Background: Our previous study showed that purified protein derivative (PPD)-stimulated pleural mononuclear cells (PMC) from tuberculous pleurisy (Tbp) produced significantly more $IFN-{\gamma}$ (10- to 70-fold) after in vitro PPD stimulation than freshly isolated pleural cells from malignant pleurisy. The present study was designed to determine whether blocking the CD40-CD40 ligand (CD40L) interaction decreases $IFN-{\gamma}$ production by altering IL-12 levels. Methods: IL-12 and $IFN-{\gamma}$ production after neutralizing anti-CD40L antibody treatment was compared to the efficacy of anti-CD80, anti-CD86, and a combination of anti-CD80 and CD86 (CD80+86) monoclonal antibodies (mAb). These activities were measured by enzyme-linked immunosorbent assays (ELISAs) and reverse transcription-polymerase chain reaction (RT-PCR), after in vitro stimulation with PPO antigen (Ag). Results: Neutralization of CD80, CD86 and CD80+86 did not decrease $IFN-{\gamma}$ and IL-12 production in Tbp-PMC, whereas neutralization of CD40L significantly depressed IL-12 p40 and $IFN-{\gamma}$. In addition, neutralization of CD40L completely inhibited IL-12 p40 and $IFN-{\gamma}$ mRNA expression. Conclusion: The CD40-CD40L interaction might play a major role in IL-12 and $IFN-{\gamma}$ production in Tbp-PMC, thus contributing to protective immunity in human tuberculosis.
Background: This study was aimed to differentiate two forms of CTLA-4 (CD152) in activated peripheral blood lymphocyte and clarify the mechanism how cytoplasmic form of this molecule is targeted to cell surface. Methods: For this purpose we generated 2 different anti-human CD152 peptide antibodies and 5 different N'-terminal deletion mutant CTLA4Ig fusion proteins and carried out a series of Western blot and ELISA analyses. Antipeptide antibodies made in this study were anti-CTLA4pB and anti-CTLA4pN. The former recognized a region on extracellular single V-like domain and the latter recognized N'-terminal sequence of leader domain of human CD152. Results: In Western blot, the former antibody recognized recombinant human CTLA4Ig fusion protein as an antigen. And this recognition was completely blocked by preincubating antipeptide antibody with the peptide used for the antibody generation at the peptide concentration of 200 ug/ml. These antibodies were recognized human CD152 as a cytoplasmic sequestered- and a membrane bound- forms in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte (PBL). These two forms of CD152 were further differentiated by using anti-CTLA4pN and anti-CTLA4pB antibodies such that former recognized cytosolic form only while latter recognized both cytoplasmic- and membraneforms of this molecule. Furthermore, in a transfection expression study of 5 different N'-terminal deletion mutant CTLA4Ig, mutated proteins were secreted out from transfected cell surface only when more than 6 amino acids from N'-terminal were deleted. Conclusion: Our results implies that cytosolic form of CTLA-4 has leader sequence while membrane form of this molecule does not. And also suggested is that at least N'-terminal 6 amino acid residues of human CTLA-4 are required for regulation of targeting this molecule from cytosolic- to membrane- area of activated human peripheral blood T lymphocyte.
Kim, Jin Sook;You, Da-Bin;Lim, Ji-Young;Lee, Sung-Eun;Kim, Yoo-Jin;Kim, Hee-Je;Chung, Nack-Gyun;Min, Chang-Ki
IMMUNE NETWORK
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v.15
no.2
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pp.66-72
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2015
Currently, detecting biochemical differences before and after allogeneic stem cell transplantation (SCT) for improved prediction of acute graft-versus-host disease (aGVHD) is a major clinical challenge. In this pilot study, we analyzed the kinetics of circulating adipokine levels in patients with or without aGVHD before and after allogeneic SCT. Serum samples were obtained and stored at $-80^{\circ}C$ within 3 hours after collection, prior to conditioning and at engraftment after transplantation. A protein array system was used to measure the levels of 7 adipokines of patients with aGVHD (n=20) and without aGVHD (n=20). The resistin level at engraftment was significantly increased (p<0.001) after transplantation, regardless of aGVHD occurrence. In the non-aGVHD group, the concentrations of the hepatocyte growth factor (HGF) (mean values${\pm}$SD; $206.6{\pm}34.3$ vs. $432.3{\pm}108.9pg/ml$, p=0.040) and angiopoietin-2 (ANG-2) (mean values${\pm}$SD; $3,197.2{\pm}328.3$ vs. $4,471.8{\pm}568.4pg/ml$, p=0.037) at engraftment were significantly higher than those of the pre-transplant period, whereas in the aGVHD group, the levels of adipokines did not change after transplantation. Our study suggests that changes in serum HGF and ANG-2 levels could be considered helpful markers for the subsequent occurrence of aGVHD.
Interleukin-23 (IL-23) is a novel pro-inflammatory cytokine which has been implicated to play a pathogenic role in rheumatoid arthritis (RA). This study was undertaken to investigate the IL-23 inductive activity of the proinflammatory cytokine IL-17, $IL-1{\beta}$ and tumor necrosis factor (TNF-${\alpha}$) in RA synovial fluid mononuclear cells (SFMC). Expression of IL-23p19, IL-17, $IL-1{\beta}$ and TNF-${\alpha}$ in joint was examined by immunohistochemistry (IHC) of patients with RA and osteoarthritis (OA). The effects of IL-17 and $IL-1{\beta}$ on expression of IL-23p19 in human SFMC from RA patients were determined by reverse transcriptase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). IL-23p19 was expressed in the RA fibroblast like synoviocyte (FLS), but not from OA FLS. Similar to the protein expression, IL-23p19 mRNA could be detected by RT-PCR in RA SFMC. IL-17 and $IL-1{\beta}$ could induce RA SFMC to produce the IL-23p19. The effects of IL-17 were much stronger than $IL-1{\beta}$ or TNF-${\alpha}$. These responses were observed in a doseresponsive manner. In addition, IL-17 or $IL-1{\beta}$ neutralizing antibody down-regulated the expression of IL-23p19 induced by LPS in RA-SFMC. Our results demonstrate that IL-23p19 is overexpressed in RA synovium and IL-17 and $IL-1{\beta}$ appears to upregulate the expression of IL-23p19 in RA-SFMC.
Background: Platelet-activating factor (PAF) induces nuclear factor $(NF)-{\kappa}B$ activation and angiogenesis and increases tumor growth and pulmonary tumor metastasis in vivo. The role of $NF-{\kappa}B$ activation in PAF-induced angiogenesis in a mouse model of Matrigel implantation, and in PAF-mediated pulmonary tumor metastasis were investigated. Methods: Angiogenesis using Matrigel and experimental pulmonary tumor metastasis were tested in a mouse model. Electrophoretic mobility shift assay was done for the assessment of $NF-{\kappa}B$ translocation to the nucleus. Expression of angiogenic factors, such as tumor necrosis factor $(TNF)-{\alpha}$, interleukin $(IL)-1{\alpha}$, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were tested by RT-PCR and ELISA. Results: PAF induced a dose- and time-dependent angiogenic response. PAF-induced angiogenesis was significantly blocked by PAF antagonist, CV6209, and inhibitors of $NF-{\kappa}B$ expression or action, including antisense oligonucleotides to p65 subunit of $NF-{\kappa}B$ (p65 AS) and antioxidants such as ${\alpha}$-tocopherol and N-acetyl-L-cysteine. In vitro, PAF activated the transcription factor, $NF-{\kappa}B$ and induced mRNA expression of $TNF-{\alpha}$, $IL-1{\alpha}$, bFGF, VEGF, and its receptor, KDR. The PAF-induced expression of the above mentioned factors was inhibited by p65 AS or antioxidants. Also, protein synthesis of VEGF was increased by PAF and inhibited by p65 AS or antioxidants. The angiogenic effect of PAF was blocked when anti-VEGF antibodies was treated or antibodies against $TNF-{\alpha}$, $IL-1{\alpha}$, and bFGF was co-administrated, but not by antibodies against $TNF-{\alpha}$, $IL-1{\alpha}$, and bFGF each alone. PAF-augmented pulmonary tumor metastasis was inhibited by p65 AS or antioxidants. Conclusion: These data indicate that PAF increases angiogenesis and pulmonary tumor metastasis through $NF-{\kappa}B$ activation and expression of $NF-{\kappa}B$-dependent angiogenic factors.
This study was conducted to determine whether CD4 T cell responses to citrullinated fibrinogen occur in patients with rheumatoid arthritis (RA), especially in HLA-DR4-positive subjects. Whole peripheral blood mononuclear cells (PBMCs) of RA patients and control subjects were stimulated with citrullinated fibrinogen peptides, and T-cell production of proliferation and proinflammatory cytokines, such as interferon-${\gamma}$(IFN-${\gamma}$) and interleukin-17A (IL-17A), were measured. In addition, CD4 T cells from RA patients were stimulated with the citrullinated fibrinogen peptide, $Fib-{\alpha}$ R84Cit, identified as a DRB1*0401-restricted T cell epitope in HLA-DR4 transgenic mice, and the degree of T cell activation was examined similarly. No proliferative responses to the citrullinated fibrinogen peptides were observed in whole PBMCs or CD4 T cells from RA patients. Furthermore, no increased production of IFN-${\gamma}$ or IL-17A was found in whole PBMCs or CD4 T cells stimulated with the citrullinated fibrinogen peptides, although these cells responded to recall antigen, a mixture of tetanus toxoid, purified protein derivative (PPD) from Mycobacterium tuberculosis, and Candida albicans. The results of this study indicate that anti-citrulline immunity in RA patients may be mediated by fibrinogen because there is no evidence of CD4 T cell-mediated immune responses to citrullinated fibrinogen peptides.
Proceedings of the Materials Research Society of Korea Conference
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2011.10a
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pp.7-7
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2011
Quantum beam technology has been expected to develop breakthroughs for nanotechnology during the third basic plan of science and technology (2006~2010). Recently, Green- or Life Innovations has taken over the national interests in the fourth basic science and technology plan (2011~2015). The NIMS (National Institute for Materials Science) has been conducting the corresponding mid-term research plans, as well as other national projects, such as nano-Green project (Global Research for Environment and Energy based on Nanomaterials science). In this lecture, the research trends in Japan and NIMS are firstly reviewed, and the typical achievements are highlighted over key nanotechnology fields. As one of the key nanotechnologies, the quantum beam research in NIMS focused on synchrotron radiation, neutron beams and ion/atom beams, having complementary attributes. The facilities used are SPring-8, nuclear reactor JRR-3, pulsed neutron source J-PARC and ion-laser-combined beams as well as excited atomic beams. Materials studied are typically fuel cell materials, superconducting/magnetic/multi-ferroic materials, quasicrystals, thermoelectric materials, precipitation-hardened steels, nanoparticle-dispersed materials. Here, we introduce a few topics of neutron scattering and ion beam nanofabrication. For neutron powder diffraction, the NIMS has developed multi-purpose pattern fitting software, post RIETAN2000. An ionic conductor, doped Pr2NiO4, which is a candidate for fuel-cell material, was analyzed by neutron powder diffraction with the software developed. The nuclear-density distribution derived revealed the two-dimensional network of the diffusion paths of oxygen ions at high temperatures. Using the high sensitivity of neutron beams for light elements, hydrogen states in a precipitation-strengthened steel were successfully evaluated. The small-angle neutron scattering (SANS) demonstrated the sensitive detection of hydrogen atoms trapped at the interfaces of nano-sized NbC. This result provides evidence for hydrogen embrittlement due to trapped hydrogen at precipitates. The ion beam technology can give novel functionality on a nano-scale and is targeting applications in plasmonics, ultra-fast optical communications, high-density recording and bio-patterning. The technologies developed are an ion-and-laser combined irradiation method for spatial control of nanoparticles, and a nano-masked ion irradiation method for patterning. Furthermore, we succeeded in implanting a wide-area nanopattern using nano-masks of anodic porous alumina. The patterning of ion implantation will be further applied for controlling protein adhesivity of biopolymers. It has thus been demonstrated that the quantum beam-based nanotechnology will lead the innovations both for nano-characterization and nano-fabrication.
COP9 signalosome (CSN), which is originally identified as the regulator of the photomorphogenic development in plant, is highly conserved protein complex in diverse eukaryotic organisms. Most eukaryotic CSN complex is composed of 8 subunits, which is structurally and functionally similar to the lid subunit of 26S proteasome and eIF3 translation initiation complex. CSN play important functions in the regulation of cell cycle and checkpoint response by controlling Cullin-Ring E3 ubiquitin ligases (CRL) activities. CSN exhibits an isopeptidase activity which cleaves the neddylated moiety of cullin components. In fission yeast, S-phase cell cycle progression was delayed and the sensitivity to g-ray or UV was increased in CSN1 and CSN2 deletion mutants, indicating that yeast CSN is also involved in the checkpoint regulation. CSN in fungal system more closely resembles that of the higher organisms in the structure and assembly of their components. Functionally, CSN is associated with the regulation of conidiation rhythms in Neurospora crassa and the sexual development in Aspsergillus nidulans. Recent studies also revealed that CSN functions as an essential cell cycle regulator, playing key roles in the regulation of DNA replication and DNA damage response in Aspergillus. Overall, CSN of microorganisms, such as fission yeast and fungi, share functionally common aspects with higher organisms, implying that they can be useful tools to study the role of CSN in the CRL-mediated diverse cellular activities.
Proceedings of the Korean Society of Near Infrared Spectroscopy Conference
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2001.06a
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pp.1062-1062
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2001
The concept of “precision agriculture” or “site-specific farming” is usually confined to the fields of soil science, crop science and agronomy. However, because plants grow in soil, animals eat plants, and humans eat animal products, it could be argued (perhaps with some poetic licence) that the fields of feed quality, animal nutrition and animal production should also be considered in this context. NIR spectroscopy has proved over the last 20 years that it can provide a firm foundation for quality measurement across all of these fields, and with the continuing developments in instrumentation, computer capacity and software, is now a major cog in the wheel of precision agriculture. There have been a few giant leaps and a lot of small steps in the impact of NIR on the animal world. These have not been confined to the amazing advances in hardware and software, although would not have occurred without them. Rapid testing of forages, grains and mixed feeds by NIR for nutritional value to livestock is now commonplace in commercial laboratories world-wide. This would never have been possible without the pioneering work done by the USDA NIR Forage Research Network in the 1980's, following the landmark paper of Norris et al. in 1976. The advent of calibration transfer between instruments, algorithms which utilize huge databases for calibration and prediction, and the ability to directly scan whole grains and fresh forages can also be considered as major steps, if not leaps. More adventurous NIR applications have emerged in animal nutrition, with emphasis on estimating the functional properties of feeds, such as in vivo digestibility, voluntary intake, protein degradability and in vitro assays to simulate starch digestion. The potential to monitor the diets of grazing animals by using faecal NIR spectra is also now being realized. NIR measurements on animal carcasses and even live animals have also been attempted, with varying degrees of success, The use of discriminant analysis in these fields is proving a useful tool. The latest giant leap is likely to be the advent of relatively low-cost, portable and ultra-fast diode array NIR instruments, which can be used “on-site” and also be fitted to forage or grain harvesters. The fodder and livestock industries are no longer satisfied with what we once thought was revolutionary: a 2-3 day laboratory turnaround for fred quality testing. This means that the instrument needs to be taken to the samples rather than vice versa. Considerable research is underway in this area, but the challenge of calibration transfer and maintenance of instrument networks of this type remains. The animal world is currently facing its biggest challenges ever; animal welfare, alleged effects of animal products on human health, environmental and economic issues are difficult enough, but the current calamities of BSE and foot and mouth disease are “the last straw” NIR will not of course solve all these problems, but is already proving useful in some of these areas and will continue to do so.
Berzaghi, Paolo;Flinn, Peter C.;Dardenne, Pierre;Lagerholm, Martin;Shenk, John S.;Westerhaus, Mark O.;Cowe, Ian A.
Proceedings of the Korean Society of Near Infrared Spectroscopy Conference
/
2001.06a
/
pp.1141-1141
/
2001
The aim of the study was to evaluate the performance of 3 calibration methods, modified partial least squares (MPLS), local PLS (LOCAL) and artificial neural network (ANN) on the prediction of chemical composition of forages, using a large NIR database. The study used forage samples (n=25,977) from Australia, Europe (Belgium, Germany, Italy and Sweden) and North America (Canada and U.S.A) with information relative to moisture, crude protein and neutral detergent fibre content. The spectra of the samples were collected with 10 different Foss NIR Systems instruments, which were either standardized or not standardized to one master instrument. The spectra were trimmed to a wavelength range between 1100 and 2498 nm. Two data sets, one standardized (IVAL) and the other not standardized (SVAL) were used as independent validation sets, but 10% of both sets were omitted and kept for later expansion of the calibration database. The remaining samples were combined into one database (n=21,696), which was split into 75% calibration (CALBASE) and 25% validation (VALBASE). The chemical components in the 3 validation data sets were predicted with each model derived from CALBASE using the calibration database before and after it was expanded with 10% of the samples from IVAL and SVAL data sets. Calibration performance was evaluated using standard error of prediction corrected for bias (SEP(C)), bias, slope and R2. None of the models appeared to be consistently better across all validation sets. VALBASE was predicted well by all models, with smaller SEP(C) and bias values than for IVAL and SVAL. This was not surprising as VALBASE was selected from the calibration database and it had a sample population similar to CALBASE, whereas IVAL and SVAL were completely independent validation sets. In most cases, Local and ANN models, but not modified PLS, showed considerable improvement in the prediction of IVAL and SVAL after the calibration database had been expanded with the 10% samples of IVAL and SVAL reserved for calibration expansion. The effects of sample processing, instrument standardization and differences in reference procedure were partially confounded in the validation sets, so it was not possible to determine which factors were most important. Further work on the development of large databases must address the problems of standardization of instruments, harmonization and standardization of laboratory procedures and even more importantly, the definition of the database population.
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