bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Improved Retroviral Gene Transfer Technique Demonstrates Inhibition of CD4−CD8− Thymocyte Development by Kinase-Inactive ZAP-70
Authors: Sugawara Takehiko , Di Bartolo Vincenzo , Miyazaki Tadaaki , Nakauchi Hiromitsu , Acuto Oreste , Takahama Yousuke 
Published: December 2022

AbstractZAP-70 is a Syk family tyrosine kinase that plays an essential role in initiating TCR signals. Deficiency in ZAP-70 causes a defect in the development at CD4+CD8+ thymocytes due to defective TCR-mediated positive and negative selection. Using a newly devised retrovirus gene transfer and an efficient green fluorescence protein detection technique in fetal thymus organ cultures, the present study shows that forced expression in developing thymocytes of a catalytically inactive mutant of ZAP-70, but not wild-type ZAP-70, inhibits T cell development at the earlier CD4−CD8− stage. The ZAP-70 mutant blocked the generation of CD4+CD8+ thymocytes even in the absence of endogenous ZAP-70. Thus, the present results demonstrate a novel technique for gene transfer into developing T cells and suggest that ZAP-70/Syk family tyrosine kinases are involved in the signals inducing the generation of CD4+CD8+ thymocytes.
The Inability of the Nonobese Diabetic Class II Molecule to Form Stable Peptide Complexes Does Not Reflect a Failure to Interact Productively with DM
Authors: Peterson Mary , Sant Andrea J. 
Published: December 2022

AbstractSequence variability in MHC class II molecules plays a major role in genetically determined susceptibility to insulin-dependent diabetes mellitus (IDDM). It is not yet clear whether MHC class II polymorphism allows selective binding of diabetogenic peptides or regulates some key intracellular events associated with class II-restricted Ag presentation. In this study, we have employed gene transfer techniques to analyze the intracellular events that control peptide acquisition by the unique class II molecule expressed by nonobese diabetic mice (I-Ag7). This structurally unique class II molecule fails to demonstrate stable binding to antigenic peptides and fails to undergo the conformational change associated with stable peptide binding to class II molecules. The experiments reported here demonstrate that I-Ag7 can productively associate with two protein cofactors important in class II-restricted Ag presentation, invariant chain (Ii) and DM. DM participates in the removal of the Ii-derived class II-associated Ii chain peptide and the p12 degradation product from the I-Ag7 molecule. In addition, I-Ag7 undergoes a conformational change when DM is expressed within the APC. Finally, DM can mediate accumulation of peptide/class II complexes on the surface of APCs. Collectively, our experiments indicate that the failure of the I-Ag7 molecule to stably bind peptide cannot be attributed to a failure to interact with the DM or Ii glycoproteins.
EBI1/CCR7 Is a New Member of Dendritic Cell Chemokine Receptor That Is Up-Regulated upon Maturation
Authors: Yanagihara Shigehiro , Komura Emiko , Nagafune Jun , Watarai Hiroshi , Yamaguchi Yasunori 
Published: December 2022

AbstractDendritic cells (DC) that are stimulated with inflammatory mediators can maturate and migrate from nonlymphoid tissues to lymphoid organs to initiate T cell-mediated immune responses. This migratory step is closely related to the maturation of the DC. In an attempt to identify chemokine receptors that might influence migration and are selectively expressed in mature DC, we have discovered that the chemokine receptor, EBI1/CCR7, is strikingly up-regulated upon maturation in three distinct culture systems: 1) mouse bone marrow-derived DC, 2) mouse epidermal Langerhans cells, and 3) human monocyte-derived DC. The EBI1/CCR7 expressed in mature DC is functional because ELC/MIP-3β, recently identified as a ligand of EBI1/CCR7, induces a rise in intracellular free calcium concentrations and directional migration of human monocyte-derived mature DC (HLA-DRhigh, CD1alow, CD14−, CD25+, CD83+, and CD86high) in a dose-dependent manner, but not of immature DC (HLA-DRlow, CD1ahigh, CD14−, CD25−, CD83−, and CD86−). In contrast, macrophage inflammatory protein-1α (MIP-1α), monocyte chemotactic protein-3 (MCP-3), and RANTES are active on immature DC but not on mature DC. Thus, it seems likely that MIP-1α, MCP-3, and RANTES can mediate the migration of immature DC located in peripheral sites, whereas ELC/MIP-3β can direct the migration of Ag-carrying DC from peripheral inflammatory sites, where DC are stimulated to up-regulate the expression of EBI1/CCR7, to lymphoid organs. It is postulated that different chemokines and chemokine receptors are involved in DC migration in vivo, depending on the maturation state of DC.
c-Fos Induces Apoptosis in Germinal Center B Cells
Authors: Inada Kunimasa , Okada Seiji , Phuchareon Janyaporn , Hatano Masahiko , Sugimoto Toyohiko , Moriya Hideshige , Tokuhisa Takeshi 
Published: December 2022

AbstractWe examined the role of c-Fos in the differentiation of mature B cells into IgG-producing cells using transgenic mice carrying the c-fos gene under the control of the IFN-α/β-inducible Mx promoter (Mx-c-fos) or the constitutive H-2Kb promoter (H2-c-fos). Splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4, and IgG1+ B cells were developed in the culture after day 3. When IFN-α/β was added to the culture from day 2, development of IgG1+ B cells was perturbed, and the number of apoptotic cells increased within 24 h, suggesting that c-Fos induces apoptosis in Ig class-switching B cells. To confirm the effect of c-Fos on B cell differentiation in vivo, H2-c-fos mice were immunized with DNP-OVA. The mice produced primary IgM, but not IgG, anti-DNP Ab in serum and failed to generate germinal centers in spleen. The perturbation of germinal center formation in H2-c-fos mice was rescued by mating them with transgenic mice carrying the bcl-2 gene with the Ig promoter. However, primary IgG1 anti-DNP Ab production was still suppressed in doubly transgenic mice, suggesting that Bcl-2 can delay the time of c-Fos-induced apoptosis in Ig class-switching B cells but cannot rescue the death. Since c-Fos is induced in mature B cells reacted with Ags, and clonal deletion of self-reactive B cells in germinal centers is insensitive to Bcl-2, these results suggest that c-Fos plays a causal role in clonal deletion of germinal center B cells.
Therapy with Monoclonal Antibodies. II. The Contribution of Fcγ Receptor Binding and the Influence of CH1 and CH3 Domains on In Vivo Effector Function
Authors: Isaacs John D. , Greenwood Judith , Waldmann Herman 
Published: December 2022

AbstractAn in vivo model is used to define Fc motifs engaged by mAbs to deplete target cells. Human IgG1 and human IgG4 were very potent, and mutations within a motif critical for FcγR binding (glutamate 233 to proline, leucine/phenylalanine 234 to valine, and leucine 235 to alanine) completely prevented depletion. Mouse IgG2b was also potent, and mutations to prevent complement activation did not impair depletion with this isotype, as previously shown for human IgG1. In contrast, a mutation that impaired binding to mouse FcγRII (glutamate 318 to alanine) eliminated effector function of mouse IgG2b and also reduced the potency of human IgG4. To reveal potential contributions of domains other than CH2, domain switch mutants were created between human IgG1 and rat IgG2a. Two hybrid mAbs were generated with potencies exceeding anything previously seen in this model. While their mechanism of depletion was not defined, their activity appeared dependent upon interdomain interactions in the Fc region.
Comparisons of the Ability of Human IgG3 Hinge Mutants, IgM, IgE, and IgA2, to Form Small Immune Complexes: A Role for Flexibility and Geometry
Authors: Roux Kenneth H. , Strelets Lioudmila , Brekke Ole Henrik , Sandlie Inger , Michaelsen Terje E. 
Published: December 2022

AbstractVarious native and hinge-modified forms of Ig with identical Ids were reacted with an anti-Id mAb, and the resultant immune complexes were analyzed by negative stain immunoelectron microscopy. Complexes were scored for their geometry (linear versus ring complexes) and size (dimer, trimer, etc.). Ring dimers are the thermodynamically most favorable configuration, unless inhibited by steric and/or flexibility constraints. We found ring dimerization to correlate with the length of the upper, but not middle or lower, hinge. In contrast, the geometry and size of complexes of those molecules lacking formal hinges were unpredictable. A hingeless IgG mutant and native IgE readily formed ring dimers. Remarkably, monomeric IgM formed more ring dimers than any of the other Igs tested, including IgG3. We also tagged the Fab arms and measured the mean Fab-Fab angles and the degree of angular variation for each type of Ig. Surprisingly, IgM proved the most flexible by this assay. In hinged Igs, there was a correlation between length of the upper hinge and Fab-Fab flexibility. In contrast, we found no correlation between the mean Fab-Fab angle in uncomplexed Igs and their ability to dimerize with anti-Id mAb. These data suggest that the physicochemical methods typically used to evaluate molecular flexibility are often of low predictive value when tested in a functional assay.
Heat-KilledListeria monocytogenesas an Adjuvant Converts Established Murine Th2-Dominated Immune Responses into Th1-Dominated Responses
Authors: Yeung V. Peter , Gieni Randall S. , Umetsu Dale T. , DeKruyff Rosemarie H. 
Published: December 2022

AbstractWe investigated the capacity of heat-killed Listeria monocytogenes (HKL), a potent stimulator of the innate immune system, as a vaccine adjuvant to modify both primary and secondary Ag-specific immune responses. Mice immunized with the Ag keyhole limpet hemocyanin (KLH) mixed with HKL generated a KLH-specific primary response characterized by production of Th1 cytokines and large quantities of KLH-specific IgG2a Ab. Moreover, administration of KLH with HKL as an adjuvant reversed established immune responses dominated by the production of Th2 cytokines and high levels of KLH-specific IgE and induced a Th1-type response with high levels of IFN-γ and IgG2a and low levels of IgE and IL-4. Neutralization of IL-12 activity at the time of HKL administration blocked the enhancement of IFN-γ and reduction of IL-4 production, indicating that IL-12, induced by HKL, was responsible for the adjuvant effects on cytokine production. These results suggest that HKL as an adjuvant during immunization can successfully bias the development of Ag-specific cytokine synthesis toward Th1 cytokine production even in the setting of an ongoing Th2-dominated response. Thus, HKL may be clinically effective in vaccine therapies for diseases such as allergy and asthma, which require the conversion of Th2-dominated immune responses into Th1-dominated responses.
Oleic Acid Increases Cell Surface Expression and Activity of CD11b on Human Neutrophils
Authors: Mastrangelo Anthony M. , Jeitner Thomas M. , Eaton John W. 
Published: December 2022

Abstract
Traumatic bone injury frequently results in the release of marrow-derived fatty material into the circulation. This may lead to the syndrome of fat embolism, associated with the generation of free fatty acids, the sequestration of neutrophils in the lungs, and the subsequent development of acute respiratory distress. Neutrophil accumulation in tissues requires their adherence to vascular endothelial cells and involves the β2 integrin, CD11b/CD18 (Mac-1). We now report that the exposure of isolated human neutrophils to oleic acid causes a rapid increase in the cell surface expression and affinity state of CD11b, particularly under acidic conditions that are typical of inflammatory sites. Oleic acid also triggers neutrophil aggregation and neutrophil adherence to both fibrinogen-coated surfaces and confluent cultures of HUVEC. These processes are blocked by CD11b-specific inhibitors, including neutrophil-inhibitory factor and mAbs to CD11b. These observations may help explain the etiology of so-called fat embolism wherein trauma-induced release of fatty material causes pulmonary neutrophil accumulation and the development of acute respiratory distress.
Evidence for a Critical Role for IL-2 in CD40-Mediated Activation of Naive B Cells by Primary CD4 T Cells
Authors: Johnson-Léger Caroline , Christenson Jillian R. , Holman Mary , Klaus Gerry G. B. 
Published: December 2022

AbstractThe interaction of CD40 on B cells with the CD40 ligand (CD40L) on preactivated CD4 T cells is critical for the initiation of T-dependent Ab responses. It is believed that signals via CD40 synergize with cytokines (e.g., IL-4 and IL-5) to drive B cell activation. However, primary T cells preactivated via CD3 alone cannot induce B cell proliferation; we have shown previously that costimulation of T cells via CD3 and CD28 stabilizes the expression of the CD40L, which we propose contributes to their capacity to act as competent helper-effector cells. Here we show that an additional, critical reason why CD3-stimulated CD40L-bearing T cells are incompetent helper cells is because they secrete insufficient IL-2. In contrast, CD28/CD3-activated T cells induce B cells to become IL-2 responsive via a combination of CD40L and IL-2-mediated signals, and these two stimuli subsequently drive B cell proliferation and IgM secretion. We therefore propose that T cells must first encounter Ag in conjunction with CD80/86 on APCs. This leads to the stable expression of CD40L and maximal secretion of IL-2, which together render primary T cells competent to activate B cells in an IL-2-dependent fashion.
Role of T Cells and Germinal Center Formation in the Generation of Immune Responses to the Thymus-Independent Carbohydrate Dextran B512
Authors: Sverremark Eva , Fernandez Carmen 
Published: December 2022

AbstractImmunization with the thymus-independent (TI) Ag native dextran (DX) B512 induces germinal center (GC) formation in the spleen. However, despite this GC formation, the anti-DX response is poor, and no affinity maturation can be observed. Using cholera toxin (CT) as an adjuvant, splenic as well as humoral responses to DX are improved. In this study, we investigated immune responses against DX in mice lacking TNF receptor I and in athymic mice. The adjuvant effect of CT on these responses was also evaluated. Mice lacking the TNF receptor I allowed us to investigate the role of follicular dendritic cell networks and GC formation in the spleen for the generation of Ab responses to DX, whereas we could investigate the role of T cells in GC development to TI Ags using athymic mice. We found that the humoral immune response to TI DX B512 was not dependent upon T cells or the presence of GCs, although GC development occurred after DX immunization. However, T cells were required for this GC formation, since athymic mice could not develop GCs after immunization with DX. We also show that even if CT is able to directly activate B cells when administered as an adjuvant, the major effect may require T cell participation; this is also the case for TI Ags. In contrast, CT adjuvancy is independent of GC formation.
Human Endothelial Cells Induce and Regulate Cytolytic T Cell Differentiation
Authors: Biedermann Barbara C. , Pober Jordan S. 
Published: December 2022

AbstractWe compared the capacity of cultured human endothelial cells (EC) vs B lymphoblastoid cells (BLC) from the same donor to stimulate allogeneic CD8+ T cells to differentiate into CTL, assaying for allorestricted cytotoxicity, T cell growth, IFN-γ secretion, and perforin expression. The input cell number affected specificity and potency of the resulting CTL. At low input (&lt;105 cells/well), anti-EC CTL were rarely detected. At high input (&gt;106 cells/well), anti-EC CTL developed that displayed unrestricted, low-titer killing and an unstable phenotype. At intermediate input (1.0–2.5 × 105 cells/well), classical class I MHC-restricted, CD8+, and perforin-positive anti-EC CTL developed with reproducible frequencies. However, under all conditions EC were less efficient stimulators than BLC from the same donor. Anti-EC CTL did not kill BLC, whereas anti-BLC CTL killed BLC and EC from the same donor with comparable efficiency. When CD8+ T lymphocytes were grown in the presence of EC and BLC together, the differentiation of anti-BLC CTL was completely suppressed, while the anti-EC response was intact. The inhibition of the allogeneic anti-BLC CTL response was independent of T cell-EC contact, and proliferation of CD8+ T cells was inhibited by EC-conditioned medium. We conclude that EC are competent but less efficient activators of CTL differentiation than are BLC and that EC actively regulate differentiation and/or expansion of allospecific CTL.
Ligation of Cell Surface CD38 Protein with Agonistic Monoclonal Antibody Induces a Cell Growth Signal in Myeloid Leukemia Cells
Authors: Konopleva Marina , Estrov Zeev , Zhao Shourong , Andreeff Michael , Mehta Kapil 
Published: December 2022

AbstractCD38 is expressed during early stages of differentiation in normal and leukemic myeloid cells. Recently, CD38 has been shown to participate in intracellular signal transduction pathways following its ligation with CD38-specific mAbs. In this study we report that ligation of CD38 by one such agonistic mAb (IB4) induced proliferation of cultured leukemic cells in vitro. In HL-60, KG-1A, NB4, and OCI-AML-3 myeloid leukemia cell lines, IB4 mAb induced an increase in the proliferating cell fraction as determined by cell number, clonogenic assay, and flow cytometric analysis. The presence of Ab caused a dose-dependent increase in the number of CFU and an increase in cell divisions. HL-60-Dox cells (a HL-60-doxorubicin-resistant cell line), which have no detectable CD38 expression, failed to respond to IB4 mAb. The effect of CD38 ligation on cell growth was also evaluated in freshly isolated leukemic cells from patients with acute myelogenous leukemia (AML). A significant increase in the proliferating cell fraction (S+G2M) was observed in 50% of the patients incubated with IB4 mAb. In five of the six AML patients, anti-CD38 mAb stimulated the proliferation of AML colony-forming cells. These results suggest that ligation of CD38 can induce the proliferation of leukemic cells and may play a role in the propagation of leukemic cell clones in certain cohorts of AML patients.
A Molecular Basis for How a Single TCR Interfaces Multiple Ligands
Authors: Boesteanu Alina , Brehm Michael , Mylin Lawrence M. , Christianson Gregory J. , Tevethia Satvir S. , Roopenian Derry C. , Joyce Sebastian 
Published: December 2022

AbstractCD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.
Two Diverged Complement Factor B/C2-Like cDNA Sequences from a Teleost, the Common Carp (Cyprinus carpio)1, 2
Authors: Nakao Miki , Fushitani Yuko , Fujiki Kazuhiro , Nonaka Masaru , Yano Tomoki 
Published: December 2022

AbstractMammalian complement components factor B and C2 act as proteolytic subunits of the C3 convertases in the alternative and the classical activation pathways, respectively, and are believed to have diverged from a common ancestor by gene duplication. However, it is unclear when the B/C2 duplication occurred. Here, we describe two diverged B/C2-like cDNA clones (B/C2-A and B/C2-B) isolated from a bony fish, the common carp (Cyprinus carpio). B/C2-A shares the same domain structure as the factor B and C2 complement components of vertebrates reported so far and shows a close similarity to zebrafish B and medaka fish B/C2. These teleost sequences show almost the same degree of similarity to C2 and B of higher vertebrates. In contrast, B/C2-B has a novel structural feature in that it contains four short consensus repeat modules and does not have a close relative upon phylogenetic analysis. Northern blotting revealed the presence of two transcripts with different sizes for both the B/C2-A and B/C2-B in the hepatopancreas of the carp. Southern blotting suggested the presence of multiple genes for B/C2-A and a single gene for B/C2-B. Although structural features of B/C2-B are slightly more C2-like than B-like, B/C2-B has a crucial amino acid substitution in the serine protease domain, which makes it unlikely that B/C2-B functions as a C3 convertase. A possible phylogenetic relationship between the two carp sequences and mammalian C2 and B is discussed.
Mechanism of Catecholamine-Mediated Destabilization of Messenger RNA Encoding Thy-1 Protein in T-Lineage Cells
Authors: Wajeman-Chao Sophie A. , Lancaster Susan A. , Graf Jr. Lloyd H. , Chambers Donald A. 
Published: December 2022

AbstractThe Ig superfamily cell surface glycoprotein Thy-1 expressed on immune cells and neurons of rodents and humans is hypothesized to function in cell adhesion and signal transduction in T cell differentiation, proliferation, and apoptosis. This study analyzes effects of cAMP and catecholamines on transcriptional Thy-1 gene expression. Incubation of murine thymocytes or S49 mouse thymoma cells with dibutyryl-cAMP, 8-bromo-cAMP, cholera toxin, norepinephrine, or isoproterenol caused time- and concentration-dependent decreases in levels of Thy-1 mRNA assayed by Northern hybridization or T2 nuclease protection. After 4 h of treatment with 500 μM dibutyryl-cAMP or 8-bromo-cAMP, 1 nM cholera toxin, 100 μM norepinephrine, or 100 μM isoproterenol, Thy-1 mRNA levels were 60 to 96% lower than those of controls. Norepinephrine-mediated decreases in Thy-1 mRNA levels were prevented by the β-adrenergic receptor antagonist propranolol (10 μM). Dibutyryl-cAMP and norepinephrine decreased the apparent half-life of S49 cell Thy-1 mRNA from ≫6 h to 2 to 3 h, whereas nuclear run-on assays showed no cAMP or norepinephrine effect on de novo transcription of the Thy-1 gene. In mutant S49 cells lacking cAMP-dependent protein kinase A, neither dibutyryl cAMP nor norepinephrine affected Thy-1 mRNA levels. These observations show that exogenous cAMP and norepinephrine can induce decreases in steady state Thy-1 mRNA levels in T-lineage cells through posttranscriptional destabilization of Thy-1 mRNA, associated with protein kinase A-mediated protein phosphorylation. Catecholamine-mediated β-adrenergic protein kinase A-dependent Thy-1 mRNA destabilization may be an example of a more general mRNA decay system regulating cellular responses to stress.