bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct Tumor Lysis by NK Cells Uses a Ras-Independent Mitogen-Activated Protein Kinase Signal Pathway
Authors: Wei Sheng , Gilvary Danielle L. , Corliss Brian C. , Sebti Said , Sun Jiazhi , Straus David B. , Leibson Paul J. , Trapani Joseph A. , Hamilton Andrew D. , Weber Michael J. , Djeu Julie Y. 
Published: April 2014

AbstractDestruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.
Phosphatidylinositol 3-Kinase and NF-κB/Rel Are at the Divergence of CD40-Mediated Proliferation and Survival Pathways
Authors: Andjelic Sofija , Hsia Constance , Suzuki Harumi , Kadowaki Takashi , Koyasu Shigeo , Liou Hsiou-Chi 
Published: April 2014

AbstractCD40 receptor ligation evokes several crucial outcomes for the fate of an activated B cell, including proliferation and survival. Although multiple signaling molecules in the CD40 pathways have been identified, their specific roles in regulating proliferation and maintaining cell viability are still obscure. In this report, we demonstrate that the activation of both phosphatidylinositol 3-kinase (PI-3K) and NF-κB/Rel transcription factors is crucial for CD40-mediated proliferation. Furthermore, our data indicate that PI-3K is indispensable for CD40-mediated NF-κB/Rel activation. This is achieved via activation of AKT and the degradation of IκBα. Furthermore, we show that PI-3K activity is necessary for the degradation of cyclin-dependent kinase inhibitor p27kip. Therefore, both of these events comprise the mechanism by which PI-3K controls cell proliferation. In contrast to the absolute requirement of PI-3K and NF-κB/Rel for proliferation, these signaling molecules are only partially responsible for CD40-mediated survival, as blocking of PI-3K activity did not lead to apoptosis of anti-CD40-treated cells. However, the PI-3K/NF-κB pathway is still required for CD40-induced Bcl-X gene expression. Taken together, our data indicate that multiple survival pathways are triggered via this receptor, whereas NF-κB/Rel and PI-3K are crucial for CD40-induced proliferation.
Functional Analysis of Ligand-Binding and Signal Transduction Domains of CD69 and CD23 C-Type Lectin Leukocyte Receptors
Authors: Sancho David , Santis Ana G. , Alonso-Lebrero José L. , Viedma Fernando , Tejedor Reyes , Sánchez-Madrid Francisco 
Published: April 2014

AbstractCD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-α synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-α was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via FcεRI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.
Restriction in Vκ Gene Use and Antigen Selection in Anti-Myeloperoxidase Response in Mice
Authors: Jethwa Hitendra S. , Clarke Stephen H. , Itoh-Lindstrom Yoshie , Falk Ronald J. , Jennette J. Charles , Nachman Patrick H. 
Published: April 2014

AbstractAnti-neutrophil cytoplasmic Abs, directed primarily toward myeloperoxidase (MPO) and proteinase 3, are detected in the majority of patients with distinct forms of small vessel vasculitides and pauci-immune necrotizing glomerulonephritis. However, the origin of these autoantibodies remains unknown. We studied the V region gene use in murine anti-MPO Abs derived from Spontaneous Crescentic Glomerulonephritis/Kinjoh mice. A total of 13 anti-MPO-producing hybridomas were generated from four unimmunized mice. Ten of the 13 hybridomas (corresponding to 3 of 4 clones) expressed Vκ1C but differed in their use of VH genes. The remaining three hybridomas expressed a Vκ5 gene. Anti-MPO hybridomas from individual mice were derived from single clones as deduced by sequence similarity and splice-site identity. We found a statistically significant bias of amino acid replacement mutations to the complementarity-determining regions (CDR) in the Vκ1C-expressing hybridomas. Intriguingly, all 10 Vκ1C hybridomas share a lysine to glutamate mutation in the CDR1. To determine the effects of somatic V gene mutations on binding to MPO, we generated an anti-MPO Ab with an unmutated Vκ1C L chain and compared its ability to bind MPO with its mutated counterpart. The mutated hybridoma-derived Ab has a 4.75-fold higher avidity for MPO than the unmutated Ab. These results suggest that: 1) the L chain plays a dominant role in determining Ab specificity to MPO, 2) the anti-MPO Ab response is oligoclonal, consistent with Ag selection, and 3) MPO is a driving Ag in the murine anti-MPO Ab response.
Identification of a Novel Cytokine Response Element in the Human IFN Regulatory Factor-1 Gene Promoter
Authors: Imanishi Daisuke , Yamamoto Kazuo , Tsushima Hideki , Miyazaki Yasushi , Kuriyama Kazutaka , Tomonaga Masao , Matsuyama Toshifumi 
Published: April 2014

AbstractThe present study investigates the regulatory mechanisms involved in the cooperation between IFN-γ and TNF-α to promote transcription from IFN regulatory factor-1 (IRF-1). A transient transfection analysis revealed that the region between −218 and −144, where +1 is the transcription start site, as well as previously reported downstream elements, ppκB and IFN-γ activation site/κB, were required for the optimal response to the two cytokines. A subsequent DNase I footprint analysis showed that the region between −171 and −144 was inducibly protected with stimulation by TNF-α, and this protection was significantly enhanced with the combination of IFN-γ and TNF-α. In an EMSA with the protected region as a probe, a TNF-α-inducible complex (C1) and an IFN-γ-inducible complex (C2), but no synergy-specific DNA-protein complexes, were recognized. The C1 complex consisted of a pre-existing factor (p65/p50), whereas the C2 complex consisted of a newly synthesized IRF-1-related factor. A methylation interference assay revealed the critical G residues (from −167 to −151) for the DNA-protein complex formation specific to the cytokine response, and within this region the novel κB sequence, the promoter distal κB (pdκB) element (5′-GGGGAAGTAC-3′), was identified. Because the base substitutions over the pdκB region (from −171 to −144) affected not only the TNF-α-response but also that of IFN-γ, this region might contribute to the cooperative action of the NF-κB subunits with the IRF-1-related factor. Finally, we demonstrated that none of the cis-acting elements, ppκB, pdκB, or IFN-γ activation site/κB, is dispensable for the optimal synergism in response to IFN-γ and TNF-α.
Distinct Effects of Surfactant Protein A or D Deficiency During Bacterial Infection on the Lung
Authors: LeVine Ann Marie , Whitsett Jeffrey A. , Gwozdz Jodie A. , Richardson Theresa R. , Fisher James H. , Burhans Michael S. , Korfhagen Thomas R. 
Published: April 2014

AbstractMice lacking surfactant protein (SP)-A (SP-A−/−) or SP-D (SP-D−/−) and wild-type mice were infected with group B streptococcus or Haemophilus influenzae by intratracheal instillation. Although decreased killing of group B streptococcus and H. influenzae was observed in SP-A−/− mice but not in SP-D−/− mice, deficiency of either SP-A or SP-D was associated with increased inflammation and inflammatory cell recruitment in the lung after infection. Deficient uptake of bacteria by alveolar macrophages was observed in both SP-A- and SP-D-deficient mice. Isolated alveolar macrophages from SP-A−/− mice generated significantly less, whereas those from SP-D−/− mice generated significantly greater superoxide and hydrogen peroxide compared with wild-type alveolar macrophages. In SP-D−/− mice, bacterial killing was associated with increased lung inflammation, increased oxidant production, and decreased macrophage phagocytosis. In contrast, in the absence of SP-A, bacterial killing was decreased and associated with increased lung inflammation, decreased oxidant production, and decreased macrophage phagocytosis. Increased oxidant production likely contributes to effective bacterial killing in the lungs of SP-D−/− mice. The collectins, SP-A and SP-D, play distinct roles during bacterial infection of the lung.
Role of Cystic Fibrosis Transmembrane Conductance Regulator in Pulmonary Clearance of Pseudomonas aeruginosa In Vivo
Authors: Chroneos Zissis C. , Wert Susan E. , Livingston Jennifer L. , Hassett Daniel J. , Whitsett Jeffrey A. 
Published: April 2014

Abstract
Cystic fibrosis (CF)2 is a fatal genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) that is commonly associated with chronic pulmonary infections with mucoid Pseudomonas aeruginosa (PA). To test the hypothesis that CFTR plays a direct role in PA adhesion and clearance, we have used mouse lines expressing varying levels of human (h) or mouse (m) CFTR. A subacute intratracheal dose of 3 × 106 bacteria was cleared with similar kinetics in control wild-type (WT) and transgenic mice overexpressing hCFTR in the lung from the surfactant protein C (SP-C) promoter (SP-C-hCFTR+/−). In a second series of experiments, the clearance of an acute intratracheal dose of 1.5 × 107 PA bacteria was also similar in WT, hemizygous SP-C-hCFTR+/−, and bitransgenic gut-corrected FABP-hCFTR+/+-mCFTR−/−, the latter lacking expression of mCFTR in the lung. However, a small but significant decrease in bacterial killing was observed in lungs of homozygote SP-C-hCFTR+/+ mice. Lung pathology in both WT and SP-C-hCFTR+/+ mice was marked by neutrophilic inflammation and bacterial invasion of perivascular and subepithelial compartments. Bacteria were associated primarily with leukocytes and were not associated with alveolar type II or bronchiolar epithelial cells, the cellular sites of SP-C-hCFTR+/+ transgene expression. The results indicate that there is no direct correlation between levels of CFTR expression and bacterial clearance or association of bacteria with epithelial cells in vivo.
Granzyme Activity in the Inflamed Lung Is Not Controlled by Endogenous Serine Proteinase Inhibitors
Authors: Tremblay Guy M. , Wolbink Angela M. , Cormier Yvon , Hack C. Erik 
Published: April 2014

AbstractNumerous lung diseases, such as hypersensitivity pneumonitis (HP), are characterized by the presence of activated alveolar CTL and NK cells. Since these cells produce granzymes, granzyme A and B levels in bronchoalveolar lavage (BAL) fluids from 14 normal subjects and 12 patients with HP were measured by ELISA. Median (range) BAL granzyme A and B levels were 4 (0–37) and 0 (0–6) pg/ml in normal subjects. BAL granzyme levels were significantly higher in HP patients, being at 74 (0–1889) and 10 (0–78) pg/ml for granzymes A and B, respectively. In vitro, neither of the three main serine protease inhibitors of the lung, namely α1-antitrypsin, secretory leukocyte protease inhibitor, and elafin, showed any effect on granzyme A or B activity. In addition, granzyme A was shown to be fully active in BAL fluids. Hence, these data show that granzyme activity may be poorly controlled by protease inhibitors in inflamed tissues. Thus, granzymes could contribute to tissue remodeling and inflammation characterizing HP.
Nitric Oxide-Dependent Ribosomal RNA Cleavage Is Associated with Inhibition of Ribosomal Peptidyl Transferase Activity in ANA-1 Murine Macrophages
Authors: Cai Charles Q. , Guo Hongtao , Schroeder Rebecca A. , Punzalan Cecile , Kuo Paul C. 
Published: April 2014

Abstract
NO can regulate specific cellular functions by altering transcriptional programs and protein reactivity. With respect to global cellular processes, NO has also been demonstrated to inhibit total protein synthesis and cell proliferation. The underlying mechanisms are unknown. In a system of ANA-1 murine macrophages, iNOS expression and NO production were induced by exposure to endotoxin (LPS). In selected instances, cells were exposed to an exogenous NO donor, S-nitroso-N-acetylpenicillamine or a substrate inhibitor of NO synthesis. Cellular exposure to NO, from both endogenous and exogenous sources, was associated with a significant time-dependent decrease in total protein synthesis and cell proliferation. Gene transcription was unaltered. In parallel with decreased protein synthesis, cells exhibited a distinctive cleavage pattern of 28S and 18S rRNA that were the result of two distinct cuts in both 28S and 18S rRNA. Total levels of intact 28S rRNA, 18S rRNA, and the composite 60S ribosome were significantly decreased in the setting of cell exposure to NO. Finally, 60S ribosome-associated peptidyl transferase activity, a key enzyme for peptide chain elongation, was also significantly decreased. Our data suggest that NO-mediated cleavage of 28S and 18S rRNA results in decreased 60S ribosome associated peptidyl transferase activity and inhibition of total protein synthesis.
GM-CSF Regulates Bleomycin-Induced Pulmonary Fibrosis Via a Prostaglandin-Dependent Mechanism
Authors: Moore Bethany B. , Coffey Michael J. , Christensen Paul , Sitterding Stephanie , Ngan Rose , Wilke Carol A. , McDonald Rod , Phare Susan M. , Peters-Golden Marc , Paine Robert , Toews Galen B. 
Published: April 2014

AbstractTo characterize the role of GM-CSF in pulmonary fibrosis, we have studied bleomycin-induced fibrosis in wild-type mice vs mice with a targeted deletion of the GM-CSF gene (GM-CSF−/− mice). Without GM-CSF, pulmonary fibrosis was worse both histologically and quantitatively. These changes were not related to enhanced recruitment of inflammatory cells because wild-type and GM-CSF−/− mice recruited equivalent numbers of cells to the lung following bleomycin. Interestingly, recruitment of eosinophils was absent in GM-CSF−/− mice. We investigated whether the enhanced fibrotic response in GM-CSF−/− animals was due to a deficiency in an endogenous down-regulator of fibrogenesis. Analysis of whole lung homogenates from saline- or bleomycin-treated mice revealed that GM-CSF−/− animals had reduced levels of PGE2. Additionally, alveolar macrophages were harvested from wild-type and GM-CSF−/− mice that had been exposed to bleomycin. Although bleomycin treatment impaired the ability of alveolar macrophages from wild-type mice to synthesize PGE2, alveolar macrophages from GM-CSF−/− mice exhibited a significantly greater defect in PGE2 synthesis than did wild-type cells. Exogenous addition of GM-CSF to alveolar macrophages reversed the PGE2 synthesis defect in vitro. Administration of the PG synthesis inhibitor, indomethacin, to wild-type mice during the fibrogenic phase postbleomycin worsened the severity of fibrosis, implying a causal role for PGE2 deficiency in the evolution of the fibrotic lesion. These data demonstrate that GM-CSF deficiency results in enhanced fibrogenesis in bleomycin-induced pulmonary fibrosis and indicate that one mechanism for this effect is impaired production of the potent antifibrotic eicosanoid, PGE2.
CD4+ T Lymphocytes with Constitutive CD40 Ligand in Preautoimmune (NZB × NZW)F1 Lupus-Prone Mice: Phenotype and Possible Role in Autoreactivity
Authors: Lettesjö Helene , Burd Gary P. , Mageed Rizgar A. 
Published: April 2014

AbstractLupus disease is marked by B lymphocyte hyperactivity and the production of Abs to dsDNA. The production of these anti-dsDNA Abs is T lymphocyte dependent. However, it is not clear how CD4+ T lymphocytes provide help for B lymphocytes to produce IgG anti-dsDNA Abs. One possible mechanism is suggested by studies showing that human patients with systemic lupus erythematosus and lupus mice have increased numbers of CD40 ligand (CD40L)+ T and B lymphocytes. The results described in this study reveal that young, clinically healthy lupus-prone New Zealand Black × New Zealand White F1 (BWF1) mice have naive CD4+ T cells with preformed CD40L. These cells contribute to a brisk response to immunization and to the production of anti-dsDNA Abs. In vitro experiments revealed that CD4+ T cells with preformed CD40L could, upon stimulation, provide antiapoptotic signals for B cells but could not induce proliferation or reduce activation threshold. These results suggest that the direct target cells for the effect of T cells with preformed CD40L in lupus may not be B lymphocytes.
Expression of a Human Coxsackie/Adenovirus Receptor Transgene Permits Adenovirus Infection of Primary Lymphocytes
Authors: Schmidt Madelyn R. , Piekos Brian , Cabatingan Mark S. , Woodland Robert T. 
Published: April 2014

AbstractReplication-defective adenoviruses are effective vehicles for gene transfer, both for the repair of defective genes and for studies of gene function in primary cells. Many cell types, including lymphocytes, are refractory to adenovirus infection because they lack the Coxsackie/adenovirus receptor (CAR) needed for virus attachment. To extend the advantages of adenovirus-mediated gene transfer to primary lymphoid populations and other cell types lacking endogenous CAR, we produced a mouse that expresses human (h) CAR as a transgene under control of a murine MHC class I promoter. hCAR protein is expressed on T and B lymphocytes from a variety of organs (spleen, lymph node, bone marrow, thymus, and peritoneum). These lymphocytes are susceptible to adenovirus infection, as demonstrated by reporter green fluorescent protein gene expression, with the fraction of expressing cells as high as 70%. Some lymphocyte subpopulations required stimulation subsequent to adenovirus infection for reporter expression. This activation requirement is a restriction imposed by the promoter used in the adenovirus construct. In subpopulations requiring activation, the elongation factor 1 promoter was far superior to a hCMV promoter for directing green fluorescent protein production. We also find that hCAR mRNA is produced in nonlymphoid tissues from all founder lines, including tissues that do not express endogenous murine CAR, suggesting the opportunity for effecting gene delivery to and testing gene function in a wide variety of primary cell types previously resistant to gene transfer.
Cutting Edge: Gab2 Mediates an Inhibitory Phosphatidylinositol 3′-Kinase Pathway in T Cell Antigen Receptor Signaling
Authors: Pratt Joanne C. , Igras Vivien E. , Maeda Hiroyuki , Baksh Shairaz , Gelfand Erwin W. , Burakoff Steven J. , Neel Benjamin G. , Gu Haihua 
Published: April 2014

AbstractPhosphatidylinositol 3′-kinase (PI3K) is a key component of multiple signaling pathways, where it typically promotes survival, proliferation, and/or adhesion. Here, we show that in TCR signaling, the scaffolding adapter Gab2 delivers an inhibitory signal via PI3K. Overexpression of Gab2 in T cell lines inhibits TCR-evoked activation of the IL-2 promoter, blocking NF-AT- and NF-κB-directed transcription. Inhibition is abrogated by mutating the Gab2 p85-binding sites, by treatment with PI3K inhibitors or by cotransfection of phosphatase homolog of tensin. Our findings provide the first evidence of a negative function for a scaffolding adapter in T cells and identify Gab2/PI3K-containing complexes as novel regulators of TCR signaling.
Phosphorothioate Backbone Modification Modulates Macrophage Activation by CpG DNA
Authors: Sester David P. , Naik Shalin , Beasley Shannon J. , Hume David A. , Stacey Katryn J. 
Published: April 2014

AbstractMacrophages respond to unmethylated CpG motifs present in nonmammalian DNA. Stabilized phosphorothioate-modified oligodeoxynucleotides (PS-ODN) containing CpG motifs form the basis of immunotherapeutic agents. In this study, we show that PS-ODN do not perfectly mimic native DNA in activation of macrophages. CpG-containing PS-ODN were active at 10- to 100-fold lower concentrations than corresponding phosphodiester ODN in maintenance of cell viability in the absence of CSF-1, in induction of NO production, and in activation of the IL-12 promoter. These enhancing effects are attributable to both increased stability and rate of uptake of the PS-ODN. By contrast, PS-ODN were almost inactive in down-modulation of the CSF-1R from primary macrophages and activation of the HIV-1 LTR. Delayed or poor activation of signaling components may contribute to this, as PS-ODN were slower and less effective at inducing phosphorylation of the extracellular signal-related kinases 1 and 2. In addition, at high concentrations, non-CpG PS-ODN specifically inhibited responses to CpG DNA, whereas nonstimulatory phosphodiester ODN had no such effect. Although nonstimulatory PS-ODN caused some inhibition of ODN uptake, this did not adequately explain the levels of inhibition of activity. The results demonstrate that the phosphorothioate backbone has both enhancing and inhibitory effects on macrophage responses to CpG DNA.