bluuebunny/crossref_metadata_2025_split huggingface dataset:
Acute IL-3 Priming Up-Regulates the Stimulus-Induced Raf-1-Mek-Erk Cascade Independently of IL-3-Induced Activation of Erk
Authors: Vilariño Natalia , Miura Katsushi , MacGlashan Donald W. 
Published: April 2014

AbstractIL-3 is a potent priming cytokine for human basophils, inducing an increase of mediator release after stimulation. The mechanism of IL-3 priming of the basophil response to FcεRI aggregating stimuli remains unknown. We explored the regulation of several elements of IgE-mediated signaling by a short priming with IL-3. Early signaling events such as phosphorylation of Syk, Shc, linker for activation of T cells, and the calcium signal were not statistically affected by acute IL-3 priming. Downstream in the signaling cascade, a point of up-regulation was found at the level of Raf-1-Mek-Erk. Although the phosphorylation of Raf-1 was not changed by IL-3 priming, IL-3-primed anti-IgE-stimulated basophils showed a strong synergism for Mek and Erk phosphorylation when compared with either IL-3 or anti-IgE alone; pre-exposure to IL-3 induced a final 13-fold average increase over anti-IgE-induced Erk phosphorylation (6-fold above the sum of anti-IgE and IL-3 alone). The kinetics, dose response, and pharmacologic characteristics of the IL-3 priming of stimulus-induced Erk phosphorylation support the involvement of a yet unknown mechanism that is independent of IL-3-induced Erk and PI3K activation. This type of preactivation can be mimicked by incubation with the Ser-Thr kinase inhibitors, Ro-81–3220, or bisindoylmaleimide II.
Expansion of Functional Endogenous Antigen-Specific CD4+CD25+ Regulatory T Cells from Nonobese Diabetic Mice
Authors: Masteller Emma L. , Warner Matthew R. , Tang Qizhi , Tarbell Kristin V. , McDevitt Hugh , Bluestone Jeffrey A. 
Published: April 2014

Abstract
CD4+CD25+Foxp3+ regulatory T cells (Treg) are critical for controlling autoimmunity. Evidence suggests that Treg development, peripheral maintenance, and suppressive function are dependent on Ag specificity. However, there is little direct evidence that the Treg responsible for controlling autoimmunity in NOD mice or other natural settings are Ag specific. In fact, some investigators have argued that polyclonal Ag-nonspecific Treg are efficient regulators of immunity. Thus, the goal of this study was to identify, expand, and characterize islet Ag-specific Treg in NOD mice. Ag-specific Treg from NOD mice were efficiently expanded in vitro using IL-2 and beads coated with recombinant islet peptide mimic-MHC class II and anti-CD28 mAb. The expanded Ag-specific Treg expressed prototypic surface markers and cytokines. Although activated in an Ag-specific fashion, the expanded Treg were capable of bystander suppression both in vitro and in vivo. Importantly, the islet peptide mimic-specific Treg were more efficient than polyclonal Treg in suppressing autoimmune diabetes. These results provide a direct demonstration of the presence of autoantigen-specific Treg in the natural setting that can be applied as therapeutics for organ-specific autoimmunity.
The TCRβ Enhancer Is Dispensable for the Expression of Rearranged TCRβ Genes in Thymic DN2/DN3 Populations but Not at Later Stages
Authors: Busse Christian E. , Krotkova Anna , Eichmann Klaus 
Published: April 2014

Abstract
The Eβ enhancer has been shown to be dispensable for germline transcription of nonrearranged TCRβ segments but appears to be required for TCRβ V to DJ rearrangement. Eβ dependency of the subsequent expression of VDJ-rearranged TCRβ genes in thymic subpopulations has so far not been analyzed. We generated transgenic mice, using a Vβ8.2Dβ1Jβ1.3-rearranged TCRβ bacterial artificial chromosome, which lacked Eβ, and monitored transgene expression by flow cytometry using Vβ-specific mAbs and an IRES-eGFP reporter. Transgene expression was found in double negative (DN)2 and DN3 but not at later stages of thymopoesis. There was no toxicity associated with the transgene given that apoptosis in DN3, DN4 was not increased, and the number of DN4 cells generated from DN3 cells in reaggregate thymic organ cultures was not diminished. The transgenic TCRβ gave rise to a pre-TCR, as suggested by its ability to suppress endogenous TCRβ rearrangement, to facilitate β-selection on a TCRβ-deficient background and to inhibit γδ T cell lineage development. The results suggest that the Vβ8.2 promoter is sufficient to drive expression of rearranged TCRβ VDJ genes Eβ independently in DN2/DN3 but not at later stages.
In Vivo Humoral Immune Responses to Isolated Pneumococcal Polysaccharides Are Dependent on the Presence of Associated TLR Ligands
Authors: Sen Goutam , Khan Abdul Q. , Chen Quanyi , Snapper Clifford M. 
Published: April 2014

Abstract
We determined whether T cell-independent Ig isotype responses to isolated pneumococcal polysaccharides (PPS) required TLR signaling in vivo. IgG anti-PPS responses to PPS3, PPS14, and C-polysaccharide (C-PS) were virtually undetectable in TLR2−/− mice, whereas specific IgM induction was variably reduced compared with wild-type mice. All PPS-containing preparations induced IL-6 and TNF-α from wild-type, but not TLR2−/−, macrophages. TLR2 activity was distinct from that of PPS, in that it was phenol extractable. Immunization of wild-type mice with phenol-extracted PPS14 also resulted in a marked reduction in the IgG, although not the IgM-anti-PPS14, response compared with untreated PPS14. The commercial 23-valent PPS vaccine, Pneumovax-23 also contained TLR ligands (TLR2 and TLR4), which were absolutely critical for the IgG-inducing activity of the vaccine in mice. Finally, the commercial pneumococcal conjugate vaccine, Prevnar, contained a TLR2 ligand(s) that substantially enhanced both the primary and secondary anti-PPS responses in mice, especially the type 1 IgG isotypes. These data strongly suggest the absolute need for a distinct, TLR-dependent second signal for inducing in vivo IgG T cell-independent humoral immune responses to isolated pneumococcal polysaccharide Ags and highlight the potential importance of previously unappreciated copurified and/or contaminating TLR ligands in PPS vaccine preparations.
NKT Cell Stimulation with Glycolipid Antigen In Vivo: Costimulation-Dependent Expansion, Bim-Dependent Contraction, and Hyporesponsiveness to Further Antigenic Challenge
Authors: Uldrich Adam P. , Crowe Nadine Y. , Kyparissoudis Konstantinos , Pellicci Daniel G. , Zhan Yifan , Lew Andrew M. , Bouillet Philippe , Strasser Andreas , Smyth Mark J. , Godfrey Dale I. 
Published: April 2014

Abstract
Activation of NKT cells using the glycolipid α-galactosylceramide (α-GalCer) has availed many investigations into their immunoregulatory and therapeutic potential. However, it remains unclear how they respond to stimulation in vivo, which costimulatory pathways are important, and what factors (e.g., Ag availability and activation-induced cell death) limit their response. We have explored these questions in the context of an in vivo model of NKT cell dynamics spanning activation, population expansion, and subsequent contraction. Neither the B7/CD28 nor the CD40/CD40L costimulatory pathway was necessary for cytokine production by activated NKT cells, either early (2 h) or late (3 days) after initial stimulation, but both pathways were necessary for normal proliferative expansion of NKT cells in vivo. The proapoptotic Bcl-2 family member Bim was necessary for normal contraction of the NKT cell population between days 3–9 after stimulation, suggesting that the pool size is regulated by apoptotic death, similar to that of conventional T cells. Ag availability was not the limiting factor for NKT cell expansion in vivo, and a second α-GalCer injection induced a very blunted response, whereby cytokine production was reduced and further expansion did not occur. This appeared to be a form of anergy that was intrinsic to NKT cells and was not associated with inhibitory NK receptor signaling. Furthermore, NKT cells from mice prechallenged with α-GalCer in vivo showed little cytokine production and reduced proliferation in vitro. In summary, this study significantly enhances our understanding of how NKT cells respond to primary and secondary antigenic challenge in vivo.
Characterization of the Murine C3a Receptor Enhancer-Promoter: Expression Control by an Activator Protein 1 Sequence and an Ets-Like Site
Authors: Martin Carol B. , Martin Brian K. 
Published: April 2014

Abstract
The complement anaphylatoxins, C3a and C5a, exert their effects by binding to their respective receptors. A number of studies have implicated these proteins in human disease, yet little is known about anaphylatoxin receptor gene regulation. In this report, we demonstrate that most of the regulatory functions in the murine C3aR gene lie within 50 bp of the transcription start site. This region is critical for macrophage expression but does not have activity in a nonexpressing melanoma cell line. Within this small region are putative consensus binding sites for AP-1, NF-κB, Ets, and GATA transcription factors. Lack of a corresponding NF-κB site in the human sequence and lack of DNA binding activity in macrophage nuclear extracts suggests that the NF-κB site is nonfunctional. Luciferase data demonstrate that the GATA site functions as a negative regulatory element in RAW 264.7 macrophages. The AP-1 and Ets sites are critical for C3aR reporter gene expression, such that when each is mutated, a significant loss of activity is observed. Furthermore, we demonstrate that these sequences cooperate to mediate both basal and LPS-induced expression of C3aR. Interestingly, EMSA analyses demonstrate that the AP-1 site binds to c-Jun, and in vivo footprinting shows a typical footprint in this site, but the Ets site does not have a “typical” Ets footprint and does not bind to Ets-1/2 proteins in RAW 264.7 extracts. These data suggest that, although the control region for C3aR is small, interaction of several transcription factors can lead to complex patterns of gene regulation.
Rap1-GTP Is a Negative Regulator of Th Cell Function and Promotes the Generation of CD4+CD103+ Regulatory T Cells In Vivo
Authors: Li Lequn , Greenwald Rebecca J. , Lafuente Esther M. , Tzachanis Dimitrios , Berezovskaya Alla , Freeman Gordon J. , Sharpe Arlene H. , Boussiotis Vassiliki A. 
Published: April 2014

Abstract
The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.
HIV Nef-Mediated CD4 Down-Regulation Is Adaptor Protein Complex 2 Dependent
Authors: Jin Yong-Jiu , Cai Catherine Yi , Zhang Xiaoping , Zhang Hui-Tang , Hirst John A. , Burakoff Steven J. 
Published: April 2014

Abstract
Nef is a crucial viral protein for HIV to replicate at high titers and in the development of AIDS. One Nef function is down-regulating CD4 from the cell surface, which correlates with Nef-enhanced viral pathogenicity. Nef down-regulates CD4 by linking CD4 to clathrin-coated pits. However, the mechanistic connection between the C-terminal dileucine motif of Nef and the component(s) of the clathrin-coated pits has not been pinpointed. In this report we used two AP-2 complex-specific inhibitors: a dominant negative mutant of Eps15 (Eps15DIII) that binds to the α subunit of AP-2 complex and a small interference RNA that is specific for the μ2 subunit of AP-2 complex. We show that both HIV Nef- and SIV Nef-mediated CD4 down-regulations were profoundly blocked by the synergistic effect of Eps15DIII and RNA interference of AP-2 expression. The results demonstrate that HIV/SIV Nef-mediated CD4 down-regulation is AP-2 dependent. We also show that the PMA-induced CD4 down-regulation was blocked by these two inhibitors. Therefore, PMA-induced CD4 down-regulation is also AP-2 dependent. The results demonstrate that, like the tyrosine sorting motif-dependent endocytosis (for which the transferrin receptor and the epidermal growth factor receptor are the two prototypes), dileucine sorting motif-dependent endocytosis of Nef and CD4 are also AP-2 dependent.
Cytokine Induction by the Hepatitis B Virus Capsid in Macrophages Is Facilitated by Membrane Heparan Sulfate and Involves TLR2
Authors: Cooper Arik , Tal Guy , Lider Ofer , Shaul Yosef 
Published: April 2014

Abstract
The hepatitis B virus (HBV) core Ag (HBcAg) serves as the structural subunit of the highly immunogenic capsid shell. HBcAg harbors a unique arginine-rich C terminus that was implicated in immune responses induced by the capsid. In this study, we examined the capacity of the HBV capsid to induce proinflammatory and regulatory cytokines in human THP-1 macrophages and the possible underlying mechanism. Full-length HBc capsids, but not ΗΒc-144 capsids lacking the arginine-rich domain of HBcAg, efficiently bound differentiated THP-1 macrophages and strongly induced TNF-α, IL-6, and IL-12p40. Capsid binding to macrophages and cytokine induction were independent of the RNA associated with the arginine-rich domain. Soluble heparin and heparan sulfate but not chondroitin sulfates greatly diminished cytokine induction through inhibition of capsid binding to THP-1 macrophages. Furthermore, serine phosphorylation in the arginine-rich domain modulates capsid binding to macrophages and the cytokine response. Induction of cytokines by the capsid involved activation of NF-κB, ERK-1/2, and p38 MAPK and did not require endosomal acidification. Finally, NF-κB activation by the capsid in HEK 293 cells specifically required expression of TLR2 and was compromised by soluble heparin. Thus, cytokine induction by the HBV capsid in macrophages is facilitated by interaction of its arginine-rich domain with membrane heparan sulfate and involves signaling through TLR2.
Mannan-Binding Protein Blocks the Activation of Metalloproteases Meprin α and β
Authors: Hirano Makoto , Ma Bruce Yong , Kawasaki Nana , Okimura Kazumichi , Baba Makoto , Nakagawa Tomoaki , Miwa Keiko , Kawasaki Nobuko , Oka Shogo , Kawasaki Toshisuke 
Published: April 2014

Abstract
Mannan-binding protein (MBP) is a C-type serum lectin that is known to be a host defense factor involved in innate immunity, and recognizes mannose, fucose, and N-acetylglucosamine residues. Although some exogenous MBP ligands have been reported, little is known about its endogenous ligands. In the present study, we found that endogenous MBP ligands are highly expressed in the brush border epithelial cells of kidney-proximal tubules by immunohistochemistry, and both meprin α and β (meprins), as novel endogenous MBP ligands, have been identified through affinity chromatography and mass spectrometry. Meprins are membrane-bound and secreted zinc metalloproteases extensively glycosylated and highly expressed in kidney and small intestinal epithelial cells, leukocytes, and certain cancer cells. Meprins are capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. Deglycosylation experiments indicated that the MBP ligands on meprins are high mannose- or complex-type N-glycans. The interaction of MBP with meprins resulted in significant decreases in the proteolytic activity and matrix-degrading ability of meprins. Our results suggest that core N-linked oligosaccharides on meprins are associated with the optimal enzymatic activity and that MBP is an important regulator for modulation of the localized meprin proteolytic activity via N-glycan binding. Because meprins are known to be some of the major matrix-degrading metalloproteases in the kidney and intestine, MBP, which functions as a natural and effective inhibitor of meprins, may contribute, as a potential therapeutic target, to tumor progression by facilitating the migration, intravasation, and metastasis of carcinoma cells, and to acute renal failure and inflammatory bowel diseases.
Upper Respiratory Tract Resistance to Influenza Infection Is Not Prevented by the Absence of Either Nasal-Associated Lymphoid Tissue or Cervical Lymph Nodes
Authors: Wiley James A. , Tighe Michael P. , Harmsen Allen G. 
Published: April 2014

Abstract
The murine nasal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLN) are involved in the generation of local immune responses within the upper respiratory tract (URT). However, their involvement in these responses does not imply the necessity for resistance to URT infections. We surgically removed NALT or CLN to address the necessity of these lymphatic tissues for the development of a local protective immune response after a URT influenza infection. No histological evidence of the re-establishment of either tissue was detected after surgery and the subsequent infection. Removal of NALT did not elicit changes in serum or nasal mucosa-associated influenza-specific Ig levels. However, increases in PR8-specific serum IgG and nasal mucosa-associated IgA were detected after removal of CLN. Recruitment of influenza-specific CD4 T cells into the nasal mucosa was not altered by removal of NALT. The removal of NALT or CLN did not alter the recruitment of influenza-specific CD8 T cells into the URT. However, increased levels of influenza-specific CD8 T cells were observed in the tracheal-bronchial lymph nodes after CLN surgery. The rate of viral clearance from nasal mucosa and lungs was not altered by removal of NALT or CLN. These studies demonstrate that despite the participation of NALT and CLN in the generation of local immunity to influenza infections, neither tissue is essential for the development of protective immunity and viral clearance in URT.
NK T Cell Activation Promotes Chlamydia trachomatis Infection In Vivo
Authors: Bilenki Laura , Wang Shuhe , Yang Jie , Fan Yijun , Joyee Antony George , Yang Xi 
Published: April 2014

Abstract
We used two approaches to examine the role of NK T cells (NKT) in an intracellular bacterial (Chlamydia trachomatis mouse pneumonitis (C. muridarum)) infection. One is to use CD1 gene knockout (KO) mice, which lack NKT, and the other is to activate NKT using α-galactosylceramide (α-GalCer), a natural ligand of these cells. The data showed a promoting effect of NKT activation on Chlamydia lung infection. Specifically, CD1 KO mice exhibited significantly lower levels of body weight loss, less severe pathological change and lower chlamydial in vivo growth than wild-type mice. Immunological analysis showed that CD1 KO mice exhibited significantly lower C. muridarum-specific IL-4 and serum IgE Ab responses as well as more pronounced delayed-type hypersensitivity response compared with wild-type controls. In line with the finding in KO mice, the in vivo stimulation of NKT using α-GalCer enhanced chlamydial growth in vivo, which were correlated with reduced delayed-type hypersensitivity response and increased C. muridarum-driven IL-4/IgE production. Moreover, neutralization of IL-4 activity in the α-GalCer-treated BALB/c mice significantly reduced the promoting effect of α-GalCer treatment on chlamydial growth in vivo. These data provide in vivo evidence for the involvement of NKT in a bacterial pathogenesis and its role in promoting Th2 responses during infection.
Infection of Dendritic Cells by a γ2-Herpesvirus Induces Functional Modulation
Authors: Flaño Emilio , Kayhan Basak , Woodland David L. , Blackman Marcia A. 
Published: April 2014

Abstract
The murine γ-herpesvirus-68 (γHV68) establishes viral latency in dendritic cells (DCs). In the present study, we examined the specific consequences of DC infection by γHV68, both in vivo and in vitro. Ex vivo analysis of infected mice showed that the virus colonizes respiratory DCs very early after infection and that all subsets of splenic DCs analyzed are viral targets. We have developed and characterized an in vitro model of γHV68 infection of DCs. Using this model, we demonstrated that viral infection neither induces full DC maturation nor interferes with exogenous activation, which is assessed by cell surface phenotypic changes. However, whereas γHV68 infection alone failed to elicit cytokine secretion, IL-10 secretion of exogenously activated DCs was enhanced. Furthermore, γHV68-infected DCs efficiently stimulated virus-specific T cell hybridomas but failed to induce alloreactive stimulation of normal T cells. These data indicate that viral infection doesn’t interfere with Ag processing and presentation but does interfere with the ability of DCs to activate T cells. The inhibition of T cell activation was partially reversed by blocking IL-10. Analysis of infected mice shows elevated levels of IL-10 expression in DCs and that lack of endogenous IL-10 is associated with decreased γHV68 long-term latency. Taken together, these observations indicate that γ2-herpesvirus infection of DCs is a mechanism of viral immune evasion, partially mediated by IL-10.
Wild-Type Measles Virus Infection in Human CD46/CD150-Transgenic Mice: CD11c-Positive Dendritic Cells Establish Systemic Viral Infection
Authors: Shingai Masashi , Inoue Naokazu , Okuno Tsuyoshi , Okabe Masaru , Akazawa Takashi , Miyamoto Yasuhide , Ayata Minoru , Honda Kenya , Kurita-Taniguchi Mitsue , Matsumoto Misako , Ogura Hisashi , Taniguchi Tadatsugu , Seya Tsukasa 
Published: April 2014

Abstract
We generated transgenic (TG) mice that constitutively express human CD46 (huCD46) and/or TLR-inducible CD150 (huCD150), which serve as receptors for measles virus (MV). These mice were used to study the spreading and pathogenicity of GFP-expressing or intact laboratory-adapted Edmonston and wild-type Ichinose (IC) strains of MV. Irrespective of the route of administration, neither type of MV was pathogenic to these TG mice. However, in ex vivo, limited replication of IC was observed in the spleen lymphocytes from huCD46/huCD150 TG and huCD150 TG, but not in huCD46 TG and non-TG mice. In huCD150-positive TG mouse cells, CD11c-positive bone marrow-derived myeloid dendritic cells (mDC) participated in MV-mediated type I IFN induction. The level and induction profile of IFN-β was higher in mDC than the profile of IFN-α. Wild-type IC induced markedly high levels of IFN-β compared with Edmonston in mDC, as opposed to human dendritic cells. We then generated huCD46/huCD150 TG mice with type I IFN receptor (IFNAR1)−/− mice. MV-bearing mDCs spreading to draining lymph nodes were clearly observed in these triple mutant mice in vivo by i.p. MV injection. Infectious lymph nodes were also detected in the double TG mice into which MV-infected CD11c-positive mDCs were i.v. transferred. This finding suggests that in the double TG mouse model mDCs once infected facilitate systemic MV spreading and infection, which depend on mDC MV permissiveness determined by the level of type I IFN generated via IFNAR1. Although these results may not simply reflect human MV infection, the huCD150/huCD46 TG mice may serve as a useful model for the analysis of MV-dependent modulation of mDC response.