expression fell to adult levels of 20 to 30%. Bcl-2 was present in peripheral T cells from lymph node, spleen, and peripheral blood at uniformly high levels. In vitro stimulation with anti-CD3 or anti-TCR antibodies increased Bcl-2 expression in total thymocyte cultures, but could not induce Bcl-2 expression in CD4+8+ cells, even with the addition of a variety of cytokines. These data suggest that early double negative thymocytes express Bcl-2 but lose Bcl-2 with differentiation to the double positive stage. Thymocytes regain Bcl-2 during selection to a single positive state and retain Bcl-2 in the periphery.
Analysis of human skin mast cell proteins by two-dimensional gel electrophoresis. Identification of tryptase as a sialylated glycoprotein.
Authors: Benyon R C , Enciso J A , Befus A D
Published: December 2022

Proteins of mast cells purified from human foreskin were separated by 2-D polyacrylamide gel electrophoresis using either nonequilibrium pH gradient electrophoresis or isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. Silver staining showed that a major feature of skin mast cell 2-D protein maps was a variety of relatively abundant proteins in the m.w. range of 29 to 37 kDa and covering a broad pH range from 5.0 to 8.5. Tryptase was identified on Western blots of 2-D-separated proteins by its binding of mAb and of 3H-diisopropylfluorophosphate. The precise distribution of tryptase varied among individuals but this protein generally occupied a continuum of molecular weights between 28 and 37 kDa and ranged in isoelectric point between 5.0 and 6.5. Tryptase was one of a number of mast cell proteins that bound the lectin concanavalin A as well as lectins specific for sialic acid, demonstrating that this enzyme is a sialylated glycoprotein. The diffuse m.w. distribution of skin mast cell tryptase (31 to 36 kDa) observed after SDS-PAGE was reduced to a single band of 30 kDa after treatment with protein-N-glycosidase F to remove asparagine-linked oligosaccharides. This finding suggests that intrinsic m.w. heterogeneity of tryptase in skin mast cells is largely a result of the addition of variable amounts of oligosaccharide to the tryptase polypeptide.
Isolation of a macrophage-like cell line defective in binding of lipopolysaccharide. Influence of serum and lipopolysaccharide chain length on macrophage activation.
Authors: Kirikae T , Schade F U , Kirikae F , Rietschel E T , Morrison D C
Published: December 2022

A mutant cell line (J7.DEF.3) derived from murine macrophage-like J774.1 cells, was isolated on the basis of defective specific 125I-labeled LPS-binding in the presence of serum. Although J7.DEF.3 cells still respond to LPS in inducing TNF-alpha release and nitric oxide (NO) formation, these cells nevertheless showed significantly decreased responsiveness to LPS relative to the J774.1 parent. Under serum-free conditions, no differences between J774.1 and J7.DEF.3 cells in response to LPS were observed. The time kinetics of responsiveness to LPS also showed a delay in the onset of TNF-alpha release and NO formation in the mutant cells relative to parent cells. Importantly, this decrease in responsiveness to LPS relative to parental cells was dependent on the length of the polysaccharide portion of LPS. The defect in the mutant cells has been shown to be specific for LPS, in that these cells respond to heat-killed Listeria monocytogenes and to zymosan to a similar extent as do the parental cells. Collectively these results suggest that the defect in the J7.DEF.3 mutant cells may be related to a cellular LPS-binding molecule that, in turn, may depend upon an LPS-binding serum component.
Human mononuclear phagocytes express adenosine A1 receptors. A novel mechanism for differential regulation of Fc gamma receptor function.
Authors: Salmon J E , Brogle N , Brownlie C , Edberg J C , Kimberly R P , Chen B X , Erlanger B F
Published: December 2022

Using monoclonal anti-adenosine A1 receptor antibodies that bind the A1 receptor ligand binding site, we demonstrate that A1 receptors are expressed on cultured monocytes and rheumatoid synovial fluid mononuclear phagocytes. This finding is associated with the acquisition of reactivity with selective adenosine A1 receptor agonists and is temporally coordinated with the induction of adenosine A2 receptors on cultured monocytes. In a rapid, concentration-dependent fashion, these two distinct adenosine receptors modulate Fc gamma receptor-mediated phagocytosis, a response critical to the pathogenesis of immune complex diseases. Occupancy of A1 receptors by N6-cyclopentyladenosine (an A1-specific adenosine analogue) or mAb AA1 (an anti-A1 mAb) results in a potent stimulation that is blocked by adenosine receptor antagonists. This A1 receptor-induced enhancement of Fc gamma receptor-mediated phagocytosis is a consequence of preferential augmentation of Fc gamma RI function, suggesting distinct mechanisms for receptor-effector coupling of Fc gamma receptor families. In contrast, ligation of A2 receptors by A2-specific agonists decreases Fc gamma receptor-mediated phagocytosis in cultured monocytes. The opposing effects of adenosine A1 and A2 receptors allow for a concentration-dependent feed-back loop that responds more rapidly than effects elicited by other endogenous modulators. Low concentrations of adenosine are proinflammatory providing enhanced Fc gamma receptor function via A1 receptors, whereas higher concentrations that can occur with tissue damage are anti-inflammatory providing inhibition via A2 receptors. This rapid and potent modulation of Fc gamma receptor-mediated function suggests that adenosine is an important local regulator of the inflammatory response.
Apoptosis in splenic B lymphocytes. Regulation by protein kinase C and IL-4.
Authors: Illera V A , Perandones C E , Stunz L L , Mower D A , Ashman R F
Published: December 2022

Small dense splenic B lymphocytes from adult specific pathogen-free mice were shown to undergo apoptosis in vitro as indicated by internucleosomal DNA fragmentation, hypodiploid DNA content of isolated nuclei, and morphologic features by electron microscopy. Unstimulated cultures showed spontaneous apoptosis increasing gradually and monotonically from < 2 to 32% of B cells by 16 h. The rate of accumulation of apoptotic cells was reduced by the addition of IL-4 or PMA, but not by the inactive phorbol ester, 4 alpha PDD. In contrast, inhibitors of protein kinase C (H7 and staurosporine) increased the percentage of cells undergoing apoptosis to > 70% by 12 h; HA 1004, genistein, and herbimycin A all had no effect on apoptosis. Thus, protein kinase C activity regulates apoptosis, but there is no evidence that protein kinases A and G and tyrosine kinases are involved. Cycloheximide increased apoptosis, indicating that apoptosis may be restrained in B cells by the presence of one or more labile protective proteins. The percentage of apoptotic cells measured by flow cytometry and the percentage of fragmented DNA measured by the diphenylamine method were nearly equal, regardless of the method of apoptotic regulation. Together with the absence of nuclei with flow cytometric properties intermediate between normal and apoptotic, these results suggest that in individual B cells apoptosis progresses rapidly to completion. These data suggest a fundamental change in our concept of the life-style of the "resting" B cell: instead of a dormant cell remaining unchanged until it receives activation signals, the mature spleen B cell appears programmed to die by apoptosis unless rescued by specific agents, such protein kinase C activators or IL-4.
Junctional diversity in the absence of N regions. Neonatal T cell receptor beta chain junctional sequences are more heterogeneous than neonatal T cell receptor gamma delta or IgH junctions.
Authors: Feeney A J
Published: December 2022

Early in ontogeny, Ig, TCR-alpha beta, and TCR-gamma delta lack N regions. In addition, Ig and TCR-gamma delta junctions preferentially occur at regions of short sequence homology, thus limiting junctional diversity for these neonatal lymphocyte populations. Here, we analyze the extent of heterogeneity in TCR-beta chain junctions made early in ontogeny. DNA and cDNA from fetal/neonatal thymocytes were amplified by polymerase chain reaction, and the V-D and D-J junctions from these randomly generated sequences were analyzed. The D-J junctions were very heterogeneous, and displayed little evidence of homology-directed recombination. The V beta 8-D and V beta 5-D junctions that we analyzed each had a particular junctional sequence that was created at the site of a two-nucleotide homology, but in each case that sequence only comprised 10 to 17% of the total sequences. This junctional heterogeneity of N region lacking TCR-beta chains can be partially explained by a relative paucity of homologies at the appropriate locations near the coding ends, particularly at the D-J junction, but other factors such as the sequence surrounding the homology may also contribute. Thus, TCR-beta chains have extensive junctional heterogeneity early in ontogeny before N regions begin to be added. Since TCR-alpha beta CDR3 plays a major role in binding antigenic peptides, this junctional heterogeneity is likely to be advantageous for establishing a diverse TCR repertoire. We suggest that the sequences of the coding ends of the TCR-alpha beta have been selected through evolution to avoid the restricted junctional diversity resulting from homology-directed recombination.
Expression cloning of a cDNA encoding a novel murine B cell activation marker. Homology to human CD38.
Authors: Harada N , Santos-Argumedo L , Chang R , Grimaldi J C , Lund F E , Brannan C I , Copeland N G , Jenkins N A , Heath A W , Parkhouse R M
Published: December 2022

A rat mAb (NIM-R5) has recently been prepared against a novel murine B cell activation marker. We report here isolation of a cDNA (1-19) encoding the B cell-derived protein recognized by NIM-R5 antibody. This cDNA contains an open reading frame that encodes a polypeptide of 304 amino acids with a predicted molecular weight of 34,500. The existence of a 22-amino acid hydrophobic region located 23 amino acids from the amino terminal of the deduced protein, together with four potential N-linked glycosylation sites, characterize the deduced protein encoded by I-19 cDNA as a typical type II transmembrane glycoprotein. Although I-19 cDNA appears to encode a novel murine protein, its nucleotide sequence and deduced amino acid sequence show approximately 70% homology to the previously reported sequence of human CD38, suggesting that I-19 cDNA encodes either the mouse homologue of CD38 or a closely related protein. Northern blot analysis of the expression of this cDNA product in a variety of cell types, together with immunoprecipitation of the recombinant protein expressed in BaF3 cells, indicated that I-19 cDNA encodes not only the epitope recognized by NIM-R5 but also a protein that is indistinguishable biochemically and in terms of distribution from the murine B cell activation marker recognized by NIM-R5 antibody. Chromosomal mapping studies have localized this locus to the proximal region of mouse chromosome 5. We anticipate that the availability of probes for the murine B cell activation marker recognized by NIM-R5, and the recombinant protein itself, will greatly aid efforts to define the role of this molecule in murine B cell development.
In vivo tumor immunotherapy by a bacterial superantigen.
Authors: Ochi A , Migita K , Xu J , Siminovitch K
Published: December 2022

We have investigated the in vivo efficacy of Staphylococcus aureus enterotoxin B (SEB) coupled to tumor-specific anti-idiotypic antibody in redirecting T cell effector activity to the growth inhibition of B lymphoma 38C13. Incubation of 38C13 lymphoma cells with syngeneic C3H/He splenic cells and SEB-anti-Id conjugate was associated with between 80 and 100% growth inhibition of the tumor cells. V beta 8+ T cells were integral for the SEB-anti-Id-induced tumor cell growth inhibition. Administration of SEB-anti-Id i.v. to mice previously inoculated with 38C13 lymphoma cells led to greater than 40% survival at 100 days compared to a mean survival of 21 days in control animals. When we compared this reagent with other targeting constructs--the anti-CD3-anti-Id and anti-TCR V beta 8-anti-Id--these more or less effectively prevented tumor growth. However, anti-CD3-anti-Id impaired almost the entire T cell response, whereas the effects of SEB-anti-Id or anti-V beta 8-anti-Id had effects limited to V beta 8+ T cells. Previous studies showed that in vivo administration of SEB caused a small change in V beta 8+ T cell numbers in contrast to anti-V beta 8 antibody, which depleted the entire population. These results together suggest that SEB-anti-tumor antibody conjugates represent a potentially powerful approach for better tumor immunotherapy.
Phosphoenolpyruvate and creatine phosphate augment ATP and magnesium-dependent, Fc epsilon RI-mediated activation of phospholipase C in RBL cell ghosts.
Authors: Dreskin S C , Kuhn D E , Huang Y
Published: December 2022

We have previously reported that Fc epsilon RI-mediated activation of PLC occurs in plasma membrane vesicles derived from RBL cells. This activity is dependent on ATP and magnesium, and is greatly enhanced by the addition of either PEP or CP. We undertook these studies to determine whether these compounds augment Fc epsilon RI-mediated activation of PLC because of their ability to form ATP. The specific findings were 1) the addition of increasing amounts of ATP to the ghost vesicles cannot substitute for the combination of ATP and either PEP or CP; 2) the addition of increasing amounts of ATP or a combination of ATP and either PEP or CP results in similar levels of intravesicular ATP; 3) many metabolically related compounds have some activity to support receptor-mediated PI hydrolysis, but only PEP, CP, and 3-PG give a maximal signal; and 4) the inability of ATP alone to support optimal receptor-mediated PI hydrolysis does not appear to be due to the accumulation of ADP is only modestly inhibitory. Taken together, these data are evidence that augmentation of Fc epsilon RI-mediated PI hydrolysis by PEP and CP is not explained simply by the ability of these compounds to generate intravesicular ATP.
IL-1 beta and IL-6 selectively induce transforming growth factor-beta isoforms in human articular chondrocytes.
Authors: Villiger P M , Kusari A B , ten Dijke P , Lotz M
Published: December 2022

Transforming growth factor-beta (TGF-beta) plays an important role in homeostasis of connective tissues, but regulation of its expression in mesenchymal cells is not well characterized. This study examines the effects of the cytokines IL-1 beta and IL-6 on expression of TGF-beta isoforms in human articular chondrocytes. IL-6 caused a fivefold increase, in the secretion of TGF-beta bioactivity by primary chondrocytes, whereas IL-1 beta showed only marginal stimulatory effects. Analysis by Northern blotting showed that IL-6 induced TGF-beta 1 gene expression but had no detectable effect on TGF-beta 2 mRNA levels and marginally increased TGF-beta 3 mRNA. However, IL-1 inhibited TGF-beta 1 mRNA expression induced by serum. In contrast, IL-1 beta strongly and selectively upregulated the TGF-beta 3 isoform. To determine whether this differential effect of IL-1 beta resulted in a corresponding change in protein synthesis, chondrocytes were metabolically labeled and analyzed by immunoprecipitation. IL-1 beta selectively induced TGF-beta 3 protein synthesis but reduced synthesis of the TGF-beta 1 and TGF-beta 2 isoforms. Consistent with the effects on TGF-beta 1 mRNA, IL-6 increased the synthesis of TGF-beta 1. These differential effects of the cytokines IL-1 beta and IL-6 provide new insight into the regulation of TGF-beta expression and may represent a protective mechanism against cytokine-induced connective tissue catabolism.
Generation of a human anti-idiotypic antibody that mimics the GD2 antigen.
Authors: Saleh M N , Stapleton J D , Khazaeli M B , LoBuglio A F
Published: December 2022

In a phase 1 trial, patients with metastatic melanoma received the anti-GD2 murine mAb 14G2a. All patients developed human anti-14G2a antibodies including anti-Id antibodies. Peripheral blood MNCs from one such patient were fused with the murine myeloma cell line Ag8. Four human anti-14G2a secreting hybridomas were generated and the mAb product of one of the hybridomas was characterized. The human mAb 4B5 (hu-IgG, lambda) binds to the variable region of murine 14G2a (anti-Id). The 4B5 binds to the antigen-combining site of 14G2a and inhibits its binding to GD2 expressing Mel-21 cells. Rabbits were immunized with the human anti-Id 4B5. Sera from the immunized rabbits demonstrated anti-4B5 antibodies and anti-Mel-21 and anti-GD2 reactivity. Furthermore, rabbit sera competitively inhibited binding of 14G2a to Mel-21 cells. Rabbits immunized with 4B5 developed a DTH response when challenged with 4B5 antibody and Mel-21 cells. These studies demonstrate that the human anti-Id 4B5 mimics the GD2 antigen and is capable of eliciting both a humoral and cellular anti-GD2 immune response. This antibody could be potentially used as a human anti-Id vaccine in patients with malignant melanoma.
Regulation of C gamma subclass germ-line transcripts in human peripheral blood B cells.
Authors: Kitani A , Strober W
Published: December 2022

In the present study we investigated the early steps of human IgG subclass differentiation by defining the conditions necessary for IgG subclass-specific production of germ-line transcripts in human peripheral blood B cells. Constant region of gamma-globulin (C gamma) subclass germ-line transcripts were measured using newly developed reverse transcription and polymerase chain reaction (RT-PCR) assays that were shown to be C gamma subclass-specific germ-line transcripts by size of the amplification products obtained before and after digestion with appropriate endonucleases (NarI and NcoI). In initial studies we found that C gamma 1 and C gamma 2 germ-line transcripts were constitutively expressed in total peripheral blood B cells, but not in high density sIgM+(sIgG-) B cells prepared with Percoll density gradients and magnetic beads separation techniques; the latter cells were therefore used throughout this study. Induction of germ-line transcripts (germ-line C gamma 1 transcript) was noted in stimulated B cells (SAC plus IL-2) at 6 h; thus, the appearance of germ-line transcripts could not be attributed to preferential growth of B cells expressing germ-line transcripts. In subsequent studies we found that induction of germ-line transcripts for the various IgG subclasses fell into two patterns. Induction of C gamma 3 and C gamma 1 germ-line transcripts, transcripts of genes in the first human duplication unit, generally required a proliferative stimulus (Staphylococcus aureus Cowan I, SAC) and was brought about by SAC plus IL-4 (C gamma 3 germ-line transcripts) and SAC alone or SAC plus IL-2 (C gamma 1 germ-line transcripts); in contrast, induction of C gamma 2 and C gamma 4 germ-line transcripts, transcripts of genes in the second duplication unit, was accomplished with cytokines alone: IFN-gamma (C gamma 2 germ-line transcripts) and IL-4 (C gamma 4 germ-line transcripts), and was not augmented by addition of a proliferative stimulus. Finally, we found that IFN-gamma inhibited IL-4-induced C gamma 3 and C gamma 4 germ-line transcripts (with or without SAC). These findings establish that the various human IgG subclasses manifest distinct requirements for the regulation of early steps in isotype differentiation. In addition, they suggest that human C gamma genes exhibit patterns of regulation related to their respective duplication units.