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ABSTRACT: We have recently demonstrated that the circulating level of LOX-1 ligand containing apoB (LAB) predicts the risk of cardiovascular events; however, as is the case in other assays measuring oxidized LDL (oxLDL), chemical unstability and inter-lot variance of standard oxLDL may limit the utility of measuring LAB. This study aimed to develop an alternative protein standard that is simultaneously recognized by LOX-1 and anti-apoB antibody instead of copper-oxidized LDL.
cDNAs encoding the variable regions of anti-LOX-1 monoclonal antibody were cloned from hybridomas and reorganized to express anti-LOX-1 single-chain variable fragment (Fv). cDNAs of four regions of human apoB (B1 to B4), which were reported to be epitopes of many anti-apoB antibodies, were also cloned. After confirming the respective reactivity of Fv and apoB fragments to LOX-1 and anti-apoB antibodies, cDNAs of Fv and apoB fragments were connected to express Fv-ApoB chimeric proteins. These fusion proteins were found to be recognized by both LOX-1 and anti-apoB antibodies. Among them, the fusion proteins of Fv-B1 and Fv-B3 gave saturable binding curves against immobilized LOX-1 when detected by anti-apoB antibodies. The binding curves of different Fv-B1 preparations to LOX-1 were almost identical while those of oxLDL varied among the preparations, suggesting better quality control of Fv-B1 preparations.
The fusion proteins composed of Fv-form anti-LOX-1 antibody and apoB fragment are useful alternatives to copper-oxidized LDL in determining LAB, which would facilitate the application of modified LDL analyses to the clinical diagnosis and risk evaluation of cardiovascular disease.
Journal of atherosclerosis and thrombosis 07/2011; 18(9):818-28. · 2.69 Impact Factor
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ABSTRACT: Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase
2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin α9β1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment
in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash
of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent
mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was
incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated
OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for α9β1, was not responsible
for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild
type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice
lacking the integrin α9 subunit in leukocytes, indicating that α9β1 is required for polymerization-induced recruitment. We
have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN.
Journal of Biological Chemistry 03/2011; 286(13):11170-11178. · 4.77 Impact Factor
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ABSTRACT: Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase 2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin α9β1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for α9β1, was not responsible for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice lacking the integrin α9 subunit in leukocytes, indicating that α9β1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN.
Journal of Biological Chemistry 02/2011; 286(13):11170-8. · 4.77 Impact Factor
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Developmental Biology 08/2010; 344(1):523. · 4.07 Impact Factor
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Nobutaka Inoue,
Tomonori Okamura,
Yoshihiro Kokubo,
Yoshiko Fujita,
Yuko Sato,
Mamoru Nakanishi,
Kazuki Yanagida,
Akemi Kakino,
Shin Iwamoto,
Makoto Watanabe,
Sayoko Ogura,
Kazunori Otsui, Haruo Matsuda,
Kagehiro Uchida,
Ryo Yoshimoto,
Tatsuya Sawamura
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ABSTRACT: Lectin-like oxidized LDL receptor 1 (LOX-1) is implicated in atherothrombotic diseases. Activation of LOX-1 in humans can be evaluated by use of the LOX index, obtained by multiplying the circulating concentration of LOX-1 ligands containing apolipoprotein B (LAB) times that of the soluble form of LOX-1 (sLOX-1) [LOX index = LAB x sLOX-1]. This study aimed to establish the prognostic value of the LOX index for coronary heart disease (CHD) and stroke in a community-based cohort.
An 11-year cohort study of 2437 residents age 30-79 years was performed in an urban area located in Japan. Of these, we included in the analysis 1094 men and 1201 women without history of stroke and CHD. We measured LAB and sLOX-1 using ELISAs with recombinant LOX-1 and monoclonal anti-apolipoprotein B antibody and with 2 monoclonal antibodies against LOX-1, respectively.
During the follow-up period, there were 68 incident cases of CHD and 91 cases of stroke (with 60 ischemic strokes). Compared with the bottom quartile, the hazard ratio (HR) of the top quartile of LOX index was 1.74 (95% CI 0.92-3.30) for stroke and 2.09 (1.00-4.35) for CHD after adjusting for sex, age, body mass index, drinking, smoking, hypertension, diabetes, non-HDL cholesterol, and use of lipid-lowering agents. Compared with the bottom quartile of LOX index, the fully adjusted HRs for ischemic stroke were consistently high from the second to the top quartile: 3.39 (95% CI 1.34-8.53), 3.15 (1.22-8.13) and 3.23 (1.24-8.37), respectively.
Higher LOX index values were associated with an increased risk of CHD. Low LOX index values may be protective against ischemic stroke.
Clinical Chemistry 04/2010; 56(4):550-8. · 7.91 Impact Factor
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Atushi Nakano,
Nobutaka Inoue,
Yuko Sato,
Norihisa Nishimichi,
Kenji Takikawa,
Yoshiko Fujita,
Akemi Kakino,
Kazunori Otsui,
Saburo Yamaguchi, Haruo Matsuda,
Tatsuya Sawamura
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ABSTRACT: Hypertension is a powerful independent risk factor for atherosclerotic cardiovascular diseases; however, the precise molecular mechanisms whereby hypertension promotes atherosclerotic formation remain to be determined. The interaction between oxidized low-density lipoprotein (oxLDL) and its receptor lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays a critical role in atherogenesis. To clarify how hypertension promotes atherosclerosis, we investigated specific roles of LOX-1 in acceleration of lipid deposition under a hypertensive state.
We employed a model of stroke-prone spontaneously hypertensive rats (SHR-SP) that exhibits acute lipid deposition in mesenteric artery induced by high fat and salt loading. These vascular lipid deposition lesions share similar characteristics with the initial lesions of human atherosclerosis.
The enhanced LOX-1 expression in SHR-SP was associated with oxidized LDL deposited in vascular wall. Anti-LOX-1 neutralizing antibody dramatically suppressed the lipid deposition in vivo in SHR-SP. Vitamin E decreased serum oxLDL-like LOX-1 ligands, and suppressed the vascular lipid deposition. The vascular permeability, evaluated by the leakage of Evans blue, was markedly enhanced by pretreatment of oxLDL. The enhancement of vascular permeability induced by oxLDL was suppressed by anti-LOX-1 antibody.
The enhanced expression and activation of LOX-1 mediated the enhancement of vascular permeability, which contributed to the vascular lipid accumulation under hypertensive states.
Journal of hypertension 03/2010; 28(6):1273-80. · 4.02 Impact Factor
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Tetsuya Matsumoto,
Masatoshi Fujita,
Tatsuya Sawamura,
Akemi Kakino,
Yuko Sato,
Yoshiko Fujita, Haruo Matsuda,
Mamoru Nakanishi,
Kagehiro Uchida,
Izuru Nakae,
Hiroshi Kanda,
Akira Yoshida,
Kunihisa Miwa,
Hideki Hayashi,
Kenichi Mitsunami,
Minoru Horie
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ABSTRACT: The aim of this study was to determine the impact of pitavastatin on low-density lipoprotein cholesterol (LDL-C) and lectin-like oxidized LDL receptor-1 (LOX-1) in patients with hypercholesterolemia. Twenty-five hypercholesterolemic patients (8 male, 17 female; age 66 +/- 13, 21-80 years) who had not received anti-dyslipidemic agents and had LDL-C levels of more than 160 mg/dL were examined. Biochemical factors were measured at baseline and after treatment with pitavastatin (2 mg/day) for 6 months. Serum levels of LOX-1 with apolipoprotein B-100 particle ligand and a soluble form of LOX-1 (sLOX-1) were measured by ELISA. All subjects completed the study with no adverse side effects. Total-C (268 +/- 26 vs. 176 +/- 17 mg/dL), LDL-C (182 +/- 21 vs. 96 +/- 14 mg/dL), and LOX-1 ligand (867 +/- 452 vs. 435 +/- 262 ng/mL) were reduced with pitavastatin treatment (P < 0.0001 for each). Significant decreases in triacylglycerols were noted (P < 0.0001), but there were no changes in high-density lipoprotein cholesterol. After 6 months, there were no significant changes in high-sensitivity CRP or soluble LOX-1. At baseline, there were no significant correlations between LOX-1 ligand and either LDL-C or sLOX-1. The decrease in LOX-1 ligand was not correlated with the decrease in LDL-C, but was correlated with the decrease in sLOX-1 (r = 0.47, P < 0.05). In conclusion, pitavastatin therapy had beneficial effects on markers of oxidative stress in hypercholesterolemic subjects. Serum levels of LOX-1 ligand may be a useful biomarker of the pleiotropic effects of statins.
Lipids 03/2010; 45(4):329-35. · 2.13 Impact Factor
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ABSTRACT: Prolonged interference or suppression of maternal antibodies of the humoral immune response of newly hatched chicks to active immunization has been documented; however, the immunological mechanisms responsible for such suppression are still unclear. Laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Purified maternal anti-DNP or non-specific IgY antibodies were transferred by yolk sac inoculation to newly hatched chicks, and they were immunized with DNP-KLH or rabbit serum albumen (RSA) at 1 and 4 weeks of age. The concentrations of anti-DNP and anti-RSA antibodies in serum samples of these chicks were measured using an enzyme-linked immunosorbent assay (ELISA). The immune responses of the chicks that received a high dose of maternal anti-DNP antibodies and were immunized with an appropriate dose of DNP-KLH were suppressed. However, those of the chicks that received the same high dose of maternal non-specific IgY antibodies and were immunized with an appropriate dose of DNP-KLH and those of the chicks that received a high dose of maternal anti-DNP antibodies and were immunized with RSA were not suppressed. On the other hand, suppression of anti-DNP antibody production would not be induced if the chicks received a high dose of antigen specific maternal antibodies and were immunized with a high dose of the same antigen. These results revealed that the immune suppressive effect of maternal antibodies on the immune response of the newly hatched chicks was antigen specific and depended mainly on the ratio of antigen/maternal antibody at the time of immunization.
Journal of Veterinary Medical Science 03/2010; 72(3):257-62. · 0.85 Impact Factor
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ABSTRACT: Although the inhibitory effect of maternal antibodies on active immunization of neonates has been extensively documented, much less attention has been devoted on the exact level of these antibodies which can induce this effect and the extent of such effect. Firstly, laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH).Then, maternal anti-DNP antibodies in chicks derived from these hens were measured by using enzyme-linked immunosorbent assay (ELISA). Chicks with high levels of maternal anti-DNP showed immune suppression, while chicks with low levels of maternal anti-DNP showed normal immune response when they immunized with the same antigen at 1 and 4 weeks of age. Then, different doses of purified maternal anti-DNP were transferred to fertile eggs at 16 days of embryogenesis by in ovo injection and all chicks were immunized with DNP-KLH at 1 and 4 weeks of age. Chicks received 1 mg of anti-DNP showed normal immune response, chicks received 3 mg of anti-DNP showed weak immune response, and chicks received 5 and 8 mg of anti-DNP showed immune suppression. Chicks received 8 mg of anti-DNP were immunized with DNP-KLH at 4 and 7 weeks of age. Their immune response was significantly lower than that of chicks of no-maternal anti-DNP. These results suggested that high levels of maternal antibodies interfere or suppress the immune response of active immunization not only at early period but also at the period in which the maternal antibodies at very low levels.
Journal of Veterinary Medical Science 05/2009; 71(4):417-24. · 0.85 Impact Factor
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ABSTRACT: Osteopontin (OPN) is a cytokine and ligand for multiple members of the integrin family. OPN undergoes the in vivo polymerization catalyzed by cross-linking enzyme transglutaminase 2, which consequently increases the bioactivity through enhanced interaction with integrins. The integrin alpha9beta1, highly expressed on neutrophils, binds to the sequence SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence appears to be cryptic in intact OPN because alpha9beta1 does not recognize intact OPN. Because transglutaminase 2-catalyzed polymers change their physical and chemical properties, we hypothesized that the SVVYGLR site might also be exposed on polymeric OPN. As expected, alpha9beta1 turned into a receptor for polymeric OPN, a result obtained by cell adhesion and migration assays with alpha9-transfected cells and by detection of direct binding of recombinant soluble alpha9beta1 with colorimetry and surface plasmon resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a ligand for alpha9beta1, has been reported to attract neutrophils, we next examined migration of neutrophils to polymeric OPN using time-lapse microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which was clearly inhibited by anti-alpha9beta1 antibody. Unexpectedly, mutagenesis studies showed that alpha9beta1 bound to polymeric OPN independently of the SVVYGLR sequence, and further, SVVYGLR sequence of polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize polymeric OPN. These results demonstrate that polymerization of OPN generates a novel alpha9beta1-binding site and that the interaction of this site with the alpha9beta1 integrin is critical to the neutrophil chemotaxis induced by polymeric OPN.
Journal of Biological Chemistry 05/2009; 284(22):14769-76. · 4.77 Impact Factor
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ABSTRACT: C-reactive protein (CRP) exerts biological activity on vascular endothelial cells. This activity may promote atherothrombosis, but the effects of this activity are still controversial. Lectin-like oxidized LDL receptor-1 (LOX-1), the oxidized LDL receptor on endothelial cells, is involved in endothelial dysfunction induced by oxidized LDL.
We used laser confocal microscopy to examine and fluorescence cell image analysis to quantify the binding of fluorescently labeled CRP to cells expressing LOX-1. We then examined the binding of unlabeled CRP to recombinant human LOX-1 in a cell-free system. Small interfering RNAs (siRNAs) against LOX-1 were applied to cultured bovine endothelial cells to analyze the role of LOX-1 in native cells. To observe its in vivo effects, we injected CRP intradermally in stroke-prone spontaneously hypertensive (SHR-SP) rats and analyzed vascular permeability.
CRP bound to LOX-1-expressing cells in parallel with the induction of LOX-1 expression. CRP dose-dependently bound to the cell line and recombinant LOX-1, with significant binding detected at 0.3 mg/L CRP concentration. The K(d) value of the binding was calculated to be 1.6 x 10(-7) mol/L. siRNA against LOX-1 significantly inhibited the binding of fluorescently labeled CRP to the endothelial cells, whereas control RNA did not. In vivo, intradermal injection of CRP-induced vascular exudation of Evans blue dye in SHR-SP rats, in which expression of LOX-1 is greatly enhanced. Anti-LOX-1 antibody significantly suppressed vascular permeability.
CRP and oxidized LDL-receptor LOX-1 directly interact with each other. Two risk factors for ischemic heart diseases, CRP and oxidized LDL, share a common molecule, LOX-1, as their receptor.
Clinical Chemistry 01/2009; 55(2):285-94. · 7.91 Impact Factor
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ABSTRACT: Antibodies can distinguish not only differences in amino acid sequences (primary structure), but also differences in three-dimensional structure and thus may be useful for detecting the conversion of prion proteins, especially in vivo. For diagnosis, we prepared chicken single chain variable fragment (scFv) antibodies that specifically recognized a prion protein using a phage display approach. As antigen, mouse prion protein (MoPrP) 138-153 containing YYR residues was conjugated with KLH. Total RNA was extracted from the splenocytes of an immunized chicken, and the cDNA of scFv was ligated in a phagemid vector. The phage display scFv library was panned against the peptide antigen four times. Twenty-three scFv phage clones that tested positive using ELISA with the peptide antigen were then reacted with recombinant mouse prion protein (23-231), mouse brain homogenate, mouse neuroblastoma Neuro-2a, recombinant human V129 and M129 prion proteins, and human glyoma T98G using ELISA, immunoblotting analysis, and immunocytochemistry. The results suggested that the scFv phage clones were useful for detecting mouse and human prion proteins.
Kokuritsu Iyakuhin Shokuhin Eisei Kenkyūjo hōkoku = Bulletin of National Institute of Health Sciences 01/2009;
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Yasushi Ishigaki,
Hideki Katagiri,
Junhong Gao,
Tetsuya Yamada,
Junta Imai,
Kenji Uno,
Yutaka Hasegawa,
Keizo Kaneko,
Takehide Ogihara,
Hisamitsu Ishihara,
Yuko Sato,
Kenji Takikawa,
Norihisa Nishimichi, Haruo Matsuda,
Tatsuya Sawamura,
Yoshitomo Oka
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ABSTRACT: Several clinical studies of statin therapy have demonstrated that lowering low-density lipoprotein (LDL) cholesterol prevents atherosclerotic progression and decreases cardiovascular mortality. In addition, oxidized LDL (oxLDL) is suggested to play roles in the formation and progression of atherosclerosis. However, whether lowering oxLDL alone, rather than total LDL, affects atherogenesis remains unclear.
To clarify the atherogenic impact of oxLDL, lectin-like oxLDL receptor 1 (LOX-1), an oxLDL receptor, was expressed ectopically in the liver with adenovirus administration in apolipoprotein E-deficient mice at 46 weeks of age. Hepatic LOX-1 expression enhanced hepatic oxLDL uptake, indicating functional expression of LOX-1 in the liver. Although plasma total cholesterol, triglyceride, and LDL cholesterol levels were unaffected, plasma oxLDL was markedly and transiently decreased in LOX-1 mice. In controls, atherosclerotic lesions, detected by Oil Red O staining, were markedly increased (by 38%) during the 4-week period after adenoviral administration. In contrast, atherosclerotic progression was almost completely inhibited by hepatic LOX-1 expression. In addition, plasma monocyte chemotactic protein-1 and lipid peroxide levels were decreased, whereas adiponectin was increased, suggesting decreased systemic oxidative stress. Thus, LOX1 expressed in the livers of apolipoprotein E-deficient mice transiently removes oxLDL from circulating blood and possibly decreases systemic oxidative stress, resulting in complete prevention of atherosclerotic progression despite the persistence of severe LDL hypercholesterolemia and hypertriglyceridemia.
OxLDL has a major atherogenic impact, and oxLDL removal is a promising therapeutic strategy against atherosclerosis.
Circulation 08/2008; 118(1):75-83. · 14.74 Impact Factor
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Yutaka Kikuchi,
Tomoshi Kakeya,
Osamu Nakajima,
Ayako Sakai,
Kikuko Ikeda,
Naoto Yamaguchi,
Takeshi Yamazaki,
Ken-ichi Tanamoto, Haruo Matsuda,
Jun-ichi Sawada,
Kosuke Takatori
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ABSTRACT: The human prion protein (PrP) is a glycoprotein with a glycosylphosphatidylinositol (GPI) anchor at its C-terminus. Here we report alternative splicing within exon 2 of the PrP gene (PRNP) in the human glioblastoma cell line T98G. The open reading frame of the alternatively spliced mRNA lacked the GPI anchor signal sequence and encoded a 230 amino acid polypeptide. Its product, GPI-anchorless PrP (GPI(-) PrPSV), was unglycosylated and soluble in non-ionic detergent, and was found in the cytosolic fraction. We also detected low levels of alternatively spliced mRNA in human brain and non-neuronal tissues. When long-term passaged T98G cells were placed in a low-oxygen environment, alternatively spliced mRNA expression increased and expression of normally spliced PrP mRNA decreased. These findings imply that oxygen tension regulates GPI(-) PrPSV expression in T98G cells.
FEBS Journal 07/2008; 275(11):2965-76. · 3.79 Impact Factor
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ABSTRACT: Chicken monoclonal antibodies are potentially useful for diagnostic research and have clinical applications, as chicken show higher potential for antibody production with mammalian-conserved biological molecules. However, the applications of chicken antibodies are limited because of their immunogenicity in mammals. To overcome this problem, we have constructed a chicken-mouse chimeric antibody containing the chicken variable region and the mouse constant region. This chimeric antibody retained similar binding affinities as the parental chicken antibody. The chimeric antibody was also producible as an ascitic antibody in BALB/c mice. Furthermore, when the chimeric antibody was administered to mice, it did not provoke the mouse anti-chicken antibody response. These results indicate that the chimeric antibody is suitable for application to preclinical mouse studies.
Journal of Veterinary Medical Science 05/2008; 70(4):397-400. · 0.85 Impact Factor
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ABSTRACT: Oxidized LDL (OxLDL) is implicated in endothelial dysfunction as well as the formation and progression of atherosclerosis. It has become evident that the atherogenic properties induced by OxLDL are mainly mediated via lectin-like OxLDL receptor-1 (LOX-1). Over the past decade, much research has been performed to investigate lipid metabolism and atherogenesis using genetically engineered mice. To understand the significance of OxLDL, methods to measure the levels of OxLDL in these experimental animals should be established. Utilizing a chicken monoclonal antibody technique, here, we generated anti-human ApoB antibodies that are able to recognize mouse VLDL/LDL. These antibodies were selected from single chain fragment of variable region (scFv) phage library constructed from chickens immunized with human LDL. One of these antibodies, HUC20, was reconstructed into IgY form. Immunohistochemical analysis revealed that this novel antibody specifically stains atherosclerotic lesions of ApoE-deficient mice, associated with Oil red O positive and macrophage-antigen-positive regions. Furthermore, in combination with recombinant LOX-1, a sandwich enzyme immunoassay was developed to measure the levels of LOX-1 ligands in mouse plasma. The sandwich enzyme immunoassay revealed a dramatic increase in the level of LOX-1 ligands in the plasma of ApoE-deficient mice fed high-fat diet, suggesting a link between the level of LOX-1-ligands and the progression of atherosclerosis in mice. Hence, the chicken anti-ApoB monoclonal antibody HUC20 developed here, could be a useful tool to analyze the role of ApoB-containing lipoprotein in atherogenesis in mice.
Atherosclerosis 03/2008; 200(2):303-9. · 3.79 Impact Factor
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ABSTRACT: Recently, we reported the application of a recombinant chicken IgY monoclonal antibody, Ab3-15, against mammalian prion protein (PrP), for the diagnosis of bovine spongiform encephalopathy in cattle. In this study, we have characterized a soluble, single-chain variable fragment (scFv) form of this antibody, sphAb3-15 using brain homogenates from mice. This sphAb3-15 antibody recognized denatured forms of both PrP(C) and PrP(Sc), and PrP(Sc) after PK-treatment, on Western blotting. In sandwich ELISAs, on dot blots and by immunoprecipitation, sphAb3-15 efficiently bound to PrP from normal brain homogenates, but weakly bound PrP from scrapie-infected brain homogenates. These results suggest that sphAb3-15 selectively recognizes PrP(C) under native conditions and that the epitope recognized by sphAb3-15 may undergo conformational changes during the conversion of PrP(C) into PrP(Sc).
Biologicals 11/2007; 35(4):303-8. · 1.70 Impact Factor
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ABSTRACT: We generated two recombinant chicken IgYs, designated Ab3-15 and Ab4-19, against mammalian prion protein (PrP) from the single chain fragment of variable region (scFv) antibodies. These two antibodies recognized PrP(Sc) from bovine spongiform encephalopathy (BSE) in cattle and were more sensitive than the corresponding scFv antibodies. These antibodies also recognized PrP(Sc) from other scrapie-infected mammals. These results indicate that Ab3-15 and Ab4-19 are useful for diagnosis of BSE as well as other prion diseases.
Biologicals 04/2007; 35(1):31-4. · 1.70 Impact Factor
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ABSTRACT: Mammalian interleukin-13 (IL-13) is an important regulatory T2 cytokine secreted by activated T lymphocytes. The IL-13 receptor (IL-13R) has two different chains, IL-13Ralpha1 and IL-13Ralpha2. Although the chicken IL-13 gene is well characterized, little is known about IL-13Rs. We cloned a cDNA encoding the 380 amino acid pro-peptide of chicken IL-13Ralpha2 (chIL-13Ralpha2) and developed a monoclonal antibody (mAb), HU13-1, against it. The chIL-13Ralpha2 amino acid sequence showed 37-39% sequence identity with mammalian homologs. High levels of chIL-13Ralpha2 mRNA were expressed in liver, testis, ovary, brain, and lipopolysaccharide (LPS)-stimulated IN24 cells. HU13-1 specifically recognized recombinant chIL-13Ralpha2 in ELISAs, and western blots identified a 45-kDa glycoprotein or a 41-kDa non-glycosylated protein in LPS-stimulated IN24 cell lysates. LPS induced a gradual increase in HU13-1-positive IN24 cells over 20 h. These results indicate that mAb HU13-1 recognizes native chIL-13Ralpha2 and will be valuable for further studies of chIL-13Rs.
Developmental & Comparative Immunology 02/2007; 31(4):394-406. · 3.27 Impact Factor
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ABSTRACT: We describe a simple method for humanizing chicken monoclonal antibody (mAb). Humanization of mAbs by simple CDR-grafting often results in loss of affinity because certain framework residues of the antibody variable regions can participate in antigen-antibody interaction. In this study, humanization of chicken mAbs was achieved by CDR-grafting, followed by framework fine-tuning using a chicken phage-displayed mAb, phAb4-31, as a model antibody. In order to fine-tune the framework, we used the phage-displayed combinatorial library with permutation of important framework residues. After panning the humanized library, the "most humanized" variants were selected and analyzed for antigen-binding activity. All of these clones retained affinity comparable to the parental chicken mAb. These results suggest that chicken mAbs can easily be humanized, and thus humanized chicken mAbs may be practically applied as therapeutic agents.
Molecular Immunology 03/2006; 43(6):634-42. · 2.90 Impact Factor
