bluuebunny/crossref_metadata_2025_split huggingface dataset:
Murine Neonatal CD4+ Lymph Node Cells Are Highly Deficient in the Development of Antigen-Specific Th1 Function in Adoptive Adult Hosts
Authors: Adkins Becky , Bu Yurong , Guevara Patricia 
Published: April 2014

Abstract
It is well established that murine neonates are biased toward Th2 responses. Th2-dominant responses are observed following immunization with a variety of Ags, using different carrier/adjuvant systems, and are seen in both BALB/c and C57BL/6 mice. Therefore, Th2 skewing appears to be a universal phenomenon unique to the neonatal period. One important question about this phenomenon is whether these responses are due to T cell intrinsic properties or are regulated by the neonatal environment. Here we have addressed this issue by transferring neonatal or adult CD4+ lymph node cells to adoptive adult recombinase-activating gene 2−/− hosts and studied the development of Th responses. Neonatal CD4+ cells were highly deficient in the development of both primary and secondary Ag-specific Th1 responses. This did not appear to be due to anergy of a developed population, since exogenous IL-2 only marginally increased production of the Th1 cytokine IFN-γ. This profound Th1 deficiency was observed despite similar proliferation by neonatal and adult cells within the recombinase-activating gene 2−/− hosts. Moreover, neonatal CD4+ cells up-regulated activation markers in a manner similar to adult CD4+ cells. Therefore, although their proliferation and phenotypic maturation proceeded normally, neonatal CD4+ cells appeared to be intrinsically deficient in the functional maturation of Th1 lineage cells. These results offer a candidate explanation for the reduced graft-vs-host responses observed following transplantation of cord blood cells or murine neonatal lymphoid cells to allogeneic adult hosts.
Age-Dependent Defects in TCR-Triggered Cytoskeletal Rearrangement in CD4+ T Cells
Authors: Garcia Gonzalo G. , Miller Richard A. 
Published: April 2014

Abstract
Previous research has shown that many of the CD4 T cells from older mice do not form functional immune synapses after conjugation with peptide-pulsed APC. We now show that the defect lies at a very early stage in the cytoskeletal reorganization that precedes movement of protein kinases and their substrates to the TCR/APC interface. Antagonist peptides presented to T cells from young mice induce migration of talin (but not paxillin, vinculin, or F-actin) to the APC contact zone, but CD4 T cells from older donors typically fail to show the talin polarization response. A spreading assay in which contact with anti-CD3-coated slides induces CD4 T cells to assume a conical shape and develop lammelopodia also shows a decline with age in the proportion of T cells that can initiate cytoskeletal changes in response to this simplified stimulus. Finally, the transition from detergent-soluble to cytoskeletal forms of the p16, p21, and p23 isoforms of CD3ζ in response to CD3/CD4/CD28 cross-linking is much stronger in young than in old T cells. Thus, defects in cytoskeletal reorganization triggered by initial contact between TCR and peptide-bearing APC precede, and presumably contribute to, defective activation of protein kinase-mediated signals in the first few minutes of the activation cascade in T cells from aged mice.
Transcriptional Regulation of the MHC Class II Trans-Activator (CIITA) Promoter III: Identification of a Novel Regulatory Region in the 5′-Untranslated Region and an Important Role for cAMP-Responsive Element Binding Protein 1 and Activating Transcription Factor-1 in CIITA-Promoter III Transcriptional Activation in B Lymphocytes
Authors: van der Stoep Nienke , Quinten Edwin , van den Elsen Peter J. 
Published: April 2014

Abstract
The class II trans-activator (CIITA), which acts as a master regulator for expression of MHC class II genes, is expressed constitutively in mature B cells. This constitutive expression of CIITA is driven by CIITA promoter III (CIITA-PIII). However, little is known about the factors that control the B cell-mediated trans-activation of CIITA-PIII. In this study using B cells we have identified several cAMP-responsive elements (CREs) in the proximal promoter and in the 5′-untranslated region (5′-UTR) that are involved in the activation of CIITA-PIII. We show that activating transcription factor (ATF)/CRE binding protein (CREB) factors bind to the CREs in vitro and in vivo. Notably, our results also reveal that the 5′-UTR of CIITA-PIII functions as an important regulatory region in B lymphocytes. Furthermore, transient cotransfections of a CIITA-PIII luciferase reporter construct with either CREB-1 or ATF-1 boost CIITA-PIII trans-activation in a dose-dependent manner, which was further enhanced by addition of general coactivator CREB-binding protein. Transient transfections using mutant CIITA-PIII luciferase reporter constructs that either lack the (5′-UTR) or abolish binding of CREB-1 and ATF-1 to the CRE located in activation response element-2, displayed severely reduced promoter activity in B cells. A similar successive deletion of the CREs resulted in a subsequent reduction of CREB-1-induced activity of CIITA-PIII in B cells. Together our results argue for an important role of ATF/CREB factors and the 5′-UTR of CIITA-PIII in the trans-activation of CIITA-PIII in B cells.
Structural Factors Contributing to DM Susceptibility of MHC Class II/Peptide Complexes
Authors: Belmares Michael P. , Busch Robert , Wucherpfennig Kai W. , McConnell Harden M. , Mellins Elizabeth D. 
Published: April 2014

AbstractPeptide loading of MHC class II (MHCII) molecules is assisted by HLA-DM, which releases invariant chain peptides from newly synthesized MHCII and edits the peptide repertoire. Determinants of susceptibility of peptide/MHCII complexes to DM remain controversial, however. Here we have measured peptide dissociation in the presence and the absence of DM for 36 different complexes of varying intrinsic stability. We found large variations in DM susceptibility for different complexes using either soluble or full-length HLA-DM. The DM effect was significantly less for unstable complexes than for stable ones, although this correlation was modest. Peptide sequence- and allele-dependent interactions along the entire length of the Ag binding groove influenced DM susceptibility. We also observed differences in DM susceptibility during peptide association. Thus, the peptide repertoire displayed to CD4+ T cells is the result of a mechanistically complicated editing process and cannot be simply predicted from the intrinsic stability of the complexes in the absence of DM.
Killer Ig-Like Receptor Haplotype Analysis by Gene Content: Evidence for Genomic Diversity with a Minimum of Six Basic Framework Haplotypes, Each with Multiple Subsets
Authors: Hsu Katharine C. , Liu Xiao-Rong , Selvakumar Annamalai , Mickelson Eric , O’Reilly Richard J. , Dupont Bo 
Published: April 2014

Abstract
Killer Ig-like receptor (KIR) genes constitute a multigene family whose genomic diversity is achieved through differences in gene content and allelic polymorphism. KIR haplotypes containing a single activating KIR gene (A-haplotypes), and KIR haplotypes with multiple activating receptor genes (B-haplotypes) have been described. We report the evaluation of KIR gene content in extended families, sibling pairs, and an unrelated Caucasian panel through identification of the presence or absence of 14 KIR genes and 2 pseudogenes. Haplotype definition included subtyping for the expressed and nonexpressed KIR2DL5 variants, for two alleles of pseudogene 3DP1, and for two alleles of 2DS4, including a novel 2DS4 allele, KIR1D. KIR1D appears functionally homologous to the rhesus monkey KIR1D and likely arose as a consequence of a 22 nucleotide deletion in the coding sequence of 2DS4, leading to disruption of Ig-domain 2D and a premature termination codon following the first amino acid in the putative transmembrane domain. Our investigations identified 11 haplotypes within 12 families. From 49 sibling pairs and 17 consanguineous DNA samples, an additional 12 haplotypes were predicted. Our studies support a model for KIR haplotype diversity based on six basic gene compositions. We suggest that the centromeric half of the KIR genomic region is comprised of three major combinations, while the telomeric half can assume a short form with either 2DS4 or KIR1D or a long form with multiple combinations of several stimulatory KIR genes. Additional rare haplotypes can be identified, and may have arisen by gene duplication, intergenic recombination, or deletions.
Ly-6 Superfamily Members Ly-6A/E, Ly-6C, and Ly-6I Recognize Two Potential Ligands Expressed by B Lymphocytes
Authors: Pflugh David L. , Maher Stephen E. , Bothwell Alfred L. M. 
Published: April 2014

AbstractMost hemopoietic cells express one or more members of the Ly-6 supergene family of small glycosylphosphatidylinositol-linked proteins. Although levels of Ly-6 proteins vary with stages of differentiation and activation, their function largely remains unknown. To ascertain whether ligands for Ly-6 proteins exist, chimeric proteins were constructed in which Ly-6E, Ly-6C, and Ly-6I were fused to the murine IgM heavy chain. These chimeras specifically stained both developing and mature B lymphocytes, as assessed by flow cytometry. Analysis of variants of the CH27 B cell lymphoma revealed that Ly-6A/E and Ly-6I recognized different molecules. CH27 cells with low levels of Ly-6A/E ligand activity also lost expression of CD22, and cells transfected with CD22 gained the ability to bind the Ly-6A/E chimera and, to a lesser extent, the Ly-6C and Ly-6I chimeric proteins. As many mature B cells coexpress Ly-6A/E and CD22, the function of Ly-6 molecules may be to associate with other membrane proteins, possibly concentrating these ligands in lipid rafts, rather than acting directly as cell:cell adhesion molecules.
C-Terminal Cysteine Residues Determine the IgE Binding ofAspergillus fumigatusAllergen Asp f 2
Authors: Banerjee Banani , Kurup Viswanath P. , Greenberger Paul A. , Kelly Kevin J. , Fink Jordan N. 
Published: April 2014

AbstractThe knowledge of the structure function relationship of the allergen is essential to design allergenic variants with reduced IgE binding capacity but intact T cell reactivity. Asp f 2 is a major allergen from the fungus Aspergillus fumigatus and &gt;90% of A. fumigatus-sensitized individuals displayed IgE binding to Asp f 2. In the present study, we evaluated the involvement of C-terminal cysteine residues in IgE binding conformation of Asp f 2. The deletion mutants were constructed by adding three C-terminal cysteines of the native Asp f 2 one at a time to the non-IgE binding Asp f 2 (68–203). The point mutants of Asp f 2 (68–268) with C204A and C257A substitutions were constructed to study the role of C-terminal cysteines in IgE binding. Immunological evaluation of reduced and alkylated Asp f 2 and its mutants were conducted to determine the contribution of free sulfhydryl groups as well as the disulfide bonds in allergen Ab interaction. Four-fold increase in IgE Ab binding of Asp f 2 (68–267) compared with Asp f 2 (68–266) and complete loss in IgE binding of C204A mutant of Asp f 2 (68–268) indicate the involvement of C204 and C267 in IgE binding conformation of Asp f 2. A significant reduction in IgE binding of wild and mutated Asp f 2 after reduction and alkylation emphasizes the importance of cysteine disulfide bonds in epitope Ab interaction. The hypoallergenic variants may be explored further to develop safe immunotherapeutic strategy for allergic disorders.
The Structure of HLA-B8 Complexed to an Immunodominant Viral Determinant: Peptide-Induced Conformational Changes and a Mode of MHC Class I Dimerization
Authors: Kjer-Nielsen Lars , Clements Craig S. , Brooks Andrew G. , Purcell Anthony W. , Fontes Marcos R. , McCluskey James , Rossjohn Jamie 
Published: April 2014

Abstract
EBV is a ubiquitous human pathogen that chronically infects up to 90% of the population. Persistent viral infection is characterized by latency and periods of viral replication that are kept in check by a strong antiviral CTL response. Despite the size of the EBV genome, CTL immunity focuses on only a few viral determinants but expands a large primary and memory response toward these epitopes. In unrelated HLA-B8+ individuals, the response to the immunodominant latent Ag FLRGRAYGL from Epstein Barr nuclear Ag 3A is largely comprised of CTL clones with identical conserved αβ TCR structures. To better understand the structural correlates of Ag immunodominance and TCR selection bias, we have solved the crystal structure of the HLA-B8-FLRGRAYGL peptide complex to a resolution of 1.9 Å. The structure confirms the importance of P3-Arg, P5-Arg, and P9-Leu as dominant anchor residues involved in peptide binding to HLA-B8. A bulged conformation of the bound peptide provides a structural basis for the critical role of the P7-Tyr residue in T cell recognition. The peptide also induces backbone and side-chain conformational changes in HLA-B8 that are transmitted along the peptide-binding groove in a domino effect. The HLA-B8-FLRGRAYGL complex crystallizes as a dimer in the asymmetric unit and is oriented such that both peptide ligands are projected in the same plane suggesting a higher order arrangement of MHC-peptide complexes that could be involved in formation of the class I Ag-loading complex or in T cell activation.
Increasing the Affinity of a Human IgG1 for the Neonatal Fc Receptor: Biological Consequences
Authors: Acqua William F. Dall’ , Woods Robert M. , Ward E. Sally , Palaszynski Susan R. , Patel Nita K. , Brewah Yambasu A. , Wu Herren , Kiener Peter A. , Langermann Solomon 
Published: April 2014

Abstract
Many biological functions, including control of the homeostasis and maternofetal transfer of serum γ-globulins, are mediated by the MHC class I-related neonatal FcR (FcRn). A correlation exists in mice between the binding affinity of IgG1/Fc fragments to FcRn at pH 6.0 and their serum t1/2. To expand this observation, phage display of mutagenized Fc fragments derived from a human IgG1 was used to increase their affinity to both murine and human FcRn. Ten variants were identified that have a higher affinity toward murine and human FcRn at pH 6.0, with ΔΔG (ΔGwild type − ΔGmutant) from 1.0 to 2.0 kcal/mol and from 0.6 to 2.4 kcal/mol, respectively. Those variants exhibit a parallel increase in binding at pH 7.4 to murine, but not human, FcRn. Although not degraded in blood in vitro, accumulated in tissues, nor excreted in urine, their serum concentration in mice is decreased. We propose that higher affinity to FcRn at pH 7.4 adversely affects release into the serum and offsets the benefit of the enhanced binding at pH 6.0.
Critical Role of Mitochondrial Damage in Determining Outcome of Macrophage Infection with Mycobacterium tuberculosis
Authors: Duan Lei , Gan Huixian , Golan David E. , Remold Heinz G. 
Published: April 2014

Abstract
Human macrophages (Mφ) respond to Mycobacterium tuberculosis (Mtb) infection by undergoing apoptosis, a cornerstone of effective antimycobacterial host defense. Virulent mycobacteria override this reaction by inducing necrosis leading to uncontrolled Mtb replication. Accordingly, Mφ death induced by inoculation with Mtb had the characteristics of apoptosis and necrosis and correlated with moderate increase of mitochondrial permeability transition (MPT), mitochondrial cytochrome c release, and caspase-9 and -3 activation. We hypothesized that changes in intramitochondrial Ca2+ concentration ([Ca2+]m) determine whether Mφ undergo either apoptosis or necrosis. Therefore, we induced mechanism(s) leading to predominant apoptosis or necrosis by modulating [Ca2+]m and examined their physiological consequences. Adding calcium ionophore A23187 to Mφ inoculated with Mtb further increased calcium flux into the cells which is thought to lead to increased [Ca2+]m, blocked necrosis, stabilized MPT, decreased mitochondrial cytochrome c release, lowered caspase activation, and accompanied effective antimycobacterial activity. In contrast, Mφ infected with Mtb in presence of the mitochondrial calcium uniporter inhibitor ruthenium red showed increased mitochondrial swelling and cytochrome c release and decreased MPT and antimycobacterial activity. Thus, in Mtb-infected Mφ, high levels of mitochondrial membrane integrity, low levels of caspase activation, and diminished mitochondrial cytochrome c release are hallmarks of apoptosis and effective antimycobacterial activity. In contrast, breakdown of mitochondrial membrane integrity and increased caspase activation are characteristic of necrosis and uncontrolled Mtb replication.
Secretion of Oncostatin M by Infiltrating Neutrophils: Regulation of IL-6 and Chemokine Expression in Human Mesothelial Cells
Authors: Hurst Suzanne M. , McLoughlin Rachel M. , Monslow James , Owens Sara , Morgan Llinos , Fuller Gerald M. , Topley Nicholas , Jones Simon A. 
Published: April 2014

Abstract
Recently, we identified that regulation of leukocyte recruitment by IL-6 requires shedding of the IL-6R from infiltrating neutrophils. In this study, experiments have examined whether other IL-6-related cytokines possess similar properties. Levels of oncostatin M (OSM) and leukemia inhibitory factor were analyzed in patients with overt bacterial peritonitis during the first 5 days of infection. Although no change in leukemia inhibitory factor was observed throughout the duration of infection, OSM was significantly elevated on day 1 and rapidly returned to baseline by days 2–3. The source of OSM was identified as the infiltrating neutrophils, and OSM levels correlated both with leukocyte numbers and i.p. soluble IL-6R (sIL-6R) levels. FACS analysis revealed that OSM receptor β expression was restricted to human peritoneal mesothelial cells. Stimulation of human peritoneal mesothelial cells with OSM induced phosphorylation of gp130 and OSM receptor β, which was accompanied by activation of STAT3 and secretion of CC chemokine ligand 2/monocyte chemoattractant protein-1 and IL-6. Although OSM itself did not modulate CXC chemokine ligand 8/IL-8 release, it effectively suppressed IL-1β-mediated expression of this neutrophil-activating CXC chemokine. Moreover, OSM synergistically blocked IL-1β-induced CXC chemokine ligand 8 secretion in combination with the IL-6/sIL-6R complex. Thus suggesting that OSM and sIL-6R release from infiltrating neutrophils may contribute to the temporal switch between neutrophil influx and mononuclear cell recruitment seen during acute inflammation.
Adenosine Up-Regulates Cyclooxygenase-2 in Human Granulocytes: Impact on the Balance of Eicosanoid Generation
Authors: Pouliot Marc , Fiset Marie-Élaine , Massé Mireille , Naccache Paul H. , Borgeat Pierre 
Published: April 2014

Abstract
Polymorphonuclear neutrophils (granulocytes; PMNs) are often the first blood cells to migrate toward inflammatory lesions to perform host defense functions. PMNs respond to specific stimuli by releasing several factors and generate lipid mediators of inflammation from the 5-lipoxygenase and the inducible cyclooxygenase (COX)-2 pathways. In view of adenosine’s anti-inflammatory properties and suppressive impact on the 5-lipoxygenase pathway, we addressed in this study the impact of this autacoid on the COX-2 pathway. We observed that adenosine up-regulates the expression of the COX-2 enzyme and mRNA. Production of PGE2 in response to exogenous arachidonic acid was also increased by adenosine and correlated with COX-2 protein levels. The potentiating effect of adenosine on COX-2 could be mimicked by pharmacological increases of intracellular cAMP levels, involving the latter as a putative second messenger for the up-regulation of COX-2 by adenosine. Specific COX-2 inhibitors were used to confirm the predominant role of the COX-2 isoform in the formation of prostanoids by stimulated PMNs. Withdrawal of extracellular adenosine strikingly emphasized the inhibitory potential of PGE2 on leukotriene B4 formation and involved the EP2 receptor subtype in this process. Thus, adenosine may promote a self-limiting regulatory process through the increase of PGE2 generation, which may result in the inhibition of PMN functions. This study identifies a new aspect of the anti-inflammatory properties of adenosine in leukocytes, introducing the concept that this autacoid may exert its immunomodulatory activities in part by modifying the balance of lipid mediators generated by PMNs.
NF-κB and TNF-α: A Positive Autocrine Loop in Human Lung Mast Cells?
Authors: Coward William R. , Okayama Yoshimichi , Sagara Hironori , Wilson Susan J. , Holgate Stephen T. , Church Martin K. 
Published: April 2014

Abstract
The generation of cytokines, particularly TNF-α, by mast cells is crucial for the initiation of the allergic response. A key transcription factor involved in the synthesis of TNF-α is NF-κB. Using a mAb specific for the activated form of NF-κB, immunocytochemistry, confocal microscopy, and gel shift assays have been used in conjunction to localize this transcription factor to human lung mast cells and to study its activation. Activation of mast cells with stem cell factor (10 ng/ml) and anti-IgE (1 μg/ml) induced maximal activation of NF-κB at 4 and 2 h, respectively. In contrast, with TNF-α (5 ng/ml) maximal activation occurred within 15 min. Parallel falls in IκB were demonstrated. Confocal microscopy demonstrated the localization of the activated form of NF-κB to the nuclei of activated mast cells. NF-κB activation was verified using a gel shift assay. A supershift assay showed mast cell NF-κB to be composed primarily of p50 with smaller amounts of p65. No interaction with Abs for Rel-A, c-Rel, Rel-B, and p52 was seen. Immunocytochemistry and ELISAs showed TNF-α to be stored within mast cells and released into the extracellular environment following activation. The possible participation of TNF-α generated by mast cells in NF-κB activation by anti-IgE was investigated using a blocking Ab for TNF-α. The blocking Ab reduced NF-κB activation by anti-IgE by &gt;50%, suggesting that the release of preformed mast cell-associated TNF-α acts as a positive autocrine feedback signal to augment NF-κB activation and production of further cytokine, including GM-CSF and IL-8.