bluuebunny/crossref_metadata_2025 huggingface dataset:
Macroscale Modeling of Multi-Physics Fields during Vacuum Arc Remelting of Ti-6Al-4V
Authors: Zhu Bin , Xue Xiang Yi , Kou Hong Chao , Xiao Cong , Li Jin Shan
Published: April 2014

A 3-D finite element model has been established using ANSYS12.0 software to simulate multi-physical interaction behavior during the Vacuum Arc Remelting (VAR) of 740-mm-diameter ingots of Ti-6Al-4V. The models of temperature field, electromagnetic and flow field were combined by progressive method. The effect of thermal contraction was considered in the simulation of temperature field and electromagnetic by setting a thin layer with different nature parameters at the ingot-crucible interface. The model results demonstrate the distributions of temperature, Lorenz force and flow velocity, and the influence of water cooling conditions, melting current and other process parameters. The molten pool behavior is mostly dominated by buoyancy force under circumstances in this case. The increase of the melting current results in an increase of the pool depth and melting rate, and causes great change of the molten pool profile, while the influence of the water cooling conditions is ignored.
Crystal Plasticity Finite-Element Simulation of Single Phase Titanium Alloy with 3D Polycrystalline Models
Authors: Xie Shao , Tang Bin , Liu Yi , Han Feng Bo , Kou Hong Chao , Li Jin Shan
Published: April 2014

Based on the rate-dependent crystal plasticity theory, a finite element code which considers crystallographic slip as deformation mechanism of material was developed to investigate the stress–strain response of the β phase of Ti-5553 during uniaxial tension. Three dimensional models with random grain shapes generated by Voronoi tessellation were used for simulations, and two discretization methods were used to disperse the models. Firstly, the parameters of material were identified by fitting simulation stress-strain curves with experimental data. Then the global stress-strain curves were calculated, and effects of mesh type and mesh density were discussed. Results show that mesh type has a relatively significant influence on overall responses, whereas the influence of mesh density is slight. Investigate of local stress-strain response in each grain was also conducted, and obvious inter-granular heterogeneities were observed. Quantitative analysis indicates that the range of stress and strain variations is affected by mesh type.
Electrochemical and Salt Spray Corrosion Behavior of Copper Alloy Contact Wires in Chloride Solution
Authors: Zhang Peng Chao , Zhang Jian , Jie Jin Chuan , Gao Yuan , Dong Yong , Wang Tong Min , Li Ting Ju
Published: April 2014

The effect of different alloying elements on corrosion behavior of copper alloys was investigated using electrochemical corrosion and salt spray corrosion test in NaCl solution. Cu-Ag has the most stable corrosion current in the potentiostatic scanning test, exhibiting a better corrosion resistant performance. It can be analyzed from corrosion surface morphologies that Cu-Ag presents exfoliation corrosion mechanism while Cu-Sn shows crevice corrosion mechanism. Cu-Mg has a complex corrosion process caused by multiple corrosion mechanism. In the salt spray corrosion test, the corrosion degree of Cu-Ag is lighter than those of Cu-Sn and Cu-Mg after 24h test. Therefore, the Cu-Ag alloy exhibits the best corrosion resistance in chloride solution.
Fractographical Investigation on High-Cycle Fatigue Behavior of Direct Aging GH4169 Superalloy
Authors: Zhang Li , Wu Xue Ren , Huang Xin Yue , Li Xu Dong
Published: April 2014

The present work is aimed at the fatigue crack initiation behavior of the direct aging superalloy GH4169 at 650 °C. Un-notched specimens were tested under high-cycle fatigue loading with the two stress ratios of R = 0.1 and 0.5. Fracture surfaces were examined using field emission scanning electron microscopy (FESEM). Special attention is paid to the crack initiation sites. Two microstructure features in the fatigue crack initiation regions have been observed. One is obviously the feature of the Ti (C,N)-inclusions, and the other is some kinds of facets. The analysis on the facets was carried out by using energy dispersive spectra (EDS). It was found that the elements at the facets are similar to the matrix. However, some inclusions, Nb (C,N), have been found in the subsurface facets in a few specimens. Sometimes, fatigue lives of the specimens for the crack initiating from the inclusions are longer than those of cracks from the facets, depending on the size of inclusions and the depth from sample surface. Crack initiation for most of the specimens occurs at inclusions, and therefore inclusion is a primary source of crack initiation for this alloy.
Study on Corrosion Resistance of Several Fe-Based Amorphous Alloys
Authors: Tian Xiu Juan , Liu Wan Fu , Xie Kun
Published: April 2014

Three ribbons were obtained by rapid solidification using the melt-spinning techniques, and annealed with different temperatures. The corrosion behavior of ribbons in 0.1mol/L NaCl solution and the effects of annealing temperatures on the corrosion resistance were studied by open circuit potential and polarization curve test. Phase structure changes of the ribbons before and after corrosion were analyzed by X-ray diffraction (XRD). The amorphous forming ability and crystallization temperature of materials were characterized by differential scanning calorimetry (DSC). The surface morphology and composition before and after corrosion were analyzed by scanning electron microscopy (SEM) and electron probe microanalysis (EPMA). The result showed that Fe90Si5.5B4.5and Fe91Si5.5B3.5had high corrosion resistance. Corrosion resistance of the three ribbons had increased along with the increase of annealing temperatures.
Synthesis and Characterization of Itaconic Acid Doped Polyaniline Nanorods
Authors: Wang Yan Min , Li Hui Min , Li Ting Xi , Gu Yi Jie
Published: April 2014

Polyaniline nanorods were synthesized via soap-free emulsion polymerization using itaconic acid as dopant and characterized by particle size and distribution measurement, scanning electron microscope (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and thermo-gravimetric analysis (TGA). The particle size of the prepared polyaniline has nearly mono-dispersed property because almost all the particle sizes distribute among the range of 14~110μm. Its morphology consists of the nanorods with the diameter of 50~100nm and the length of 0.1~1μm. FT-IR spectrum indicated that polyaniline of half oxidation state was successfully synthesized and obviously doped with itaconic acid. Moreover, the polymer possesses excellent thermal stability with little weight loss up to 208°C.
Research of Crystallization Kinetics and Thermal Stability of W48.5Ni34Fe14.6B2.9 Amorphous Ribbons
Authors: Liu Wen Sheng , Zhang Jia Jia , Ma Yun Zhu , Ye Xiao Shan
Published: April 2014

The W48.5Ni34Fe14.6B2.9 (wt.%) amorphous ribbons were prepared using melt spinning method. The crystallization kinetics of amorphous ribbons was investigated by differential scanning calorimeter (DSC) at different heating rates from 10 to 40 K/min. The activation energy of amorphous ribbons was simulated by Kissinger and Ozawa equations, respectively. The precipitated phases at different annealing temperatures were analyzed by X-ray diffraction (XRD). The results showed that the glass transition temperature (Tg), the crystallization temperature (Tx) and the peak temperature (Tp) were found to increase during enhancing heating rate, indicating the obvious kinetic effects of glass transition and crystallization. The consistency of values of the activation energy calculated by Kissinger and Ozawa equations suggested the good accuracy about experiment results. The specific temperatures of Tx and Tp detected by DSC and the deduced crystallization activation energy are generally higher than these of other amorphous alloys, which suggests a higher resistance to crystallization. The W48.5Ni34Fe14.6B2.9 alloy substantially remained at amorphous structure when annealed at 923K for 60 min, showing that the alloy possessed a higher thermal stability.
Cigarette Smoke Exposure Exacerbates Lung Inflammation and Compromises Immunity to Bacterial Infection
Authors: Lugade Amit A. , Bogner Paul N. , Thatcher Thomas H. , Sime Patricia J. , Phipps Richard P. , Thanavala Yasmin
Published: April 2014

The detrimental impact of tobacco on human health is clearly recognized, and despite aggressive efforts to prevent smoking, close to one billion individuals worldwide continue to smoke. People with chronic obstructive pulmonary disease are susceptible to recurrent respiratory infections with pathogens, including nontypeable Haemophilus influenzae (NTHI), yet the reasons for this increased susceptibility are poorly understood. Because mortality rapidly increases with multiple exacerbations, development of protective immunity is critical to improving patient survival. Acute NTHI infection has been studied in the context of cigarette smoke exposure, but this is the first study, to our knowledge, to investigate chronic infection and the generation of adaptive immune responses to NTHI after chronic smoke exposure. After chronic NTHI infection, mice that had previously been exposed to cigarette smoke developed increased lung inflammation and compromised adaptive immunity relative to air-exposed controls. Importantly, NTHI-specific T cells from mice exposed to cigarette smoke produced lower levels of IFN-γ and IL-4, and B cells produced reduced levels of Abs against outer-membrane lipoprotein P6, with impaired IgG1, IgG2a, and IgA class switching. However, production of IL-17, which is associated with neutrophilic inflammation, was enhanced. Interestingly, cigarette smoke–exposed mice exhibited a similar defect in the generation of adaptive immunity after immunization with P6. Our study has conclusively demonstrated that cigarette smoke exposure has a profound suppressive effect on the generation of adaptive immune responses to NTHI and suggests the mechanism by which prior cigarette smoke exposure predisposes chronic obstructive pulmonary disease patients to recurrent infections, leading to exacerbations and contributing to mortality.
Cutting Edge: Inflammatory Responses Can Be Triggered by TREM-1, a Novel Receptor Expressed on Neutrophils and Monocytes
Authors: Bouchon Axel , Dietrich Jes , Colonna Marco
Published: April 2014

We have identified new activating receptors of the Ig superfamily expressed on human myeloid cells, called TREM (triggering receptor expressed on myeloid cells). TREM-1 is selectively expressed on blood neutrophils and a subset of monocytes and is up-regulated by bacterial LPS. Engagement of TREM-1 triggers secretion of IL-8, monocyte chemotactic protein-1, and TNF-α and induces neutrophil degranulation. Intracellularly, TREM-1 induces Ca2+ mobilization and tyrosine phosphorylation of extracellular signal-related kinase 1 (ERK1), ERK2 and phospholipase C-γ. To mediate activation, TREM-1 associates with the transmembrane adapter molecule DAP12. Thus, TREM-1 mediates activation of neutrophil and monocytes, and may have a predominant role in inflammatory responses.
Cutting Edge: A Role for CD1 in the Pathogenesis of Lupus in NZB/NZW Mice
Authors: Zeng Defu , Lee Mi-Kyeong , Tung James , Brendolan Andrea , Strober Samuel
Published: April 2014

Since anti-CD1 TCR transgenic T cells can activate syngeneic B cells via CD1 to secrete IgM and IgG and induce lupus in BALB/c mice, we studied the role of CD1 in the pathogenesis of lupus in NZB/NZW mice. Approximately 20% of B cells from the spleens of NZB/NZW mice expressed high levels of CD1 (CD1high B cells). The latter subset spontaneously produced large amounts of IgM anti-dsDNA Abs in vitro that was up to 25-fold higher than that of residual CD1int/low B cells. T cells in the NZB/NZW spleen proliferated vigorously to the CD1-transfected A20 B cell line, but not to the parent line. Treatment of NZB/NZW mice with anti-CD1 mAbs ameliorated the development of lupus. These results suggest that the CD1high B cells and their progeny are a major source of autoantibody production, and activation of B cells via CD1 may play an important role in the pathogenesis of lupus.
Cutting Edge: The IgG Response to the Circumsporozoite Protein Is MHC Class II-Dependent and CD1d-Independent: Exploring the Role of GPIs in NK T Cell Activation and Antimalarial Responses
Authors: Molano Alberto , Park Se-Ho , Chiu Ya-Hui , Nosseir Sandy , Bendelac Albert , Tsuji Moriya
Published: April 2014

Biochemical analysis has suggested that self GPI anchors are the main natural ligand associated with mouse CD1d molecules. A recent study reported that Vα14+ NK T cells responded to self as well as foreign (parasite-derived) GPIs in a CD1d-dependent manner. It further reported that the IgG response to the Plasmodium berghei malarial circumsporozoite (CS) protein was severely impaired in CD1d-deficient mice, leading to a model whereby NK T cells, upon recognition of CD1d molecules presenting the CS-derived GPI anchor, provide help for B cells secreting anti-CS Abs. We tested this model by comparing the anti-CS Ab responses of wild-type, CD1d-deficient, and MHC class II-deficient mice. We found that the IgG response to the CS protein was solely MHC class II-dependent. Furthermore, by measuring the response of a broad panel of CD1d-autoreactive T cells to GPI-deficient CD1d-expressing cells, we found that GPIs were not required for autoreactive responses.
Cutting Edge: Stromal Cell-Derived Factor-1 Is a Costimulator for CD4+ T Cell Activation
Authors: Nanki Toshihiro , Lipsky Peter E.
Published: April 2014

Stromal cell-derived factor (SDF)-1 is a chemoattractant for T cells, precursor B cells, monocytes, and neutrophils. SDF-1α was also found to up-regulate expression of early activation markers (CD69, CD25, and CD154) by anti-CD3-activated CD4+ T cells. In addition, SDF-1α costimulated proliferation of CD4+ T cells and production of IL-2, IFN-γ, IL-4, and IL-10. Stimulation with SDF-1α alone did not induce activation marker expression, proliferation, or cytokine production by the CD4+ T cells. SDF-1α-mediated costimulation was blocked by anti-CXC chemokine receptor-4 mAb. RANTES also increased activation marker expression by anti-CD3-stimulated peripheral CD4+ T cells, but less effectively than SDF-1α did, and did not up-regulate IL-2 production and proliferation. These results indicate that SDF-1 and CXC chemokine receptor-4 interactions not only play a role in T cell migration but also provide potent costimulatory signals to Ag-stimulated T cells.
Cutting Edge: The Human CytomegalovirusUL40Gene Product Contains a Ligand for HLA-E and Prevents NK Cell-Mediated Lysis
Authors: Ulbrecht Matthias , Martinozzi Silvia , Grzeschik Mariola , Hengel Hartmut , Ellwart Joachim W. , Pla Marika , Weiss Elisabeth H.
Published: April 2014

Human CMV has evolved multiple strategies to interfere with immune recognition of the host. A variety of mechanisms target Ag presentation by MHC class I molecules resulting in a reduced class I cell-surface expression. This down-regulation of class I molecules is expected to trigger NK cytotoxicity, which would have to be counteracted by the virus to establish long-term infection. Here we describe that the human CMV open reading frame UL40 encodes a canonical ligand for HLA-E, identical with the HLA-Cw03 signal sequence-derived peptide. Expression of UL40 in HLA-E-positive target cells conferred resistance to NK cell lysis via the CD94/NKG2A receptor. Generation of the UL40-derived HLA-E ligand was also observed in TAP-deficient cells. The presence of a functional TAP-independent HLA-E ligand in the UL40 signal sequence implicates this viral gene as an important negative regulator of NK activity.
Quantification of Cell Turnover Kinetics Using 5-Bromo-2′-deoxyuridine1
Authors: Bonhoeffer Sebastian , Mohri Hiroshi , Ho David , Perelson Alan S.
Published: April 2014

5-Bromo-2′-deoxyuridine (BrdU) is frequently used to measure the turnover of cell populations in vivo. However, due to a lack of detailed mathematical models that describe the uptake and loss of BrdU in dividing cell populations, assessments of cell turnover kinetics have been largely qualitative rather than quantitative. In this study, we develop a mathematical framework for the analysis of BrdU-labeling experiments. We derive analytical expressions for the fraction of labeled cells within cell populations that are growing, declining, or at equilibrium. Fitting the analytical functions to data allows us to quantify the rates of cell proliferation and cell loss, as well as the rate of cell input from a source. We illustrate this for the BrdU labeling of T lymphocytes of uninfected and SIV-infected rhesus macaques.
CD40 Engagement on Synovial Fibroblast Up-Regulates Production of Vascular Endothelial Growth Factor
Authors: Cho Chul-Soo , Cho Mi-La , Min So-Youn , Kim Wan-Uk , Min Do-June , Lee Shin-Seok , Park Sung-Hwan , Choe Jongseon , Kim Ho-Youn
Published: April 2014

We tested the impact of CD40 engagement on the production of vascular endothelial growth factor (VEGF) from rheumatoid synovial fibroblasts. Fibroblast-like synovial cells (FLS) were prepared from the synovial tissues of rheumatoid arthritis patients and cultured in the presence of CD40 ligand-transfected (CD40L+) L cells. VEGF levels were determined in the culture supernatants by ELISA. Stimulation of FLS by CD40L+ L cells increased the production of VEGF by 4.1-fold over the constitutive levels of unstimulated FLS. The CD40L on activated T cells from rheumatoid synovial fluid also up-regulated VEGF production from FLS. Neither indomethacin nor Abs to IL-1β, TNF-α, and TGF-β did affect CD40L-induced VEGF production. Stimulation of FLS with TNF-α, IL-1β, and TGF-β increased VEGF production by 1.6-, 2.0-, and 5.2-fold, respectively, and displayed an additive effect on the production of VEGF by CD40L. VEGF mRNA expression was also up-regulated by the stimulation of FLS with membranes from the CD40L+ L cells. Dexamethasone completely abrogated CD40L-induced VEGF production. In addition, pyrrolidine dithiocarbamate partially down-regulated CD40L-induced VEGF production, showing that the NF-κB pathway was partly involved in the signaling of CD40L leading to VEGF production. Collectively, these results suggest that the interaction between CD40 on synovial fibroblasts and CD40L expressed on activated T lymphocytes may be directly involved in the neovascularization in rheumatoid synovitis by enhancing the production of VEGF.
Bioactivities of Fas Ligand-Expressing Retroviral Particles
Authors: Jodo Satoshi , Strehlow David , Ju Shyr-Te
Published: April 2014

Culture supernatants from retroviral packaging cells carrying the human Fas ligand (FasL) gene killed both human (Jurkat) and mouse (LB27.4) targets within 5 h of incubation. Cytotoxicity was found both in a fraction ≥500 kDa and a fraction between 50 and 500 kDa. Following ultracentrifugation, the activity in the ≥500-kDa fraction was concentrated in the pellet (FasL vector preparation (VP)), which was also infective when added to NIH-3T3 cells. Both Polybrene and poly-l-lysine significantly enhanced the cytotoxicity of FasL VP but not anti-Fas mAb, soluble FasL (sFasL), and cell-associated FasL. In the presence of Polybrene, FasL VP killed targets that are resistant to anti-Fas mAb and sFasL. The infectivity but not FasL cytotoxicity of FasL VP was sensitive to irradiation and heat shock. By contrast, cytotoxicity of FasL VP could be enhanced or inhibited depending on the doses of anti-FasL mAb. Interestingly, the infectivity of FasL VP was specifically enhanced by anti-FasL mAb, suggesting that a nonviral gene product could be used to regulate the behavior of the retroviral vector. Thus, in addition to expressing potent FasL cytotoxicity, the FasL VP exhibits unique properties heretofore not attributed to anti-Fas mAb, sFasL, and cell-associated FasL. Our study raises the possibility of using the retroviral gene-packaging technology to make powerful, versatile, and regulatable bioactive vesicles expressing a predetermined function of the protein encoded by the target gene.
The Mouse Tumor Cell Lines EL4 and RMA Display Mosaic Expression of NK-Related and Certain Other Surface Molecules and Appear to Have a Common Origin
Authors: Gays Frances , Unnikrishnan Meera , Shrestha Sunil , Fraser Karen P. , Brown Adam R. , Tristram Colin M. G. , Chrzanowska-Lightowlers Zosia M. A. , Brooks Colin G.
Published: April 2014

As a potential means for facilitating studies of NK cell-related molecules, we examined the expression of these molecules on a range of mouse tumor cell lines. Of the lines we initially examined, only EL4 and RMA expressed such molecules, both lines expressing several members of the Ly49 and NKRP1 families. Unexpectedly, several of the NK-related molecules, together with certain other molecules including CD2, CD3, CD4, CD32, and CD44, were often expressed in a mosaic manner, even on freshly derived clones, indicating frequent switching in expression. In each case examined, switching was controlled at the mRNA level, with expression of CD3ζ determining expression of the entire CD3-TCR complex. Each of the variable molecules was expressed independently, with the exception that CD3 was restricted to cells that also expressed CD2. Treatment with drugs that affect DNA methylation and histone acetylation could augment the expression of at least some of the variable molecules. The striking phenotypic similarity between EL4 and RMA led us to examine the state of their TCRβ genes. Both lines had identical rearrangements on both chromosomes, indicating that RMA is in fact a subline of EL4. Overall, these findings suggest that EL4 is an NK-T cell tumor that may have retained a genetic mechanism that permits the variable expression of a restricted group of molecules involved in recognition and signaling.
Potassium Regulates IL-1β Processing Via Calcium-Independent Phospholipase A2
Authors: Walev Iwan , Klein Jochen , Husmann Matthias , Valeva Angela , Strauch Susanne , Wirtz Heiner , Weichel Oksana , Bhakdi Sucharit
Published: April 2014

We report that potassium leakage from cells leads to activation of the Ca2+-independent phospholipase A2 (iPLA2), and the latter plays a pivotal role in regulating the cleavage of pro-IL-1β by the IL-converting enzyme caspase-1 in human monocytes. K+ efflux led to increases of cellular levels of glycerophosphocholine, an unambiguous indicator of phospholipase A2 activation. Both maturation of IL-1β and formation of glycerophosphocholine were blocked by bromoenol lactone, the specific iPLA2 inhibitor. Bromoenol lactone-dependent inhibition of IL-1β processing was not due to perturbation of the export machinery for pro-IL-1β and IL-1β or to caspase-1 suppression. Conspicuously, activation of Ca2+-dependent phospholipase A2 did not support but rather suppressed IL-1β processing. Thus, our findings reveal a specific role for iPLA2 activation in the sequence of events underlying IL-1β maturation.
Mechanisms of Graft Acceptance: Evidence That Plasminogen Activator Controls Donor-Reactive Delayed-Type Hypersensitivity Responses in Cardiac Allograft Acceptor Mice
Authors: Bickerstaff Alice A. , Xia Dongyuan , Pelletier Ronald P. , Orosz Charles G.
Published: April 2014

We have used delayed-type hypersensitivity (DTH) responses to probe the mechanisms of drug-induced cardiac allograft acceptance in mice. DBA/2→C57BL/6 cardiac allograft recipients treated transiently with gallium nitrate accept their grafts for >90 days and fail to display DBA/2-reactive DTH responses. These DTH responses are restored when anti-TGF-β Abs are included at the challenge site, and cell depletion studies showed that this DTH inhibition is mediated by CD4+ cells. Real-time PCR analysis revealed that allograft acceptor mice produce no more than background levels of TGF-β mRNA at DTH challenge sites. This suggests that DTH regulation in allograft acceptor mice may involve TGF-β activation, rather than TGF-β production. The protease, plasmin, can activate TGF-β, and activated T cells can express a receptor for the plasmin-producing enzyme urokinase-type plasminogen activator (uPA), and can also produce both uPA and tissue-type plasminogen activator (tPA). We observed that Abs to tPA or uPA can replace anti-TGF-β mAb for the restoration of donor-reactive DTH responses in allograft acceptor mice. Histologic analysis revealed that accepted cardiac allografts express uPA, tPA, and active TGF-β, whereas accepted cardiac isografts express only tPA, but not uPA or activated TGF-β. These data demonstrate that local tPA and uPA contribute to DTH regulation in allograft acceptor mice and suggest that these elements of the fibrinolytic pathway are used to control donor-reactive cell-mediated immunity in allograft acceptor mice.
An Altered Peptide Ligand Antagonizes Antigen-Specific T Cells of Patients with Human T Lymphotropic Virus Type I-Associated Neurological Disease
Authors: Kubota Ryuji , Soldan Samantha S. , Martin Roland , Jacobson Steven
Published: April 2014

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurologic disease associated with HTLV-I infection, in which chronically activated, HTLV-I-specific CD8+ CTL have been suggested to be immunopathogenic. In HLA-A2 HAM/TSP patients, CD8+ HTLV-I-specific CTLs recognize an immunodominant peptide of the HTLV-I Tax protein, Tax11–19. We examined the functional outcome on activation of both cloned peripheral blood and cerebrospinal spinal fluid-derived CTL and bulk PBMC from HAM/TSP patients by altered peptide ligands (APL) derived from HTLV-I Tax11–19. In CTL clones generated from PBMC and CSF of HLA-A2 HAM/TSP patients, an APL substituted at position 5 significantly decreased CTL responses when compared with the native peptide. Moreover, these ligands were also shown to inhibit CTL responses to the native peptide in bulk PBMC of HLA-A2 HAM/TSP patients. These data suggest that a modification of an antigenic peptide at the central position can manipulate the T cell responses in bulk PBMC from different individuals with an inflammatory disease. Additionally, these results have implications for the potential use of APL-based immunotherapy in this T cell-mediated CNS disease.
Cloning and Characterization of Human Lnk, an Adaptor Protein with Pleckstrin Homology and Src Homology 2 Domains that Can Inhibit T Cell Activation
Authors: Li Yijin , He Xiaoqing , Schembri-King Josephine , Jakes Scott , Hayashi Jun
Published: April 2014

Lnk was originally cloned from a rat lymph node cDNA library and shown to participate in T cell signaling. Human Lnk (hLnk) was cloned by screening a Jurkat cell cDNA library. hLnk has a calculated molecular mass of 63 kDa, and its deduced amino acid sequence indicates the presence of an N-terminal proline-rich region, a pleckstrin homology domain, and a Src homology 2 domain. When expressed in COS cells, hLnk migrates with an apparent molecular mass of 75 kDa. Confocal fluorescence microscope analysis indicates that in COS cells transfected with an expression vector encoding a chimeric Lnk-green fluorescent protein, hLnk is found at the juxtanuclear compartment and also appears to be localized at the plasma membrane. Lnk is tyrosine-phosphorylated by p56lck. Following phosphorylation, p56lck binds to tyrosine-phosphorylated hLnk through its Src homology 2 domain. In COS cells cotransfected with hLnk, p56lck, and CD8-ζ, hLnk associated with tyrosine-phosphorylated TCR ζ-chain through its Src homology 2 domain. The overexpression of Lnk in Jurkat cells led to an inhibition of anti-CD3 mediated NF-AT-Luc activation. Our study reveals a potentially new mechanism of T cell-negative regulation.
Retargeting T Cell-Mediated Inflammation: A New Perspective on Autoantibody Action
Authors: Lou Ya-Huan , Park Kwan-Kyu , Agersborg Sally , Alard Pascale , Tung Kenneth S. K.
Published: April 2014

To understand the pathogenesis of organ-specific autoimmune disease requires an appreciation of how the T cell-mediated inflammation is targeted, and how the organ function is compromised. In this study, autoantibody was documented to influence both of these parameters by modulating the distribution of T cell-mediated inflammation. The murine autoimmune ovarian disease is induced by immunization with the ZP3330–342 peptide of the ovarian zona pellucida 3 glycoprotein, ZP3. Passively transferred or actively induced Ab to ZP3335–342 bound to the zona pellucida in the functional and degenerative ovarian follicles, and the ovaries remained histologically normal. Transfer of ZP3330–342 peptide-specific T cells targeted the degenerative follicles and spared the functional follicles, and the resultant interstitial oophoritis was associated with unimpaired ovarian function. Unexpectedly, the coexistence of ZP3330–342 peptide-specific T cells and zona-bound autoantibody led to a dramatic translocation of the ovarian inflammation to the growing and mature ovarian follicles, with destruction of the ovarian functional unit. Ab retargeted both Th1-induced mononuclear inflammation and Th2-induced eosinophilic inflammation, and retargeting was induced by murine and rat polyclonal Abs to multiple distinct native B cell determinants of the zona pellucida. Therefore, by reacting with the native determinants in tissue Ag, Ab alters the distribution of T cell-mediated inflammation, and results in destruction of the functional units of the target organ. We propose that this is a clinically important and previously unappreciated element of Ab action in autoimmune disease.
IL-1 Signaling Cascade in Liver Cells and the Involvement of a Soluble Form of the IL-1 Receptor Accessory Protein
Authors: Jensen Liselotte E. , Muzio Marta , Mantovani Alberto , Whitehead Alexander S.
Published: April 2014

The proinflammatory cytokine IL-1 induces the biosynthesis of a number of immunologically important proteins during infection, tissue damage, and/or stress, in part through the activation of the transcription factor NF-κB. Signal transduction is initiated at the cell membrane by complex formation between extracellular IL-1 and the transmembrane IL-1R type I (IL-1RI) and IL-1R accessory protein (IL-1RAcP). The intracellular signaling cascade involves recruitment of two IL-1R-associated kinases, IRAK1 and IRAK2, and the adapter protein MyD88, events which are dependent on the intracellular domain of membrane-bound IL-1RAcP (mIL-1RAcP). In mouse liver, IL-1RAcP is expressed as a soluble protein (sIL-1RAcP), the function of which is unknown. We have cloned the human sIL-1RAcP and established by sequence analysis that the human sIL-1RAcP mRNA arises from alternative splicing of the IL-1RAcP gene (shown here to encompass 12 exons spanning more than 56 kb). Furthermore, we demonstrate that human HepG2 hepatoma cells express both mIL-1RAcP and sIL-1RAcP and that signal transduction in these cells is mediated through IRAK1, IRAK2, and MyD88. We show that phorbol esters induce a change in the pre-mRNA splice pattern such that sIL-1RAcP mRNA becomes the dominant form. Overexpression of a membrane-anchored fusion protein of sIL-1RAcP and MHC in HepG2 cells inhibits IL-1-mediated NF-κB activation, whereas coexpression of IL-1RI with membrane-anchored sIL-1RAcP restores the capacity of the cells to respond to IL-1. This suggests that sIL-1RAcP may act as an inhibitor of IL-1 by directly interacting with IL-1RI to abolish its capacity to transduce signal.
Role of OCA-B in 3′-IgH Enhancer Function
Authors: Stevens Sean , Ong Jane , Kim Unkyu , Eckhardt Laurel A. , Roeder Robert G.
Published: April 2014

OCA-B (alternately called Bob1 and OBF-1) is a B cell-specific coactivator that interacts with the ubiquitously expressed Oct-1 and the B cell-restricted Oct-2 to activate transcription via the octamer site (5′-ATGCAAAT-3′). OCA-B−/− mice appear to undergo normal Ag-independent B cell maturation. However, Ag-dependent B cell differentiation, including germinal center formation, production of secondary Ig isotypes, and proliferation in response to surface Ig cross-linking, is greatly affected. We demonstrate that the observed reductions in expression of class-switched isotypes in OCA-B−/− mice may be due in part to deficiencies in the function of the 3′-IgH enhancer elements. Furthermore, we find that surface Ig cross-linking represses all the Ig enhancers and that this repression is absent in OCA-B−/− B cells. These results suggest an important role for OCA-B in Ig enhancer function in vivo.
Human C4b-Binding Protein Has Overlapping, But Not Identical, Binding Sites for C4b and Streptococcal M Proteins
Authors: Blom Anna M. , Berggård Karin , Webb Joanna H. , Lindahl Gunnar , Villoutreix Bruno O. , Dahlbäck Björn
Published: April 2014

Many strains of Streptococcus pyogenes bind C4b-binding protein (C4BP), an inhibitor of complement activation. The binding is mediated by surface M proteins in a fashion that has been suggested to mimic the binding of C4b. We have previously shown that a positively charged cluster at the interface between complement control protein domains 1 and 2 of C4BP α-chain is crucial for the C4b-C4BP interaction. To extend this observation, and to investigate the interaction with M proteins, we constructed and characterized a total of nine mutants of C4BP. We identified a key recognition surface for M proteins that overlaps with the C4b binding site because substitution of R64 and H67 by Gln dramatically reduces binding to both ligands. However, the analysis of all mutants indicates that the binding sites for C4b and M proteins are only overlapping, but not identical. Furthermore, M proteins were able to displace C4BP from immobilized C4b, whereas C4b only weakly affected binding of C4BP to immobilized M proteins. We found that the molecular mechanisms involved in these two interactions differ because the binding between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b binding. In addition, six mAbs directed against the α-chain interfered with C4b-C4BP interaction, whereas only two of them efficiently inhibited binding of C4BP to M proteins. Collectively, our results suggest that binding between C4b and C4BP is governed mostly by electrostatic interactions, while additional noncovalent forces cause tight binding of C4BP to streptococcal M proteins.
Repeated Exposure Induces Periportal Fibrosis inSchistosoma mansoni-Infected Baboons: Role of TGF-β and IL-4
Authors: Farah Idle O. , Mola Paul W. , Kariuki Thomas M. , Nyindo Mramba , Blanton Ronald E. , King Christopher L.
Published: April 2014

Recently, we observed that repeated Schistosoma mansoni infection and treatment boost Th2-associated cytokines and TGF-β production in baboons. Other studies have shown that some chronically infected baboons develop hepatic fibrosis. Because TGF-β, IL-2, and IL-4 have been shown to participate in development of fibrosis in murine schistosomiasis, the present study examined whether repeated exposure stimulates hepatic fibrosis in olive baboons. To test this hypothesis, animals were exposed to similar numbers of S. mansoni cercariae given once or repeatedly. After 19 wk of infection, animals were cured with praziquantel and reinfected once or multiple times. Hepatic granulomatous inflammation and fibrosis were assessed from serial liver biopsies taken at weeks 6, 9, and 16 after reinfection and egg Ag (schistosome egg Ag)-specific cytokine production by PBMC were measured simultaneously. Periportal fibroblast infiltration and extracellular matrix deposition (fibrosis), angiogenesis, and biliary duct hyperplasia developed in some animals. The presence and amount of fibrosis directly correlated with the frequency of exposure. Fibrosis was not associated with adult worm or tissue egg burden. The amount of fibrosis correlated with increased schistosome egg Ag-driven TGF-β at 6, 9, and 16 wk postinfection (rs = 0.9, 0.8, and 0.54, respectively, all p < 0.01) and IL-4 production (p = 0.02) at 16 wk postinfection and not IFN-γ, IL-2, IL-5, or IL-10. These data suggest that repeated exposure is a risk factor for periportal fibrosis by a mechanism that primes lymphocytes to produce increased levels of profibrotic molecules that include TGF-β and IL-4.
Cellular Regulation of Cytosolic Group IV Phospholipase A2 by Phosphatidylinositol Bisphosphate Levels
Authors: Balsinde Jesús , Balboa María A. , Li Wen-Hong , Llopis Juan , Dennis Edward A.
Published: April 2014

Cytosolic group IV phospholipase A2 (cPLA2) is a ubiquitously expressed enzyme with key roles in intracellular signaling. The current paradigm for activation of cPLA2 by stimuli proposes that both an increase in intracellular calcium and mitogen-activated protein kinase-mediated phosphorylation occur together to fully activate the enzyme. Calcium is currently thought to be needed for translocation of the cPLA2 to the membrane via a C2 domain, whereas the role of cPLA2 phosphorylation is less clearly defined. Herein, we report that brief exposure of P388D1 macrophages to UV radiation results in a rapid, cPLA2-mediated arachidonic acid mobilization, without increases in intracellular calcium. Thus, increased Ca2+ availability is a dispensable signal for cPLA2 activation, which suggests the existence of alternative mechanisms for the enzyme to efficiently interact with membranes. Our previous in vitro data suggested the importance of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in the association of cPLA2 to model membranes and hence in the regulation of cPLA2 activity. Experiments described herein show that PtdInsP2 also serves a similar role in vivo. Moreover, inhibition of PtdInsP2 formation during activation conditions leads to inhibition of the cPLA2-mediated arachidonic acid mobilization. These results suggest that cellular PtdInsP2 levels are involved in the regulation of group IV cPLA2 activation.
Anti-Inflammatory Effect of Heat Shock Protein Induction Is Related to Stabilization of IκBα Through Preventing IκB Kinase Activation in Respiratory Epithelial Cells
Authors: Yoo Chul-Gyu , Lee Seunghee , Lee Choon-Taek , Kim Young Whan , Han Sung Koo , Shim Young-Soo
Published: April 2014

Heat shock protein (HSP) induction confers protection against diverse forms of cellular and tissue injury. However, the mechanism by which HSP exerts cytoprotective effects is unclear. Because HSP induction inhibits genetic expression of pro-inflammatory cytokines, the transcription of which is dependent on NF-κB activation, we explored the relationship between the anti-inflammatory effect of HSP induction and the NF-κB/IκBα pathway. Both HS and sodium arsenite treatment increased HSP70 expression time dependently at mRNA and protein levels. Prior induction of HSP suppressed cytokine-induced IL-8 and TNF-α expression at both mRNA and protein levels. Although HSP induction did not affect total cellular expression of NF-κB, TNF-α-induced increase in NF-κB-DNA binding activity and nuclear translocation of the p65 subunit of NF-κB were inhibited by prior HSP induction, suggesting that activation of NF-κB was blocked. Cytokine-induced IκBα phosphorylation and its degradation were blocked in HSP-induced cells. Immune complex kinase assays demonstrated that TNF-α induced increase in IκB kinase activity was suppressed by prior HSP induction. These results suggest that the anti-inflammatory effect of HSP induction in respiratory epithelial cells is related to stabilization of IκBα, possibly through the prevention of IκB kinase activation, which thereby inhibits activation of NF-κB.
Effect of CD14 Blockade in Rabbits with Escherichia coli Pneumonia and Sepsis
Authors: Frevert Charles W. , Matute-Bello Gustavo , Skerrett Shawn J. , Goodman Richard B. , Kajikawa Osamu , Sittipunt Chanchai , Martin Thomas R.
Published: April 2014

CD14, a pattern recognition receptor found on myeloid cells, is a critical component of the innate immune system that mediates local and systemic host responses to Gram-negative and Gram-positive bacterial products. Previous studies in normal animals have tested the effect of CD14 blockade on the systemic response to i.v. LPS. The goals of the study were to determine whether CD14 blockade protected against the deleterious systemic response associated with Escherichia coli pneumonia and to determine whether this strategy affected the pulmonary response to tissue infection. Rabbits were pretreated with either anti-CD14 mAb or isotype control mAb at 2.5 mg/kg. E. coli (1 × 109 CFU) was inoculated into the lungs, and the animals were observed for either 4 or 24 h. The blockade of CD14 improved the mean arterial blood pressure (p = 0.001) and decreased the i.v. fluid requirements (p = 0.01). Although this therapy protected the vascular compartment, rabbits treated with anti-CD14 mAb had increased bacterial burdens in the bronchoalveolar lavage fluid recovered from the instilled lung (p = 0.005) and widened alveolar-arterial oxygen difference. Blockade of CD14 prevents the deleterious systemic responses that occur in sepsis; however, other measures are necessary to control bacterial proliferation at the primary site of infection.
Induction of Functional Anaphylatoxin C5a Receptors on Hepatocytes by In Vivo Treatment of Rats with IL-6
Authors: Schieferdecker Henrike L. , Schlaf Gerald , Koleva Milena , Götze Otto , Jungermann Kurt
Published: April 2014

In normal rat liver, anaphylatoxin C5a receptors (C5aR) are only expressed by nonparenchymal cells, mainly Kupffer cells and hepatic stellate cells, but not by parenchymal cells, i.e., hepatocytes (HC). Nevertheless, C5a stimulates glucose output by HC. This HC-specific defense reaction is induced indirectly via prostanoids secreted by the C5aR-expressing Kupffer cells and hepatic stellate cells. It is shown here that under inflammatory conditions simulated by in vivo treatment of rats with IL-6 C5aR mRNA and protein were induced in HC in a time-dependent manner. Maximal mRNA and protein expression were observed at 4–8 h and 8–10 h, respectively, after IL-6 injection. The newly expressed receptors were functional, because recombinant rat C5a significantly activated glycogen phosphorylase in HC isolated from IL-6-treated but not in HC from control rats. In perfused livers of IL-6-treated animals in contrast to control animals, recombinant rat C5a-induced glucose output was not impaired by inhibition of prostanoid synthesis and function with the cyclooxygenase inhibitor indomethacin and the thromboxane receptor antagonist daltroban. These results indicate that HC-specific defense reactions might be differently regulated under normal and inflammatory conditions as shown here for the indirect prostanoid-dependent or direct C5a-induced activation of hepatocellular glycogen phyosphorylase and glucose output in control or IL-6-treated rats, respectively.
Thymic and Postthymic Regulation of Diabetogenic CD8 T Cell Development in TCR Transgenic Nonobese Diabetic (NOD) Mice
Authors: Kanagawa Osami , Shimizu Jun , Vaupel Barbara A.
Published: April 2014

Natural development of diabetes in nonobese diabetic (NOD) mice requires both CD4 and CD8 T cells. Transgenic NOD mice carrying αβ TCR genes from a class I MHC (Kd)-restricted, pancreatic β cell Ag-specific T cell clone develop diabetes significantly faster than nontransgenic NOD mice. In these TCR transgenic mice, a large fraction of T cells express both transgene derived and endogenous TCR β chains. Only T cells expressing two TCR showed reactivity to the islet Ag. Development of diabetogenic T cells is inhibited in mice with no endogenous TCR expression due to the SCID mutation. These results demonstrate that the expression of two TCRs is necessary for the autoreactive diabetogenic T cells to escape thymic negative selection in the NOD mouse. Further analysis with MHC congenic NOD mice revealed that diabetes development in the class I MHC-restricted islet Ag-specific TCR transgenic mice is still dependent on the presence of the homozygosity of the NOD MHC class II I-Ag7.
Human Autoreactive CD4+ T Cells from Naive CD45RA+ and Memory CD45RO+ Subsets Differ with Respect to Epitope Specificity and Functional Antigen Avidity
Authors: Muraro Paolo A. , Pette Martin , Bielekova Bibiana , McFarland Henry F. , Martin Roland
Published: April 2014

T cells with specificity for self-Ags are normally present in the peripheral blood, and, upon activation, may target tissue Ags and become involved in the pathogenesis of autoimmune processes. In multiple sclerosis, a demyelinating disease of the CNS, it is postulated that inflammatory damage is initiated by CD4+ T cells reactive to myelin Ags. To investigate the potential naive vs memory origin of circulating myelin-reactive cells, we have generated myelin basic protein (MBP)- and tetanus toxoid-specific T cell clones from CD45RA+/RO− and CD45RO+/RA− CD4+ T cell subsets from the peripheral blood of multiple sclerosis patients and controls. Our results show that 1) the response to MBP, different from that to TT, predominantly emerges from the CD45RA+ subset; 2) the reactivity to immunodominant MBP epitopes mostly resides in the CD45RA+ subset; 3) in each individual, the recognition of single MBP epitopes is skewed to either subset, with no overlap in the Ag fine specificity; and 4) in spite of a lower expression of costimulatory and adhesion molecules, CD45RA+ subset-derived clones recognize epitopes with higher functional Ag avidity. These findings point to a central role of the naive CD45RA+ T cell subset as the source for immunodominant, potentially pathogenic effector CD4+ T cell responses in humans.
Impact of HIV-1 Infection on VH3 Gene Repertoire of Naive Human B Cells
Authors: Scamurra Ronald W. , Miller Darren J. , Dahl Linda , Abrahamsen Mitchell , Kapur Vivek , Wahl Sharon M. , Milner Eric C. B. , Janoff Edward N.
Published: April 2014

B cells of the largest Ig variable heavy chain gene (VH) family, VH3, are reportedly decreased in patients with late stage HIV-1 disease. This deficit may contribute to their impaired responses to infections and vaccines. We confirmed that the VH3 family was underrepresented in serum IgM proteins, with a 45% decrease in patients with advanced HIV-1 disease. However, the proportion of VH3 within VH(1–6) IgM mRNA from peripheral B cells did not differ from that of control subjects (mean ± SD, 57.1 ± 9.7 vs 61.1 ± 8.7%). Similarly, within VH(1–6) IgD mRNA, which even more closely represents the unstimulated naive repertoire, the relative expression of VH3 mRNA was comparable in the two groups. Moreover, the frequency of individual genes within the VH3 family for IgD, particularly genes which encode putative HIV-1 gp120 binding sites, also was normal in HIV-1-infected patients. However, VH3 family expression for IgG mRNA was significantly decreased (17%) and VH4 IgG was increased (33%) relative to other VH families in advanced HIV-1-infected patients. Thus, the changes in VH family expression were more readily apparent in previously activated IgG “memory” B cell populations and, likely, in cells actively producing IgM rather than in resting naive cells. The presence of a relatively normal naive VH3 IgM and IgD mRNA repertoire in resting cells supports the prospect that with proper stimulation, particularly in conjunction with effective antiviral therapy, vigorous humoral immune responses to infections and vaccines may be elicited in this high-risk population.