bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pro-Carboxypeptidase R is an Acute Phase Protein in the Mouse, Whereas Carboxypeptidase N Is Not
Authors: Sato Tomoo , Miwa Takashi , Akatsu Hiroyasu , Matsukawa Noriyuki , Obata Kyoko , Okada Noriko , Campbell William , Okada Hidechika 
Published: April 2014

Abstract
Carboxypeptidase R (EC 3.4.17.20; CPR) and carboxypeptidase N (EC 3.4.17.3; CPN) cleave carboxyl-terminal arginine and lysine residues from biologically active peptides such as kinins and anaphylatoxins, resulting in regulation of their biological activity. Human proCPR, also known as thrombin-activatable fibrinolysis inhibitor, plasma pro-carboxypeptidase B, and pro-carboxypeptidase U, is a plasma zymogen activated during coagulation. CPN, however, previously termed kininase I and anaphylatoxin inactivator, is present in a stable active form in plasma. We report here the isolation of mouse proCPR and CPN cDNA clones that can induce their respective enzymatic activities in culture supernatants of transiently transfected cells. Potato carboxypeptidase inhibitor can inhibit carboxypeptidase activity in culture medium of mouse proCPR-transfected cells. The expression of proCPR mRNA in murine liver is greatly enhanced following LPS injection, whereas CPN mRNA expression remains unaffected. Furthermore, the CPR activity in plasma increased 2-fold at 24 h after LPS treatment. Therefore, proCPR can be considered a type of acute phase protein, whereas CPN is not. An increase in CPR activity may facilitate rapid inactivation of inflammatory mediators generated at the site of Gram-negative bacterial infection and may consequently prevent septic shock. In view of the ability of proCPR to also inhibit fibrinolysis, an excess of proCPR induced by LPS may contribute to hypofibrinolysis in patients suffering from disseminated intravascular coagulation caused by sepsis.
Distal Recognition Site for Classical Pathway Convertase Located in the C345C/Netrin Module of Complement Component C5
Authors: Sandoval Ana , Ai Rong , Ostresh John M. , Ogata Ronald T. 
Published: April 2014

AbstractPrevious studies focused on indels in the complement C345 protein family identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the α-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSFRYIYPLD. Alanine substitutions affected activities in the highly variable manner characteristic of binding sites. Substitutions at the lysine or either phenylalanine residue in the central KYNFSF sequence had the greatest effects, yielding mutants with &lt;20% of the normal activity. These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to their hemolytic activities, but had normal susceptibilities to the cobra venom factor (CVF)-dependent convertase. Synthetic peptide MGKEALQIKYNFS-NH2 was found similarly to inhibit CP but not CVF convertase activation, and the effects of alanine substitutions in this peptide largely reflected those of the equivalent mutations in C5. These results indicate that residues KYNFSF form a novel, distal binding site for the CP, but not CVF convertase. This site lies ∼880 residues downstream of the convertase cleavage site within a module that has been independently named C345C and NTR; this module is found in diverse proteins including netrins and tissue inhibitors of metalloproteinases.
HLA-DR-Promiscuous T Cell Epitopes fromPlasmodium falciparumPre-Erythrocytic-Stage Antigens Restricted by Multiple HLA Class II Alleles
Authors: Doolan Denise L. , Southwood Scott , Chesnut Robert , Appella Ettore , Gomez Eduardo , Richards Allen , Higashimoto Yuichiro I. , Maewal Ajesh , Sidney John , Gramzinski Robert A. , Mason Carl , Koech Davy , Hoffman Stephen L. , Sette Alessandro 
Published: April 2014

AbstractPreviously, we identified and established the antigenicity of 17 CD8+ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN-γ responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8.7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.
Killer Cell Activating Receptors Function as Costimulatory Molecules on CD4+CD28null T Cells Clonally Expanded in Rheumatoid Arthritis
Authors: Namekawa Takashi , Snyder Melissa R. , Yen Jeng-Hsien , Goehring Brenda E. , Leibson Paul J. , Weyand Cornelia M. , Goronzy Jörg J. 
Published: April 2014

AbstractExpansion of CD4+CD28null T cells is a characteristic finding in patients with rheumatoid arthritis. Despite lacking CD28 molecules, these unusual CD4 T cells undergo clonal proliferation and form large and long-lived clonal populations. They produce high levels of IFN-γ, exhibit autoreactivity, and have cytolytic function. The mechanisms facilitating the expansion and longevity of CD4+CD28null T cell clones in vivo are unknown. Here, we report that CD4+CD28null, but not CD4+CD28+, T cells express MHC class I-recognizing receptors normally found on NK cells. CD4+CD28null T cells preferentially expressed killer cell activating receptors (KAR), often in the absence of killer cell inhibitory receptors. Cross-linking of KAR molecules enhanced the proliferative response to TCR-mediated stimulation, but not the cytolytic function of CD4+CD28null T cells, suggesting different signaling pathways in CD4 T cells and NK cells. Triggering of KAR signaling led to the phosphorylation of several cellular targets, although the pattern of phosphorylation differed from that induced by the TCR. Aberrant expression of KAR molecules in the absence of inhibitory receptors and in the appropriate HLA setting may lead to the clonal outgrowth of autoreactive CD4+CD28null T cells commonly seen in rheumatoid arthritis.
Identification of CD4+ T Cell Epitopes from NY-ESO-1 Presented by HLA-DR Molecules
Authors: Zeng Gang , Touloukian Christopher E. , Wang Xiang , Restifo Nicholas P. , Rosenberg Steven A. , Wang Rong-Fu 
Published: April 2014

Abstract
In previous studies, the shared cancer-testis Ag, NY-ESO-1, was demonstrated to be recognized by both Abs and CD8+ T cells. Gene expression of NY-ESO-1 was detected in many tumor types, including melanoma, breast, and lung cancers, but was not found in normal tissues, with the exception of testis. In this study, we describe the identification of MHC class II-restricted T cell epitopes from NY-ESO-1. Candidate CD4+ T cell peptides were first identified using HLA-DR4 transgenic mice immunized with the NY-ESO-1 protein. NY-ESO-1-specific CD4+ T cells were then generated from PBMC of a patient with melanoma stimulated with the candidate peptides in vitro. These CD4+ T cells recognized NY-ESO-1 peptides or protein pulsed on HLA-DR4+ EBV B cells, and also recognized tumor cells expressing HLA-DR4 and NY-ESO-1. A 10-mer peptide (VLLKEFTVSG) was recognized by CD4+ T cells. These studies provide new opportunities for developing more effective vaccine strategies by using tumor-specific CD4+ T cells. This approach may be applicable to the identification of CD4+ T cell epitopes from many known tumor Ags recognized by CD8+ T cells.
Cutting Edge: In Situ Tetramer Staining of Antigen-Specific T Cells in Tissues
Authors: Skinner Pamela J. , Daniels Mark A. , Schmidt Clint S. , Jameson Stephen C. , Haase Ashley T. 
Published: April 2014

AbstractStaining Ag-specific T cells with fluorescently labeled tetrameric MHC/peptide complexes has provided a powerful experimental approach to characterizing the immune response. In this report, we describe an extension of this method to directly visualize Ag-specific T cells in tissues. We successfully stained transgenic T cells with MHC tetramers in spleen sections from both 2C and OT-1 TCR transgenic mice. In addition, with the in situ tetramer staining technique, we detected a very small population of Ag-specific T cells in tissue after adoptive transfer of transgenic TCR T cells to a syngeneic nontransgenic mouse. We also show that the in situ tetramer technique can be applied to lightly fixed as well as frozen tissue, thus extending the method to archived tissue collections. This in situ tetramer staining technique offers a general approach to tracking the Ag-specific T cells in tissues.
Cutting Edge: IL-12 Is Required for the Maintenance of IFN-γ Production in T Cells Mediating Chronic Resistance to the Intracellular Pathogen,Toxoplasma gondii
Authors: Yap George , Pesin Michael , Sher Alan 
Published: April 2014

AbstractIL-12 is required for the development of IFN-γ-dependent resistance to intracellular pathogens but is not thought to play a major role in its maintenance. To directly assess the requirement for continuous IL-12 signaling in long-term cell-mediated immunity, recombinant cytokine was transiently administered to IL-12 p40-deficient mice during the first 2 wk of infection with the intracellular pathogen Toxoplasma gondii. As expected, these animals survived the acute phase and established chronic infections. However, 4–6 wk after IL-12 withdrawal, the mice exhibited increased brain cyst burdens and succumbed to toxoplasmic encephalitis. Reactivation was associated with a loss of T-dependent IFN-γ production without a concomitant increase in Th2 cytokine expression. Importantly, parasite Ag-induced IFN-γ synthesis by purified T cells from these animals could be restored by in vitro exposure to IL-12. These results argue that endogenous IL-12 is required for the long-term maintenance of IFN-γ-dependent resistance against intracellular pathogens.
The Abrogation of Allosensitization Following the Induction of Mixed Allogeneic Chimerism
Authors: Colson Yolonda L. , Schuchert Matthew J. , Ildstad Suzanne T. 
Published: April 2014

AbstractThe association of preformed anti-donor Abs with the hyperacute rejection of bone marrow and solid organ allografts and the persistence of the anti-donor immune response secondary to immunologic memory make allosensitization an absolute contraindication to transplantation. Mixed allogeneic (A + B→A) bone marrow chimerism has been demonstrated to confer donor-specific tolerance in nonsensitized recipients, but has not been evaluated in the setting of allosensitization. The current study documents that despite significant anti-donor sensitization, mixed allogeneic engraftment is possible and provides a marked advantage over fully allogeneic (B→A) models. Moreover, the acceptance of donor skin grafts and loss of circulating anti-donor Abs suggest that allosensitization can be abrogated with the induction of stable mixed allogeneic chimerism.
Langerhans Cells That Have Matured In Vivo in the Absence of T Cells Are Fully Capable of Inducing a Helper CD4 as Well as a Cytotoxic CD8 Response
Authors: De Creus An , Van Beneden Katrien , Taghon Tom , Stolz Frank , Debacker Veronique , Plum Jean , Leclercq Georges 
Published: April 2014

AbstractLangerhans cells (LCs) are immature dendritic cells (DCs) present in the skin epithelium. Upon Ag exposure, they migrate to the draining lymph nodes where they mature into potent stimulators of naive T cells. The aim of this study was to investigate the influence of T cells on LC migration and maturation. Therefore, the in vivo migration and maturation of LCs after sensitization with the hapten FITC was compared between C57BL/6 or BALB/c mice used as positive controls, and recombination activating gene (RAG) 1 knockout (−/−) mice or SCID mice used as T cell-deficient mice. Phenotypically, there was no difference between migrated LCs from RAG1−/− or SCID mice vs normal C57BL/6 or BALB/c mice: both populations of FITC+ cells had a dendritic morphology and a mature phenotype as they expressed high levels of MHC class II molecules and costimulatory molecules CD80, CD86, and CD54. Sorted migrated LCs of RAG1−/− or SCID mice were efficient stimulators of allogeneic T cells and Ag-specific CD4+ T cells. The same results were found if migrated LCs were fixed instead of irradiated, excluding the possibility that LCs derived from RAG1−/− or SCID mice would mature in the presence of T cells during the stimulation tests. Importantly, fixed migrated LCs of RAG1−/− mice were also efficient stimulators of cytotoxic CD8+ T cells. These data suggest that T cells are not required for full maturation of LCs.
Stimulation of the B Cell Receptor, CD86 (B7-2), and the β2-Adrenergic Receptor Intrinsically Modulates the Level of IgG1 and IgE Produced per B Cell
Authors: Kasprowicz Deborah J. , Kohm Adam P. , Berton Michael T. , Chruscinski Andrezj J. , Sharpe Arlene , Sanders Virginia M. 
Published: April 2014

AbstractOur findings using B cells from either wild-type, CD86-deficient, or β2-adrenergic receptor (β2AR)-deficient mice suggest three mechanisms by which the level of IgG1 and IgE production can be increased on a per cell basis. Trinitrophenyl-specific B cells enriched from unimmunized mouse spleens were pre-exposed to Ag and/or the β2AR ligand terbutaline for 24 h before being activated by either a β2AR-negative Th2 cell clone or CD40 ligand/Sf9 cells and IL-4 in the presence or absence of an anti-CD86 Ab. Data suggest that the first mechanism involves a B cell receptor (BCR)-dependent up-regulation of CD86 expression that, when CD86 is stimulated, increases the amount of IgG1 and IgE produced in comparison to unstimulated cells. The second mechanism involves a BCR- and β2AR-dependent up-regulation of CD86 to a level higher than that induced by stimulation of either receptor alone that, when CD86 is stimulated, further increases the amount of IgG1 and IgE produced. The third mechanism is BCR-independent and involves a β2AR-dependent increase in the ability of a B cell to respond to IL-4. Flow cytometric and limiting dilution analyses suggest that the increase in IgG1 and IgE occurs independently from the isotype switching event. These findings suggest that the BCR, the β2AR, and CD86 are involved in regulating IL-4-dependent IgG1 and IgE production.
Secondary V(D)J Rearrangements and B Cell Receptor-Mediated Down-Regulation of Recombination Activating Gene-2 Expression in a Murine B Cell Line
Authors: Maës Jérôme , Caspi Yael , Rougeon François , Haimovich Joseph , Goodhardt Michele 
Published: April 2014

AbstractIt has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD− murine B cell line, 38C-13, which has previously been found to undergo κ light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vκ-Jκ and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD− B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination.
Overexpression of the Proteasome Subunits LMP2, LMP7, and MECL-1, But Not PA28α/β, Enhances the Presentation of an Immunodominant Lymphocytic Choriomeningitis Virus T Cell Epitope
Authors: Schwarz Katrin , van den Broek Maries , Kostka Susanne , Kraft Regine , Soza Andrea , Schmidtke Gunter , Kloetzel Peter-M. , Groettrup Marcus 
Published: April 2014

AbstractThe proteasome is a large protease complex that generates most of the peptide ligands of MHC class I molecules either in their final form or in the form of N-terminally extended precursors. Upon the stimulation of cells with IFN-γ, three constitutively expressed subunits of the 20S proteasome are replaced by the inducible subunits LMP2 (low-molecular mass polypeptide 2), LMP7, and MECL-1 (multicatalytic endopeptidase complex-like-1) to form so-called immunoproteasomes. We show in this study that overexpression of these three subunits in triple transfectants led to a marked enhancement in the H-2Ld-restricted presentation of the immunodominant nonameric epitope NP118, which is derived from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. Overexpression of the α and β subunits of the IFN-γ-inducible proteasome regulator PA28, in contrast, did not have a comparable effect. In vitro, immunoproteasomes as compared with constitutive proteasomes generated higher amounts of 11- and 12-mer fragments containing the NP118 epitope. These are likely to be cytosolic precursors of NP118, as a proline anchor residue in the second position of NP118 may interfere with TAP-mediated transport of the nonameric epitope itself. In conclusion, we provide evidence that up-regulation of the three inducible subunits, LMP2, LMP7, and MECL-1, can result in a marked improvement of Ag presentation and that, depending on the epitope, PA28 and immunoproteasomes may differentially affect Ag processing.
A Role for the Region Encompassing the c″ Strand of a TCR Vα Domain in T Cell Activation Events
Authors: Qadri Ayub , Radu Caius G. , Thatte Jayant , Cianga Petru , Ober Bertram T. , Ober Raimund J. , Ward E. Sally 
Published: April 2014

AbstractThe distinct strand topology of TCR Vα domains results in a flatter surface in the region encompassing the c″ strand than the corresponding region in Ig V domains. In the current study a possible role for this region in T cell activation has been investigated by inserting a potential glycosylation site at Vα residue 82. This residue is in proximity to the c″ strand and distal to the putative interaction site for cognate peptide:MHC ligand. An additional N-linked carbohydrate at this position would create a protrusion on the Vα domain surface, and this may interfere with TCR aggregation and/or recruitment of signaling molecules. The modified TCR has been expressed in transfected T cells, and the phenotype following stimulation has been compared with that of cells expressing the wild-type TCR. The mutation has significant effects on activation-induced cell death and TCR internalization, but, unexpectedly, does not affect IL-2 secretion. Furthermore, analyses with tetrameric, peptide:MHC class II complexes suggest that the mutation decreases the ability of the TCR to aggregate into a configuration compatible with avid binding by these multivalent ligands.
B Cell Receptor Engagement and T Cell Contact Inducebcl-6Somatic Hypermutation in Human B Cells: Identity with Ig Hypermutation
Authors: Zan Hong , Li Zongdong , Yamaji Kozaburo , Dramitinos Patricia , Cerutti Andrea , Casali Paolo 
Published: April 2014

AbstractThe human bcl-6 proto-oncogene has been found to be mutated in both neoplastic and normal B cells. We used CL-01 cells, our monoclonal model of germinal center differentiation, and normal human B cells to explore the induction requirements and the modalities of bcl-6 hypermutation. As we have previously shown, CL-01 cells are IgM+ IgD+ and effectively mutate the expressed Ig VHDJH and VλJλ genes and switch to IgG, IgA, and IgE upon B cell receptor engagement and contact with CD4+ T cells through CD40:CD154 and CD80:CD28 coengagement. In this paper we showed that the same stimuli induce somatic hypermutation of bcl-6 in CL-01 and normal IgM+ IgD+ B cells. bcl-6 hypermutation was not accompanied by translocation of this proto-oncogene or hypermutation of the β-actin gene, and it did mimic Ig hypermutation. It was associated with transcription initiation, in that it targeted the first exon and a 696-bp sequence immediately downstream (∼0.6 kb) of the transcription initiation site while sparing further downstream (∼2.5 kb) and upstream (∼0.1 kb) areas. bcl-6 hypermutation displayed an overall rate of 2.2 × 10−4 changes/base/cell division with characteristic nucleotide preferences and showed strand polarity. These findings show that B cell receptor engagement promotes hypermutation in genes other than Ig, and suggest that cis-regulating elements similar to those of the Ig locus exist in bcl-6.
Underutilization of the Vκ10C Gene in the B Cell Repertoire Is Due to the Loss of Productive VJ Rearrangements During B Cell Development
Authors: Fitzsimmons Sean P. , Clark Kathleen J. , Mostowski Howard S. , Shapiro Marjorie A. 
Published: April 2014

AbstractThe Vκ10 family of murine light chain Ig genes is composed of three members, two of which (Vκ10A and Vκ10B) are well used. Vκ10C, the third member of this family, is not detected in any expressed Abs. Our previous work showed that Vκ10C is structurally functional and can recombine, but mRNA levels in spleen were extremely low relative to those of Vκ10A and Vκ10B. Furthermore, while the Vκ10C promoter was efficient in B cells, it was shown to work inefficiently in pre-B cell lines. Here, we extend our analysis of the Vκ10 family and examine Vκ10 gene accessibility, their representation in Vκ cDNA phage libraries, and the frequency and nature of rearrangements during different stages of B cell development. We demonstrate that Vκ10C is under-represented in Vκ cDNA libraries, but that the frequency of its sterile transcripts in pre-B cells surpasses both Vκ10A and Vκ10B, indicating that the gene is as accessible as Vκ10A and Vκ10B to the recombination machinery. We also demonstrate that Vκ10C recombines at a frequency equal to that of Vκ10A in pre-B cells and has a normal nonproductive to productive recombination ratio. As B cells develop, however, both the frequency of Vκ10C rearrangements and the presence of productive rearrangements decline, indicating that these cells are in some fashion being eliminated.