Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 59
Filter
1.
PLoS Pathog ; 16(8): e1008823, 2020 08.
Article in English | MEDLINE | ID: mdl-32845931

ABSTRACT

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.


Subject(s)
Influenza A virus/metabolism , Lung , Macrophages , Orthomyxoviridae Infections , PrPC Proteins/metabolism , Signal Transduction , Animals , Antibodies, Monoclonal, Murine-Derived/pharmacology , Lung/metabolism , Lung/pathology , Lung/virology , Macrophages/metabolism , Macrophages/pathology , Macrophages/virology , Mice , Mice, Mutant Strains , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/metabolism , Orthomyxoviridae Infections/pathology , PrPC Proteins/antagonists & inhibitors , src-Family Kinases/genetics , src-Family Kinases/metabolism
2.
Parasitol Res ; 115(11): 4123-4128, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27384177

ABSTRACT

In the poultry industry, Eimeria spp. is one of the important pathogens which cause significant economic losses. We have previously generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina and with cross-reactive among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium spp. Furthermore, the protein of Cryptosporidium parvum recognized by the 6D-12-G10 has been identified as elongation factor-1α (EF-1α). In the present study, to identify the target molecule of E. acervulina by the mAb, we performed two-dimensional Western blotting analysis. Finally, we found two positive molecules which are identified as EF-1α and a related protein. Our previous finding using C. parvum and the results in this study suggest that EF-1α could be associated with the invasion facilitated by the cytoskeleton at the apical region of zoites.


Subject(s)
Antigens, Protozoan/immunology , Chickens/parasitology , Coccidiosis/veterinary , Eimeria/immunology , Peptide Elongation Factor 1/metabolism , Poultry Diseases/parasitology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Protozoan/immunology , Blotting, Western/veterinary , Coccidiosis/parasitology , Cross Reactions , Cryptosporidium parvum/immunology , Cryptosporidium parvum/isolation & purification , Eimeria/isolation & purification , Enzyme-Linked Immunosorbent Assay/veterinary , Neospora/immunology , Neospora/isolation & purification , Sporozoites , Toxoplasma/immunology , Toxoplasma/isolation & purification
3.
J Biol Chem ; 288(47): 34111-34120, 2013 Nov 22.
Article in English | MEDLINE | ID: mdl-24085304

ABSTRACT

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-ß- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.


Subject(s)
Antigens, Protozoan/immunology , Cryptosporidium parvum/immunology , Peptide Elongation Factor 1/immunology , Protozoan Proteins/immunology , Sporozoites/immunology , Animals , Antibodies, Protozoan/immunology , Antigens, Protozoan/genetics , Antigens, Protozoan/metabolism , Cell Line, Tumor , Cell Membrane/immunology , Cell Membrane/metabolism , Cryptosporidiosis/genetics , Cryptosporidiosis/immunology , Cryptosporidiosis/metabolism , Cryptosporidiosis/prevention & control , Cryptosporidium parvum/metabolism , Cryptosporidium parvum/pathogenicity , Male , Mice , Mice, SCID , Peptide Elongation Factor 1/genetics , Peptide Elongation Factor 1/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Protozoan Vaccines/immunology , Sporozoites/metabolism
4.
J Biol Chem ; 287(27): 22593-608, 2012 Jun 29.
Article in English | MEDLINE | ID: mdl-22549775

ABSTRACT

DNA and protein arrays are commonly accepted as powerful exploratory tools in research. This has mainly been achieved by the establishment of proper guidelines for quality control, allowing cross-comparison between different array platforms. As a natural extension, glycan microarrays were subsequently developed, and recent advances using such arrays have greatly enhanced our understanding of protein-glycan recognition in nature. However, although it is assumed that biologically significant protein-glycan binding is robustly detected by glycan microarrays, there are wide variations in the methods used to produce, present, couple, and detect glycans, and systematic cross-comparisons are lacking. We address these issues by comparing two arrays that together represent the marked diversity of sialic acid modifications, linkages, and underlying glycans in nature, including some identical motifs. We compare and contrast binding interactions with various known and novel plant, vertebrate, and viral sialic acid-recognizing proteins and present a technical advance for assessing specificity using mild periodate oxidation of the sialic acid chain. These data demonstrate both the diversity of sialic acids and the analytical power of glycan arrays, showing that different presentations in different formats provide useful and complementary interpretations of glycan-binding protein specificity. They also highlight important challenges and questions for the future of glycan array technology and suggest that glycan arrays with similar glycan structures cannot be simply assumed to give similar results.


Subject(s)
Glycolipids/metabolism , Glycomics , N-Acetylneuraminic Acid/metabolism , Polysaccharides/metabolism , Protein Array Analysis , Acetylation , Antibodies/immunology , Antibody Specificity , Antigens, CD/metabolism , Cell Adhesion Molecules/metabolism , Glycolipids/immunology , Glycomics/instrumentation , Glycomics/methods , Glycomics/standards , Lectins/metabolism , N-Acetylneuraminic Acid/immunology , Oxidation-Reduction , Periodic Acid/metabolism , Plant Lectins/metabolism , Polysaccharides/immunology , Protein Array Analysis/instrumentation , Protein Array Analysis/methods , Protein Array Analysis/standards , Reproducibility of Results , Sialic Acid Binding Immunoglobulin-like Lectins
5.
Biochem Biophys Res Commun ; 418(4): 628-33, 2012 Feb 24.
Article in English | MEDLINE | ID: mdl-22290229

ABSTRACT

Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary. In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions.


Subject(s)
N-Acetylgalactosaminyltransferases/immunology , Peptides/immunology , Single-Chain Antibodies/biosynthesis , Two-Hybrid System Techniques , Amino Acid Sequence , Animals , Cell Line , Chickens , Humans , Molecular Sequence Data , Peptide Library , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics
6.
J Atheroscler Thromb ; 18(9): 818-28, 2011.
Article in English | MEDLINE | ID: mdl-21727756

ABSTRACT

AIM: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.


Subject(s)
Apolipoprotein B-48/metabolism , Scavenger Receptors, Class E/metabolism , Amino Acid Sequence , Apolipoprotein B-48/genetics , Base Sequence , Blotting, Western , DNA Primers , Enzyme-Linked Immunosorbent Assay , Humans , Ligands , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Scavenger Receptors, Class E/chemistry , Scavenger Receptors, Class E/immunology
7.
Cell Mol Neurobiol ; 31(7): 999-1008, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21516351

ABSTRACT

We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.


Subject(s)
Brain/cytology , Microglia/physiology , Microglia/transplantation , Prions/immunology , Scrapie/physiopathology , Single-Chain Antibodies/immunology , Animals , Cell Line , Genetic Vectors , HEK293 Cells , Humans , Mice , Microglia/cytology , Prions/pathogenicity , Prions/physiology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Scrapie/therapy , Single-Chain Antibodies/genetics , Single-Chain Antibodies/therapeutic use , Survival Rate
8.
J Biol Chem ; 286(13): 11170-8, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21321126

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 α9ß1. 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 α9ß1, 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 α9ß1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN.


Subject(s)
Chemotaxis/physiology , Integrins/metabolism , Neutrophil Infiltration/physiology , Neutrophils/metabolism , Osteopontin/metabolism , Protein Multimerization/physiology , Animals , Cell Line, Tumor , Chemotaxis/drug effects , Female , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Humans , Integrins/genetics , Mice , Neutrophil Infiltration/drug effects , Osteopontin/genetics , Osteopontin/pharmacology , Protein Glutamine gamma Glutamyltransferase 2 , Thrombin/chemistry , Thrombin/genetics , Thrombin/metabolism , Transglutaminases/genetics , Transglutaminases/metabolism
9.
J Hypertens ; 28(6): 1273-80, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20216085

ABSTRACT

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The enhanced expression and activation of LOX-1 mediated the enhancement of vascular permeability, which contributed to the vascular lipid accumulation under hypertensive states.


Subject(s)
Blood Vessels/metabolism , Hypertension/metabolism , Lipid Metabolism , Scavenger Receptors, Class E/physiology , Animals , Immunohistochemistry , In Vitro Techniques , Male , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Reverse Transcriptase Polymerase Chain Reaction
10.
Lipids ; 45(4): 329-35, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20229124

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.


Subject(s)
Hypercholesterolemia/blood , Quinolines/pharmacology , Scavenger Receptors, Class E/blood , Adult , Aged , Aged, 80 and over , Anticholesteremic Agents/pharmacology , Anticholesteremic Agents/therapeutic use , Biomarkers/blood , Biomarkers/metabolism , Cholesterol, LDL/blood , Down-Regulation/drug effects , Female , Humans , Hypercholesterolemia/drug therapy , Ligands , Male , Middle Aged , Quinolines/therapeutic use , Young Adult
11.
J Immunol ; 184(6): 3269-75, 2010 Mar 15.
Article in English | MEDLINE | ID: mdl-20173026

ABSTRACT

The generation of pigs devoid of Galalpha1,3Galbeta1,4GlcNAc (Gal) residues has stimulated interest in non-Gal Ags as potentially important targets for Ab binding leading to rejection of pig organ xenografts in humans. Although N-glycolylneuraminic acid (NeuGc) epitopes, which are widely expressed on the endothelial cells of all mammals except humans, are likely targets of anti-non-Gal Abs, this aspect has not been investigated intensively owing to the absence of an appropriate animal model. In this study, we used CMAH(-/-) mice, which are completely deficient in NeuGc and thus produce anti-NeuGc Abs. Sera obtained from CMAH(-/-) mice and healthy human volunteers having anti-NeuGc Abs initiated complement-mediated lysis against CMAH(+/+) cells in vitro. The cytotoxic activity of anti-NeuGc Abs was also determined in vivo (i.e., NeuGc-expressing CMAH(+/+) mouse splenocytes that had been i.v. injected were completely eliminated in syngeneic CMAH(-/-) mice). CMAH(-/-) mice rejected the islets transplanted from syngeneic CMAH(+/+) mice. Thus, the anti-NeuGc Ab-mediated response may be crucially involved in xenograft loss. This is the first direct demonstration of the immunogenic property of NeuGc determinants as targets of the corresponding Abs in CMAH(+/+)-to-CMAH(-/-) transplantation setting.


Subject(s)
Antibodies/toxicity , Cytotoxicity Tests, Immunologic , Epitopes/immunology , Mixed Function Oxygenases/deficiency , Mixed Function Oxygenases/genetics , Neuraminic Acids/immunology , Animals , Binding Sites, Antibody , Diabetes Mellitus, Experimental/immunology , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Epitopes/biosynthesis , Female , Galactosyltransferases/deficiency , Galactosyltransferases/genetics , Graft Rejection/immunology , Heart Transplantation/immunology , Humans , Immunoglobulin G/toxicity , Immunoglobulin M/toxicity , Islets of Langerhans Transplantation/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Neuraminic Acids/metabolism , Rats , Rats, Inbred F344 , Swine , Transplantation, Heterologous
12.
Clin Chem ; 56(4): 550-8, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20093560

ABSTRACT

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Higher LOX index values were associated with an increased risk of CHD. Low LOX index values may be protective against ischemic stroke.


Subject(s)
Biomarkers/blood , Coronary Disease/blood , Scavenger Receptors, Class E/blood , Stroke/blood , Adult , Aged , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Cohort Studies , Female , Humans , Male , Middle Aged , Recombinant Proteins/blood , Recombinant Proteins/immunology , Risk Factors , Scavenger Receptors, Class E/immunology
13.
J Vet Med Sci ; 72(3): 257-62, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20035117

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.


Subject(s)
Antigens/immunology , Chickens/immunology , Hemocyanins/immunology , Immunosuppression Therapy/methods , Animals , Antigen-Antibody Reactions , Female , Immunity, Humoral , Immunoglobulins/immunology , Immunosuppression Therapy/veterinary , Oviposition , Rabbits/immunology , Serum Albumin/immunology , Yolk Sac/immunology
14.
Expert Opin Ther Pat ; 19(7): 907-17, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19514955

ABSTRACT

BACKGROUND: There has been a dramatic decrease in the risk of transmission of bovine spongiform encephalopathy to humans. In contrast, the risk of human-to-human transmission of variant Creutzfeldt-Jakob disease (vCJD) via medical treatments became potentially high since 4 vCJD cases were reported to be possibly transmitted through blood transfusion in the UK. However, no treatments are yet available for curing prion diseases. OBJECTIVE: Conversion of the normal prion protein, PrP(C), to the amyloidogenic PrP, PrP(Sc), plays a pivotal role in the pathogenesis. Recently, certain anti-PrP or anti-37/67-kDa laminin receptor (LRP/LR) antibodies were shown to have the potential to cure chronically infected cells, clearing PrP(Sc) from the cells. This has raised the possibility of antibody based-immunotherapy for prion diseases. This article aims to introduce and discuss the recently published attempts of immunotherapy in prion diseases. METHODS: Bibliographic research was carried out using the PubMed database. Patent literature was searched using the UK Intellectual Property Office website. RESULTS/CONCLUSION: No satisfying consequences in animals could be detected with anti-PrP antibodies directly infused into the brains of animals by the intraventricular route or by anti-PrP or anti-LRP/LR single chain fragment antibodies directly delivered into the brain by virus vector-mediated gene transfer. This is probably because such delivery systems failed to deliver the antibodies to the neurons relevant for the treatments.


Subject(s)
Antibodies/therapeutic use , Immunotherapy/methods , Prion Diseases/therapy , Animals , Antibodies/administration & dosage , Antibodies/immunology , Cattle , Disease Models, Animal , Drug Delivery Systems , Gene Transfer Techniques , Humans , Patents as Topic , Prion Diseases/immunology , Prion Diseases/transmission , Prions/immunology
15.
J Vet Med Sci ; 71(4): 417-24, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19420843

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.


Subject(s)
Antibodies/immunology , Chickens/immunology , Immunity, Maternally-Acquired/immunology , Immunization/veterinary , Animals , Antibodies/blood , Antibody Formation/immunology , Enzyme-Linked Immunosorbent Assay/veterinary , Epitopes , Female , Hemocyanins/administration & dosage , Hemocyanins/immunology , Immunization/standards , Immunoglobulins/immunology
16.
J Biol Chem ; 284(22): 14769-76, 2009 May 29.
Article in English | MEDLINE | ID: mdl-19346516

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.


Subject(s)
Biopolymers/metabolism , Integrins/metabolism , Neutrophils/cytology , Osteopontin/metabolism , Amino Acid Sequence , Animals , Antibody Specificity , Binding Sites , Cell Adhesion , Cell Line, Tumor , Cell Movement , Humans , Molecular Sequence Data , Neutrophils/metabolism , Osteopontin/chemistry , Protein Binding , Recombinant Proteins/metabolism , Transfection
17.
MAbs ; 1(4): 357-63, 2009.
Article in English | MEDLINE | ID: mdl-20073134

ABSTRACT

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is the major receptor for oxidized LDL (oxLDL), and plays a key role in the pathogenesis of atherosclerosis and cardiovascular diseases. Monoclonal antibodies (mAbs) specific for human LOX-1 (hLOX-1) were generated by a phage display technique using chickens immunized with recombinant hLOX-1 (rhLOX-1). A total of 53 independent scFv clones reactive for rhLOX-1 were obtained. Of the 53 clones, 49 recognized the C-type lectin-like domain (CTL domain), which contributes to the binding of oxLDL. Of these, 45 clones inhibited oxLDL-binding with LOX-1. Furthermore, some of these clones cross-reacted with rabbit, pig and/or mouse LOX-1. For possible application as therapeutic agents in the future, two cross-reactive mAbs were re-constructed as chicken-human chimeric antibodies. The chimeric antibodies showed similar characteristics compared to the original antibodies, and inhibited oxLDL binding to LOX-1 expressed on CHO cells. The results obtained in this study indicate that anti-LOX-1 mAbs might be useful tools for functional analyses and development of therapeutic agents for cardiovascular indications such as atherosclerosis.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Scavenger Receptors, Class E/immunology , Animals , Antibodies, Monoclonal/immunology , Chickens , Electrophoresis, Polyacrylamide Gel , Humans , Mice , Rabbits , Recombinant Proteins/genetics , Scavenger Receptors, Class E/antagonists & inhibitors , Scavenger Receptors, Class E/genetics , Swine
18.
Clin Chem ; 55(2): 285-94, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19074514

ABSTRACT

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.


Subject(s)
C-Reactive Protein/metabolism , Endothelial Cells , Endothelium, Vascular , Hypertension/metabolism , Scavenger Receptors, Class E/metabolism , Animals , C-Reactive Protein/pharmacology , CHO Cells , COS Cells , Cattle , Chlorocebus aethiops , Cricetinae , Cricetulus , Endothelial Cells/enzymology , Endothelial Cells/metabolism , Endothelium, Vascular/enzymology , Endothelium, Vascular/metabolism , Enzyme-Linked Immunosorbent Assay , Gene Expression , Immunohistochemistry , Male , Microscopy, Confocal , Oxidation-Reduction , Permeability , Protein Binding , RNA, Small Interfering/pharmacology , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Scavenger Receptors, Class E/biosynthesis , Scavenger Receptors, Class E/genetics , Surface Plasmon Resonance
19.
Article in Japanese | MEDLINE | ID: mdl-20306703

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.


Subject(s)
Peptide Library , Prions/immunology , Single-Chain Antibodies/immunology , Amino Acid Sequence , Animals , Chickens , DNA, Complementary , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoblotting , Mice , Molecular Sequence Data , Mutation , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics
20.
Circulation ; 118(1): 75-83, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18559699

ABSTRACT

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: OxLDL has a major atherogenic impact, and oxLDL removal is a promising therapeutic strategy against atherosclerosis.


Subject(s)
Apolipoproteins E/deficiency , Atherosclerosis/prevention & control , Lipoproteins, LDL/metabolism , Liver/metabolism , Receptors, Oxidized LDL/metabolism , Scavenger Receptors, Class E/metabolism , Adenoviridae/genetics , Adiponectin/metabolism , Animals , Atherosclerosis/blood , Atherosclerosis/genetics , Disease Models, Animal , Genetic Therapy/methods , Lipid Peroxides/metabolism , Lipoproteins, LDL/blood , Liver/virology , Mice , Mice, Knockout , Oxidative Stress/drug effects , Receptors, Oxidized LDL/genetics , Reverse Transcriptase Polymerase Chain Reaction , Scavenger Receptors, Class E/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...