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1.
PLoS Pathog ; 16(8): e1008823, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845931

RESUMO

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.


Assuntos
Vírus da Influenza A/metabolismo , Pulmão , Macrófagos , Infecções por Orthomyxoviridae , Proteínas PrPC/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais Murinos/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/virologia , Camundongos , Camundongos Mutantes , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Proteínas PrPC/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismo
2.
Parasitol Res ; 115(11): 4123-4128, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27384177

RESUMO

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.


Assuntos
Antígenos de Protozoários/imunologia , Galinhas/parasitologia , Coccidiose/veterinária , Eimeria/imunologia , Fator 1 de Elongação de Peptídeos/metabolismo , Doenças das Aves Domésticas/parasitologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Western Blotting/veterinária , Coccidiose/parasitologia , Reações Cruzadas , Cryptosporidium parvum/imunologia , Cryptosporidium parvum/isolamento & purificação , Eimeria/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/veterinária , Neospora/imunologia , Neospora/isolamento & purificação , Esporozoítos , Toxoplasma/imunologia , Toxoplasma/isolamento & purificação
3.
J Biol Chem ; 288(47): 34111-34120, 2013 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-24085304

RESUMO

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.


Assuntos
Antígenos de Protozoários/imunologia , Cryptosporidium parvum/imunologia , Fator 1 de Elongação de Peptídeos/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/metabolismo , Criptosporidiose/genética , Criptosporidiose/imunologia , Criptosporidiose/metabolismo , Criptosporidiose/prevenção & controle , Cryptosporidium parvum/metabolismo , Cryptosporidium parvum/patogenicidade , Masculino , Camundongos , Camundongos SCID , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Vacinas Protozoárias/imunologia , Esporozoítos/metabolismo
4.
J Biol Chem ; 287(27): 22593-608, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22549775

RESUMO

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.


Assuntos
Glicolipídeos/metabolismo , Glicômica , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Análise Serial de Proteínas , Acetilação , Anticorpos/imunologia , Especificidade de Anticorpos , Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Glicolipídeos/imunologia , Glicômica/instrumentação , Glicômica/métodos , Glicômica/normas , Lectinas/metabolismo , Ácido N-Acetilneuramínico/imunologia , Oxirredução , Ácido Periódico/metabolismo , Lectinas de Plantas/metabolismo , Polissacarídeos/imunologia , Análise Serial de Proteínas/instrumentação , Análise Serial de Proteínas/métodos , Análise Serial de Proteínas/normas , Reprodutibilidade dos Testes , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico
5.
J Biol Chem ; 286(13): 11170-8, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21321126

RESUMO

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.


Assuntos
Quimiotaxia/fisiologia , Integrinas/metabolismo , Infiltração de Neutrófilos/fisiologia , Neutrófilos/metabolismo , Osteopontina/metabolismo , Multimerização Proteica/fisiologia , Animais , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Integrinas/genética , Camundongos , Infiltração de Neutrófilos/efeitos dos fármacos , Osteopontina/genética , Osteopontina/farmacologia , Proteína 2 Glutamina gama-Glutamiltransferase , Trombina/química , Trombina/genética , Trombina/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo
6.
Biochem Biophys Res Commun ; 418(4): 628-33, 2012 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-22290229

RESUMO

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.


Assuntos
N-Acetilgalactosaminiltransferases/imunologia , Peptídeos/imunologia , Anticorpos de Cadeia Única/biossíntese , Técnicas do Sistema de Duplo-Híbrido , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , Humanos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
7.
J Immunol ; 184(6): 3269-75, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20173026

RESUMO

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.


Assuntos
Anticorpos/toxicidade , Testes Imunológicos de Citotoxicidade , Epitopos/imunologia , Oxigenases de Função Mista/deficiência , Oxigenases de Função Mista/genética , Ácidos Neuramínicos/imunologia , Animais , Sítios de Ligação de Anticorpos , Diabetes Mellitus Experimental/imunologia , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Epitopos/biossíntese , Feminino , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Humanos , Imunoglobulina G/toxicidade , Imunoglobulina M/toxicidade , Transplante das Ilhotas Pancreáticas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácidos Neuramínicos/metabolismo , Ratos , Ratos Endogâmicos F344 , Suínos , Transplante Heterólogo
8.
Cell Mol Neurobiol ; 31(7): 999-1008, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21516351

RESUMO

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.


Assuntos
Encéfalo/citologia , Microglia/fisiologia , Microglia/transplante , Príons/imunologia , Scrapie/fisiopatologia , Anticorpos de Cadeia Única/imunologia , Animais , Linhagem Celular , Vetores Genéticos , Células HEK293 , Humanos , Camundongos , Microglia/citologia , Príons/patogenicidade , Príons/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Scrapie/terapia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/uso terapêutico , Taxa de Sobrevida
9.
Clin Chem ; 56(4): 550-8, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20093560

RESUMO

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.


Assuntos
Biomarcadores/sangue , Doença das Coronárias/sangue , Receptores Depuradores Classe E/sangue , Acidente Vascular Cerebral/sangue , Adulto , Idoso , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/sangue , Proteínas Recombinantes/imunologia , Fatores de Risco , Receptores Depuradores Classe E/imunologia
10.
J Vet Med Sci ; 72(3): 257-62, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20035117

RESUMO

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.


Assuntos
Antígenos/imunologia , Galinhas/imunologia , Hemocianinas/imunologia , Terapia de Imunossupressão/métodos , Animais , Reações Antígeno-Anticorpo , Feminino , Imunidade Humoral , Imunoglobulinas/imunologia , Terapia de Imunossupressão/veterinária , Oviposição , Coelhos/imunologia , Albumina Sérica/imunologia , Saco Vitelino/imunologia
11.
Circulation ; 118(1): 75-83, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18559699

RESUMO

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.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Lipoproteínas LDL/metabolismo , Fígado/metabolismo , Receptores de LDL Oxidado/metabolismo , Receptores Depuradores Classe E/metabolismo , Adenoviridae/genética , Adiponectina/metabolismo , Animais , Aterosclerose/sangue , Aterosclerose/genética , Modelos Animais de Doenças , Terapia Genética/métodos , Peróxidos Lipídicos/metabolismo , Lipoproteínas LDL/sangue , Fígado/virologia , Camundongos , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Receptores de LDL Oxidado/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Depuradores Classe E/genética
12.
Clin Chem ; 55(2): 285-94, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19074514

RESUMO

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.


Assuntos
Proteína C-Reativa/metabolismo , Células Endoteliais , Endotélio Vascular , Hipertensão/metabolismo , Receptores Depuradores Classe E/metabolismo , Animais , Proteína C-Reativa/farmacologia , Células CHO , Células COS , Bovinos , Chlorocebus aethiops , Cricetinae , Cricetulus , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Imuno-Histoquímica , Masculino , Microscopia Confocal , Oxirredução , Permeabilidade , Ligação Proteica , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Receptores Depuradores Classe E/biossíntese , Receptores Depuradores Classe E/genética , Ressonância de Plasmônio de Superfície
13.
J Vet Med Sci ; 71(4): 417-24, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19420843

RESUMO

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.


Assuntos
Anticorpos/imunologia , Galinhas/imunologia , Imunidade Materno-Adquirida/imunologia , Imunização/veterinária , Animais , Anticorpos/sangue , Formação de Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Epitopos , Feminino , Hemocianinas/administração & dosagem , Hemocianinas/imunologia , Imunização/normas , Imunoglobulinas/imunologia
14.
Artigo em Japonês | MEDLINE | ID: mdl-20306703

RESUMO

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.


Assuntos
Biblioteca de Peptídeos , Príons/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Animais , Galinhas , DNA Complementar , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Immunoblotting , Camundongos , Dados de Sequência Molecular , Mutação , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
15.
FEBS J ; 275(11): 2965-76, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18445040

RESUMO

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.


Assuntos
Regulação da Expressão Gênica , Glicosilfosfatidilinositóis/metabolismo , Hipóxia , Príons/química , Príons/genética , Processamento Alternativo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Citosol/metabolismo , Éxons , Glicoproteínas/química , Humanos , Íons , Dados de Sequência Molecular , Neurônios/metabolismo , Estrutura Terciária de Proteína
16.
J Vet Med Sci ; 70(4): 397-400, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18460836

RESUMO

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.


Assuntos
Anticorpos Monoclonais/imunologia , Galinhas , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Camundongos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
17.
Dev Comp Immunol ; 31(4): 394-406, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-16926048

RESUMO

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.


Assuntos
Anticorpos Monoclonais/biossíntese , Especificidade de Anticorpos , Galinhas/imunologia , Clonagem Molecular , Subunidade alfa2 de Receptor de Interleucina-13/genética , Subunidade alfa2 de Receptor de Interleucina-13/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Galinhas/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia
18.
Mol Immunol ; 43(6): 634-42, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16360012

RESUMO

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.


Assuntos
Anticorpos Monoclonais/genética , Biblioteca de Peptídeos , Animais , Afinidade de Anticorpos , Galinhas , Clonagem Molecular , Regiões Determinantes de Complementaridade/genética , Humanos , Fragmentos Fab das Imunoglobulinas , Região Variável de Imunoglobulina , Métodos , Engenharia de Proteínas
19.
Dev Comp Immunol ; 30(4): 419-29, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16153708

RESUMO

Interleukin-6 (IL-6) is a multifunctional cytokine that plays roles in regulating immune responses, acute phase reactions and hematopoiesis. IL-6 signaling is regulated by two receptors, a specific alpha chain (IL-6Ralpha) and a signal transducer, gp130. In this study, cDNA encoding the 445 amino acid propeptide of chicken IL-6Ralpha (chIL-6Ralpha) was identified. The predicted 445 amino acids showed approximately 40% sequence identity with mammalian homologues. In a domain search, chIL-6Ralpha had a signal peptide of 20 residues, an immunoglobulin-like (IG) domain of 71 residues and a fibronectin-type III (FN III) domain of 85 residues. On comparison with mammalian homologues, four conserved cysteine residues and the WSXWS motif were observed in the N- and C-terminal regions of the FN III domain, respectively. Expression analysis revealed that chIL-6Ralpha is strongly expressed in liver and the chicken hepatoma cell line LMH. These findings indicate that the identified chicken cDNA sequence encodes a chIL-6Ralpha homologue.


Assuntos
Galinhas/genética , Perfilação da Expressão Gênica , Receptores de Interleucina-6/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA Complementar/genética , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , Receptores de Interleucina-6/química , Alinhamento de Sequência
20.
Dev Comp Immunol ; 30(5): 513-22, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16150488

RESUMO

Leukemia-inhibitory factor (LIF) is indispensable for maintaining the undifferentiated state when propagating mouse embryonic stem (ES) cells. We previously cloned chicken LIF (chLIF) cDNA and demonstrated that it maintained chicken ES cell cultures in an undifferentiated state. Here, we developed two monoclonal antibodies, HUL-1 and HUL-2, against chLIF, which specifically recognized recombinant chLIF (rchLIF) produced by Escherichia coli and Chinese hamster ovary K1 cells, in enzyme-linked immunosorbent assays and Western blot analysis. In addition, HUL-2 inhibited the phosphorylation of signal transducer and activator of transcription 3 by rchLIF in chicken blastodermal cells (CBCs), but not that of mitogen-activated protein kinase kinase. Furthermore, the addition of HUL-2 to CBC cultures resulted in embryoid bodies forming earlier than in normal cultures. These results indicated that HUL-2 recognized not only rchLIF but also native chLIF, and suggested that CBCs in culture produce LIF, which functions in autocrine signaling.


Assuntos
Anticorpos Monoclonais/imunologia , Blastoderma/metabolismo , Interleucina-6/metabolismo , Animais , Blastoderma/citologia , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Cricetinae , Cricetulus , Escherichia coli/metabolismo , Interleucina-6/imunologia , Fator Inibidor de Leucemia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Fosforilação , Proteínas Recombinantes/imunologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
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