bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ectopic Human Telomerase Catalytic Subunit Expression Maintains Telomere Length But Is Not Sufficient for CD8+ T Lymphocyte Immortalization
Authors: Migliaccio Marco , Amacker Mario , Just Tom , Reichenbach Patrick , Valmori Danila , Cerottini Jean-Charles , Romero Pedro , Nabholz Markus 
Published: April 2014

Abstract
Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8+ T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8+ lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.
Differentiation of Murine NK Cells into Distinct Subsets Based on Variable Expression of the IL-12Rβ2 Subunit
Authors: Chakir Habiba , Camilucci Allison A. , Filion Lionel G. , Webb John R. 
Published: April 2014

Abstract
The cytokine IL-12 manifests its biological activity via interaction with a heterodimeric receptor (IL-12R) present on activated T and NK cells. The cDNAs for two IL-12R subunits have been cloned from human and mouse and designated IL-12Rβ1 and IL-12Rβ2. The expression of IL-12Rβ2 on T cells is influenced by cytokines, particularly IL-4, IL-12, and IFN-γ; however, little is known regarding regulation of IL-12R expression on NK cells. In this study we show that murine NK cells differentiate into IL-12Rβ2low and IL-12Rβ2high subsets after in vitro stimulation with IL-2 in the absence of exogenous polarizing cytokines. Subset development occurs gradually as NK cells expand in vitro and is generally complete by 8–12 days of culture. Once established, IL-12Rβ2low and IL-12Rβ2high subsets are highly stable in vitro and can be maintained for at least 20 days after FACS sorting. Formation of these NK subsets appears to be strain independent. Flow cytometric analyses demonstrate that both subsets express a number of NK-associated markers, including NK1.1, DX-5, Ly-49A, and Ly-49C, but that the Ly-49G2 class I inhibitory receptor is expressed predominantly on the IL-12Rβ2high population. Both IL-12Rβ2low and IL-12Rβ2high NK cells respond to exogenous IL-12 by rapid production of high levels of IFN-γ and increased lytic activity against NK-sensitive YAC-1 target cells. Analyses of cytokine gene expression by RNase protection assay indicated that similar to the recently described human NK1 subset, both IL-12Rβ2high and IL-12Rβ2low murine NK subsets expressed high levels of IFN-γ, whereas neither subset expressed mRNA for the NK2-associated cytokines IL-5 and IL-13.
NF-κB Is Required for the Positive Selection of CD8+ Thymocytes
Authors: Hettmann Thore , Leiden Jeffrey M. 
Published: April 2014

Abstract
To examine the role of NF-κB in T cell development, we analyzed thymocyte ontogeny in transgenic (mutant I-κBα (mI-κBα)) mice that express a superinhibitory form of the NF-κB inhibitory protein, I-κBα (I-κBαA32/36), under the control of the T cell-specific CD2 promoter and enhancer. Thymi from mI-κBα mice contained increased numbers of double-positive (DP) and decreased numbers of both CD4+ and CD8+ single-positive cells, consistent with a block in DP thymocyte maturation. In addition, expression of CD69, a marker of positive selection, was decreased on DP thymocytes from the mI-κBα mice. To test directly whether NF-κB was required for positive or negative selection, we generated mI-κBα mice expressing the H-Y or 2C αβ TCR transgenes. Expression of the I-κBαA32/36 transgene caused a block in the positive selection of CD8+ single-positive cells in both strains of TCR transgenic animals. In contrast, negative selection was unaffected by expression of the I-κBαA32/36 transgene. Taken together, these results identified a NF-κB-dependent transcriptional pathway that is selectively required for the positive selection of CD8+ thymocytes.
Microbial and T Cell-Derived Stimuli Regulate Antigen Presentation by Dendritic Cells In Vivo
Authors: Manickasingham Shivanthi , Reis e Sousa Caetano 
Published: April 2014

Abstract
B cells and dendritic cells (DC) internalize and degrade exogenous Ags and present them as peptides bound to MHC class II molecules for scrutiny by CD4+ T cells. Here we use an Ab specific for a processed form of the model Ag, hen egg lysozyme (HEL), to demonstrate that this protein is not efficiently presented by lymph node DC following s.c. immunization. HEL presentation by the DC can be dramatically enhanced upon coinjection of a microbial adjuvant, which appears to act by enhancing peptide loading onto MHC class II. CD40 cross-linking or the presence of a high frequency of T cells specific for HEL can similarly improve presentation by DC in vivo. For any of these activating stimuli, CD8α+ DC consistently display the highest proportion of HEL-loaded MHC class II molecules. These data indicate that exogenous Ags can be displayed to T cells in lymphoid tissues by a large cohort of resident DC whose presentation is regulated by innate and adaptive stimuli. Our data further reveal the existence of a feedback mechanism that augments Ag presentation during cognate APC-T cell interactions.
Receptor for Activated C-Kinase (RACK-1), a WD Motif-Containing Protein, Specifically Associates with the Human Type I IFN Receptor
Authors: Croze Ed , Usacheva Anna , Asarnow David , Minshall Richard D. , Perez H. D. , Colamonici Oscar 
Published: April 2014

Abstract
The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-αRβL) was used as bait in a yeast two-hybrid system to identify novel proteins interacting with this region of the receptor. We report here a specific interaction between the cytoplasmic domain of IFN-αRβL and a previously identified protein, RACK-1 (receptor for activated C kinase). Using GST fusion proteins encoding different regions of the cytoplasmic domain of IFN-αRβL, the minimum site for RACK-1 binding was mapped to aa 300–346. RACK-1 binding to IFN-αRβL did not require the first 91 aa of RACK-1, which includes two WD domains, WD1 and WD2. The interaction between RACK-1 and IFN-αRβL, but not the human IFN receptor chain 1 (IFNAR1 or IFN-αRα), was also detected in human Daudi cells by coimmunoprecipitation. RACK-1 was shown to be constitutively associated with IFN-αRβL, and this association was not effected by stimulation of Daudi cells with type I IFNs (IFN-β1b). RACK-1 itself did not become tyrosine phosphorylated upon stimulation of Daudi cells with IFN-β1b. However, stimulation of cells with either IFN-β1b or PMA did result in an increase in detectable immunofluorescence and intracellular redistribution of RACK-1.
DNA Vaccination Against Rat Her-2/Neu p185 More Effectively Inhibits Carcinogenesis Than Transplantable Carcinomas in Transgenic BALB/c Mice
Authors: Rovero Stefania , Amici Augusto , Carlo Emma Di , Bei Roberto , Nanni Patrizia , Quaglino Elena , Porcedda Paola , Boggio Katia , Smorlesi Arianna , Lollini Pier-Luigi , Landuzzi Lorena , Colombo Mario P. , Giovarelli Mirella , Musiani Piero , Forni Guido 
Published: April 2014

AbstractThe ability of vaccination with plasmids coding for the extracellular and the transmembrane domain of the product of transforming rat Her-2/neu oncogene (r-p185) to protect against r-p185+ transplantable carcinoma (TUBO) cells and mammary carcinogenesis was evaluated. In normal BALB/c mice, DNA vaccination elicits anti-r-p185 Ab, but only a marginal CTL reactivity, and protects against a TUBO cell challenge. Massive reactive infiltration is associated with TUBO cell rejection. In BALB/c mice transgenic for the rat Her-2/neu gene (BALB-neuT), DNA vaccination elicits a lower anti-r-p185 Ab response, no CTL activity and only incompletely protects against TUBO cells, but markedly hampers the progression of carcinogenesis. At 33 wk of age, when control BALB-neuT mice display palpable tumors in all mammary glands, about 60% of immunized mice are tumor free, and tumor multiplicity is markedly reduced. Tumor-free mammary glands still display the atypical hyperplasia of the early stages of carcinogenesis, and a marked down-modulation of r-p185, along with a massive reactive infiltrate. However, BALB-neuT mice protected against mammary carcinogenesis fail to efficiently reject a TUBO cell challenge. This suggests that the mechanisms required for the rejection of transplantable tumors may not coincide with those that inhibit the slow progression of carcinogenesis.
Archaeosomes Induce Long-Term CD8+ Cytotoxic T Cell Response to Entrapped Soluble Protein by the Exogenous Cytosolic Pathway, in the Absence of CD4+ T Cell Help
Authors: Krishnan Lakshmi , Sad Subash , Patel Girishchandra B. , Sprott G. Dennis 
Published: April 2014

AbstractThe unique ether glycerolipids of Archaea can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8+ T cell response, as measured by IFN-γ production and cytolytic activity toward the immunodominant CTL epitope OVA257–264. In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8+ T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4+ T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8+ T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8+ T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8+ T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.
Brucella abortus Lipopolysaccharide in Murine Peritoneal Macrophages Acts as a Down-Regulator of T Cell Activation
Authors: Forestier Claire , Deleuil Fabienne , Lapaque Nicolas , Moreno Edgardo , Gorvel Jean-Pierre 
Published: April 2014

Abstract
Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs. We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway. LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4+ T cells but not those of OVA to specific CD8+ T cells. This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL. In addition, B. abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides. At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4+ T cell activation. This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.
Role of p38 Mitogen-Activated Protein Kinase in Rhinovirus-Induced Cytokine Production by Bronchial Epithelial Cells
Authors: Griego Sandra D. , Weston Cynthia B. , Adams Jerry L. , Tal-Singer Ruth , Dillon Susan B. 
Published: April 2014

Abstract
The stress-activated protein kinase p38 plays a central role in the regulation of cytokine biosynthesis by various cell types in response to a wide range of stimuli. Because the local inflammatory response and the infiltration of neutrophils is thought to contribute to the symptoms and sequelae of rhinovirus infection, we investigated the role of p38 kinase in cytokine and chemokine elaboration in airway epithelial cells infected with human rhinovirus. Rhinovirus-39 infection of BEAS-2B cells resulted in synthesis of cytokines (IL-1, IL-6, G-CSF, and GM-CSF) and CXC chemokines (IL-8, epithelial neutrophil-activating protein-78, and growth-related oncogene-α), evident 24–72 h postinfection. Rhinovirus infection induced a time- and dose-dependent increase in tyrosine phosphorylation of p38 kinase, which peaked 30 min postinfection and remained elevated for 1 h. Treatment of infected cells with SB 239063, a potent pyridinyl imidazole inhibitor of p38 kinase, resulted in up to 100% inhibition of mediator production and partially reduced levels of IL-8 mRNA as determined by quantitative RT-PCR. Treatment with SB 239063 had no effect on virus replication and was not cytotoxic at concentrations ≤ 70 μM. These studies provide the first evidence that early activation of p38 kinase by rhinovirus infection is a key event in regulation of virus-induced cytokine transcription, and may provide a new target for inhibition of symptoms and airway inflammation associated with rhinovirus infection.
Regulation of Endothelial CD73 by Adenosine: Paracrine Pathway for Enhanced Endothelial Barrier Function
Authors: Narravula Sailaja , Lennon Paul F. , Mueller Beatrice U. , Colgan Sean P. 
Published: April 2014

Abstract
During episodes of inflammation, multiple cell types release adenine nucleotides in the form of ATP, ADP, 5′-AMP, and adenosine. In particular, following activation, polymorphonuclear leukocytes release larger quantities of 5′-AMP. Extracellular 5′-AMP is metabolized to adenosine by surface-expressed 5′-ectonucleotidase (CD73). Adenosine liberated by this process activates surface adenosine A2B receptors, results in endothelial junctional reorganization, and promotes barrier function. We hypothesized that adenosine signaling to endothelia provides a paracrine loop for regulated expression of CD73 and enhanced endothelial barrier function. Using an in vitro microvascular endothelial model, we investigated the influence of 5′-AMP; adenosine; and adenosine analogues on CD73 transcription, surface expression, and function. Initial experiments revealed that adenosine and adenosine analogues induce CD73 mRNA (RT-PCR), surface expression (immunoprecipitation of surface biotinylated CD73), and function (HPLC analysis of etheno-AMP conversion to ethenoadenosine) in a time- and concentration-dependent fashion. Subsequent studies revealed that similar exposure conditions increase surface protein through transcriptional induction of CD73. Analysis of DNA-binding activity by EMSA identified a functional role for CD73 cAMP response element and, moreover, indicated that multiple cAMP agonists induce transcriptional activation of functional CD73. Induced CD73 functioned to enhance 5′-AMP-mediated promotion of endothelial barrier (measured as a paracellular flux of 70-kDa FITC-labeled tracer). These results provide an example of transcriptional induction of enzyme (CD73) by enzymatic product (adenosine) and define a paracrine pathway for the regulated expression of vascular endothelial CD73 and barrier function.
A Genome Scan Using a Novel Genetic Cross Identifies New Susceptibility Loci and Traits in a Mouse Model of Rheumatoid Arthritis
Authors: Otto Jeffrey M. , Chandrasekeran Raman , Vermes Csaba , Mikecz Katalin , Finnegan Alison , Rickert Sarah E. , Enders Jill T. , Glant Tibor T. 
Published: April 2014

Abstract
Proteoglycan-induced arthritis (PGIA) is a murine model for rheumatoid arthritis (RA) both in terms of its pathology and its genetics. PGIA can only be induced in susceptible mouse strains and their F2 progeny. Using the F2 hybrids resulting from an F1 intercross of a newly identified susceptible (C3H/HeJCr) and an established resistant (C57BL/6) strain of mouse, our goals were to: 1) identify the strain-specific loci that confer PGIA susceptibility, 2) determine whether any pathophysiological parameters could be used as markers that distinguish between nonarthritic and arthritic mice, and 3) analyze the effect of the MHC haplotype on quantitative trait loci (QTL) detection. To identify QTLs, we performed a genome scan on the F2 hybrids. For pathophysiological analyses, we measured pro- and antiinflammatory cytokines such as IL-1, IL-6, IFN-γ, IL-4, IL-10, IL-12, Ag-specific T cell proliferation and IL-2 production, serum IgG1 and IgG2 levels of both auto- and heteroantibodies, and soluble CD44. We have identified four new PGIA-linked QTLs (Pgia13 through Pgia16) and confirmed two (Pgia5, Pgia10) from our previous study. All new MHC-independent QTLs were associated with either disease onset or severity. Comprehensive statistical analysis demonstrated that while soluble CD44, IL-6, and IgG1 vs IgG2 heteroantibody levels differed significantly between the arthritic and nonarthritic groups, only Ab-related parameters colocalized with the QTLs. Importantly, the mixed haplotype (H-2b and H-2k) of the C3H × C57BL/6 F2 intercross reduced the detection of several previously identified QTLs to suggestive levels, indicating a masking effect of unmatched MHCs.
Monocyte Chemotactic Protein-1 Receptor CCR2B Is a Glycoprotein That Has Tyrosine Sulfation in a Conserved Extracellular N-Terminal Region
Authors: Preobrazhensky Alexander A. , Dragan Sofya , Kawano Tomonori , Gavrilin Mikhail A. , Gulina Irina V. , Chakravarty Leena , Kolattukudy P. E. 
Published: April 2014

Abstract
Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21–26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca2+ influx into cytoplasm. Mutations that prevented adenylate cyclase inhibition and Ca2+ influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr26; this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp25, a part of the consensus sulfation site. The expressed CCR2B was found to be N-glycosylated, as N-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational modifications probably have significant biological functions.
Surges of Increased T Cell Reactivity to an Encephalitogenic Region of Myelin Proteolipid Protein Occur More Often in Patients with Multiple Sclerosis Than in Healthy Subjects
Authors: Pender Michael P. , Csurhes Peter A. , Greer Judith M. , Mowat Paul D. , Henderson Robert D. , Cameron Kaye D. , Purdie David M. , McCombe Pamela A. , Good Michael F. 
Published: April 2014

Abstract
We have previously shown that patients with multiple sclerosis (MS) have increased T cell responses to the immunodominant region (residues 184–209) of myelin proteolipid protein (PLP). The present study investigated whether this reactivity fluctuates over time and correlates with disease activity. We performed monthly limiting dilution assays for 12–16 mo in four healthy subjects and five patients with relapsing-remitting MS to quantify the frequencies of circulating T cells proliferating in response to PLP41–58, PLP184–199, PLP190–209, myelin basic protein (MBP), MBP82–100, and tetanus toxoid. Disease activity was monitored by clinical assessment and gadolinium-enhanced magnetic resonance imaging of the brain. There were fluctuations in the frequencies of autoreactive T cells in all subjects. Compared with healthy controls, MS patients had significantly more frequent surges of T cells reactive to the 184–209 region of PLP, but infrequent surges of T cell reactivity to MBP82–100. There was temporal clustering of the surges of T cell reactivity to MBP82–100 and MBP, suggesting T cell activation by environmental stimuli. Some clinical relapses were preceded by surges of T cell reactivity to PLP184–209, and in one patient there was significant correlation between the frequency of T cells reactive to PLP184–199 and the total number of gadolinium-enhancing magnetic resonance imaging lesions. However, other relapses were not associated with surges of T cell reactivity to the Ags tested. T cells reactive to PLP184–209 may contribute to the development of some of the CNS lesions in MS.