RESUMO
At present, the most reliable method for creating genetically modified chickens is the modification of the DNA sequence of primordial germ cells (PGCs). However, during embryogenesis, only a small number of chicken PGCs can be obtained. Therefore, in vitro PGC culturing is necessary to obtain sufficient cells for further genetic engineering. Previously reported PGC culturing methods lack versatility. We report here a new protocol for stable and efficient culturing of chicken PGCs using small-molecule inhibitors. The growth rate of PGCs was investigated following the addition of three small-molecule inhibitors, including blebbistatin, into the culture medium. Chicken PGC survival and proliferation rates increased after the addition of small-molecule inhibitors, compared with the untreated control. Blebbistatin was shown to be the most effective inducer of PGC growth. Long-term culturing of PGCs with blebbistatin maintained the morphology of typical PGCs, and these cells expressed marker proteins such as chicken vasa homolog (CVH) and NANOG. Additionally, PGCs transfected with a fluorescent protein gene were shown to migrate into the gonads of the recipient embryo, and progeny derived from PGCs cultured by this method were efficiently obtained. These results demonstrate that small-molecule inhibitors represent a useful tool for stable and efficient chicken PGC culturing.
RESUMO
BACKGROUND: Birds harbor specific sex determination and differentiation mechanisms. Although the molecular mechanisms associated with sex determination in somatic cells have been elucidated, those for germ cells remain unclear. RESULTS: Here, we characterized the chicken forkhead box L3 (foxl3)-like gene as a sex-determination factor in sexually indifferent medaka germline stem cells. The foxl3-like gene was cloned by rapid amplification of cDNA ends, and the nucleotide sequence was analyzed. The deduced amino acid sequence was compared with FOXL3 sequences from other species, revealing low identity and similarity scores. Expression analysis of foxl3-like mRNA during gonadogenesis showed female left-gonad-specific temporal expression in an egg incubated from 10 to 16 days, as well as low general expression in certain hatched female chicken organs. Moreover, the amino acid sequence deduced for the FOXL3-like protein displayed low identity with medaka FOXL3, with the FOXL3-like protein specifically localized in the oogonia, whereas medaka FOXL3 was found in sexually indifferent germline stem cells. Furthermore, the timing of expression differed between the foxl3-like gene and that of medaka foxl3. CONCLUSIONS: These results suggest that chicken FOXL3-like protein and medaka FOXL3 differ in terms of their functions as female sex-determination factors.
Assuntos
Fatores de Transcrição Forkhead/genética , Células Germinativas/fisiologia , Processos de Determinação Sexual , Sequência de Aminoácidos , Animais , Sequência de Bases , Aves , Galinhas/genética , Clonagem Molecular , Feminino , Peixes , Oryzias/genéticaRESUMO
Vaccination is the most effective method for controlling the infectious diseases that threaten the poultry industry worldwide. The use of adjuvants or immunostimulants is often necessary to improve vaccine efficacy, particularly for vaccines based on recombinant protein or inactivated pathogens. The adjuvant effects of zymosan A on antigen-specific antibody production were investigated in chickens. First, the optimal adjuvant dose of zymosan A was determined. Chicks were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) at a dosage of 2 mg/kg body weight (BW) with or without zymosan A (at a dosage of 0.5 mg/kg BW) co-administration at 4, 5 and 6 weeks of age. Different routes of immunization (oral, intranasal (i.n.), intraocular (i.o.), subcutaneous (s.c.), intramuscular (i.m.) and intraperitoneal (i.p.) were tested. Anti-DNP IgY and IgA concentrations in serum samples from all chicks were measured by an enzyme-linked immunosorbent assay. The results revealed that co-administration of zymosan A with DNP-KLH significantly increased anti-DNP IgY concentrations in chicks immunized by the oral and s.c. routes of administration when compared with control groups. In addition, co-administration of zymosan A with DNP-KLH significantly increased anti-DNP IgA concentrations in chicks immunized by the oral, i.o. and s.c. routes compared with control groups. In conclusion, zymosan A is a useful immune-potentiator adjuvant in chickens, and its co-administration with vaccine antigens enhances humoral immune responses.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Galinhas/imunologia , Hemocianinas/imunologia , Imunização/veterinária , Zimosan/administração & dosagem , Zimosan/farmacologia , Animais , Anticorpos/sangue , Formação de Anticorpos/efeitos dos fármacos , Vias de Administração de Medicamentos , Ensaio de Imunoadsorção Enzimática , Imunidade Humoral , Imunização/métodosRESUMO
Plecoglossus altivelis (ayu) is one of the most important fish species in the Japanese islands and in internal fish hatcheries. Living in open aquatic environments exposes fish to many pathogens. Therefore, they require rapid and strong immune defenses. We investigated in vivo the direct association between the ayu innate immune response, represented by the relative transcription of genes encoding the cathelicidin and hepcidin antimicrobial peptides, and lipopolysaccharide (LPS), a conventional pathogen-associated molecular patterns (PAMPs) of Gram-negative bacteria. Different concentrations of LPS (1, 10 and 100 µg/fish) were injected intraperitoneally into young (sexually immature) and adult (fully sexually mature) ayu. The relative expression of the antimicrobial peptide genes was measured 6 hr, 24 hr and 1 week after stimulation with LPS. We found a direct association between the expression of the antimicrobial peptide genes investigated and LPS stimulation. This relationship was time-, dose- and age-dependent. Further research is required to determine the cell-specific transcriptional regulation and posttranscriptional regulation of these antimicrobial peptides.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Lipopolissacarídeos/farmacologia , Osmeriformes/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Expressão Gênica/efeitos dos fármacos , Hepcidinas/metabolismo , Imunidade Inata/efeitos dos fármacos , Osmeriformes/fisiologia , CatelicidinasRESUMO
Specific immune suppression in newly hatched chicks induced by specific maternal antibodies has been reported. Laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Purified maternal anti-DNP and non-specific immunoglobulin (Ig) Y antibodies were transferred by yolk sac inoculation to newly hatched chicks, and then, they were immunized with an optimum immunogenic dose of DNP-KLH at 1 and 4 weeks of age. Concentrations of anti-DNP antibodies in serum samples of these chicks were measured by using Enzyme-linked immunosorbent assay (ELISA). Proportions of T-cell subsets in peripheral blood of these chicks were also measured by flow cytometric analysis at 5 weeks of age (one week after the second immunization). Suppression of anti-DNP antibody response and down-regulation of CD3(+)CD4(+) cells were observed in the chicks received high dose of maternal anti-DNP antibodies and immunized with DNP-KLH. On the other hand, normal anti-DNP antibody response and normal proportion of CD3(+)CD4(+) cells were observed in the chicks received high dose of non-specific IgY antibodies and immunized with DNP-KLH. Furthermore, when chicks received high dose of maternal anti-DNP antibodies and immunized with DNP-KLH at 1 and 4 weeks of age and then with rabbit serum albumin (RSA) at 5 and 8 weeks of age, their primary anti-RSA response was also significantly suppressed. We indicate here that specific maternal antibodies can affect both B and T cell responses and induce non-specific suppression against different antigens. However, this non-specific suppression does not continue for a long time.
Assuntos
Anticorpos/imunologia , Hemocianinas/imunologia , Imunidade Materno-Adquirida , Subpopulações de Linfócitos T/fisiologia , Adjuvantes Imunológicos , Animais , Reações Antígeno-Anticorpo , Galinhas , Terapia de Imunossupressão/métodos , Terapia de Imunossupressão/veterinária , Coelhos , Albumina Sérica/imunologiaRESUMO
Peripheral blood cells from ayu, Plecoglossus altivelis altivelis, were separated using a density gradient. Blood cells were then smeared using Shandon Cytospin and subjected to cytochemical staining. Blood cells were categorized based on morphological and cytochemical characteristics, and the density fractionation range and nucleus area/cell area ratio were observed. Lymphocytes are distinguished from neutrophils by their basophilic cytoplasm and Golgi-like field. The features of chromatin in thrombocytes are different from those of lymphocytes or neutrophils, but some small neutrophils have similar chromatin. Therefore, it is necessary to perform peroxidase staining to distinguish small neutrophils from thrombocytes. Basophils have large basophilic granules in cytoplasm. Based on density fractionation of blood cells, thrombocytes in the low-density area were separated from other blood cells. Identification of peripheral blood cells from ayu was possible with these staining methods. Monocytes/macrophages from spleen are specifically positive for esterase staining by α-naphthyl butyrate. As a result, thrombocytes, lymphocytes, neutrophils, basophils and monocytes/macrophages were identified in smears from peripheral blood or spleen tissue. In this paper, we confirmed that the peripheral blood corpuscles of ayu are able to be identified using the present staining methods.
Assuntos
Células Sanguíneas/química , Células Sanguíneas/citologia , Osmeriformes/sangue , Animais , Aquicultura/métodos , Células Sanguíneas/classificação , Fracionamento Celular/veterinária , Cromatina/química , Técnicas Citológicas/veterinária , Processamento de Imagem Assistida por Computador , Japão , Coloração e Rotulagem/veterináriaRESUMO
Bacterial cold water disease (BCWD) in ayu (Plecoglossus altivelis altivelis) has a serious impact on aquaculture and fisheries. There is known to be a significant difference among ayu strains with regard to mortality caused by BCWD. In this study, the immune response of different ayu strains against Flavobacterium psychrophilum infection was observed. One strain was resistant to infection by F. psychrophilum, and the other was susceptible to infection by the same bacteria. The number of bacteria in the body was observed in each ayu strain, and the change in bacterial counts was similar. However, there was a significant difference in bacterial count in the spleen between the two strains on days 6, 9, 12 and 15 after exposure. To observe the immune response against F. psychrophilum, agglutination assay using serum was performed. An agglutination reaction in the resistant ayu strain was observed in 4 out of 6 ayu on day 6 after exposure, while no reactions in the susceptible ayu strain were observed in any sampled fish until day 12. However, some reactions in the susceptible ayu strain were observed in surviving ayu. These results indicate that there is a correlation between the presence of bacterial multiplication and agglutination reaction against F. psychrophilum.
Assuntos
Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Flavobacteriaceae/veterinária , Flavobacterium , Osmeriformes , Testes de Aglutinação/veterinária , Análise de Variância , Animais , Aquicultura , Carga Bacteriana/veterinária , Infecções por Flavobacteriaceae/imunologia , Mortalidade , Especificidade da Espécie , Baço/microbiologia , Fatores de TempoRESUMO
Interaction between interleukin (IL)-5 and its receptor (IL-5R) is important for the regulation of immunity against worm infections, allergic reactions and B cell response in mammals. In this study, we identified a full-length cDNA encoding chicken IL-5R α-chain (chIL-5Rα). The deduced amino acid sequence showed 41-43% identity to mammalian homologues. It has four well-conserved cysteines and a WSXWS motif in the extracellular region, and a PPXP motif in the cytoplasmic region. Quantitative RT-PCR analysis revealed that chIL-5Rα mRNA expression was markedly high in bone marrow and relatively high in spleen and lung. Recombinant proteins of soluble chIL-5Rα and cytokines (artificially produced chIL-5 (achIL-5) and another IL-5-like molecule KK34) were expressed by 293F cells to examine the cytokine-receptor interactions. Interaction assay using a Biacore biosensor showed that chIL-5Rα has the capability to bind with monomeric achIL-5, but not with KK34. In conclusion, chicken has an IL-5Rα homologue but KK34 does not complement the IL-5/IL-5R system.
Assuntos
Galinhas/genética , Clonagem Molecular , Subunidade alfa de Receptor de Interleucina-5/genética , Subunidade alfa de Receptor de Interleucina-5/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Galinhas/metabolismo , Feminino , Humanos , Interleucina-5/genética , Interleucina-5/metabolismo , Subunidade alfa de Receptor de Interleucina-5/química , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
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/imunologiaRESUMO
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/imunologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais/biossíntese , Receptores Depuradores Classe E/imunologia , Animais , Anticorpos Monoclonais/imunologia , Galinhas , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Coelhos , Proteínas Recombinantes/genética , Receptores Depuradores Classe E/antagonistas & inibidores , Receptores Depuradores Classe E/genética , SuínosRESUMO
Indomethacin (IND) suppresses the T-dependent antibody response (TDAR) in juvenile males when it is administered to pregnant rats during late gestation. In this study, the effect of IND on cytokine production in juvenile rats was examined to investigate the mechanism behind the suppression of antibody production. IND was orally administered to pregnant SD rats on days 18-21 of gestation. After parturition, the spleen cells isolated from 3-week-old pups were incubated with concanavalin A (Con A) or lipopolysaccharide (LPS). The level of cytokines in the culture supernatant was measured. IL-10 decreased significantly in the males, and IL-6 and TNF-alpha tended to decrease in both sexes. In order to examine the effect of IND on cytokine production in juvenile rats in vitro, spleen cells isolated from untreated 3-week-old rats were exposed to IND and a mitogen (Con A or LPS) simultaneously, and then the levels of cytokines were measured. IL-4 decreased in the males, and IL-6 tended to decrease in both sexes. These results indicated that treating dams with IND during late gestation causes a change in the release of Th2 cytokine, and suggested that this change involves the suppression of antibody production.
Assuntos
Citocinas/biossíntese , Feto/efeitos dos fármacos , Indometacina/toxicidade , Células Th2/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Feminino , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Masculino , Gravidez , Ratos , Ratos Sprague-Dawley , Células Th2/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
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/genéticaRESUMO
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).
Assuntos
Príons/imunologia , Animais , Especificidade de Anticorpos , Western Blotting , Bovinos , Galinhas , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Camundongos , Proteínas PrPC/imunologia , Proteínas PrPSc/imunologia , Doenças Priônicas/diagnóstico , Doenças Priônicas/imunologia , Scrapie/diagnóstico , Scrapie/imunologiaRESUMO
In order to investigate the effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the development of rat immunity, indomethacin (IND; 0.25, 0.5, or 1.0 mg/kg/day), acetyl salicylic acid (ASA; 90, 180, or 360 mg/kg/day), or diclofenac sodium salt (DSS; 0.5, 1.0, or 2.0 mg/kg/day) suspended in 0.5% methylcellulose aqueous solution, was orally administered once daily to five pregnant Sprague-Dawley (IGS) rats per group on days 18-21 of gestation. After parturition, the serum IgM and IgG levels, the spleen weight, and the number of spleen cells were measured in 3- and 8-week-old pups. Afterwards, immunophenotyping analysis of splenocytes or peripheral blood lymphocytes and T-dependent antibody response were performed. The number of spleen cells in 3-week-olds increased when 1.0 mg/kg of IND and 180 mg/kg of ASA were administered. Immunophenotyping analysis using flow cytometry (FCM) indicated that the proportion and number of CD45RA(+) cells increased, and the proportion of CD3(-) NKR-P1A(+) cells decreased in males when dosed with IND at 1.0 mg/kg or ASA at 180 mg/kg. The serum anti-KLH IgG antibody titer decreased in the males of the IND 1.0 mg/kg dosing group, the serum levels of anti-KLH IgM, total IgM, and IgG were not changed at all. These changes disappeared in 8-week-old pups. There were no effects on any of the parameters in the 3- and 8-week-olds of the DSS treatment group. These results suggest that IND or ASA administration to dams during late gestation either causes a change in the lymphocyte subsets, or that they suppress the T-dependent antibody response in juvenile males. Both of these changes eventually recover to intact levels later on during development. These results will contribute to the development of a technique for the assessment of developmental immunotoxicity and generate data on the effect of prenatal administration of NSAIDs on the developmental immune system in pups.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Efeitos Tardios da Exposição Pré-Natal , Baço/imunologia , Animais , Anti-Inflamatórios não Esteroides/imunologia , Anti-Inflamatórios não Esteroides/toxicidade , Formação de Anticorpos/efeitos dos fármacos , Aspirina/imunologia , Aspirina/farmacologia , Aspirina/toxicidade , Diclofenaco/imunologia , Diclofenaco/farmacologia , Diclofenaco/toxicidade , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Hemocianinas/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Imunofenotipagem , Indometacina/imunologia , Indometacina/farmacologia , Indometacina/toxicidade , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Exposição Materna , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Organismos Livres de Patógenos Específicos , Baço/anatomia & histologia , Baço/efeitos dos fármacosRESUMO
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/imunologiaRESUMO
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.
Assuntos
Galinhas/imunologia , Encefalopatia Espongiforme Bovina/diagnóstico , Região Variável de Imunoglobulina/imunologia , Imunoglobulinas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Animais , Especificidade de Anticorpos , Bovinos , Encefalopatia Espongiforme Bovina/imunologia , Ensaio de Imunoadsorção EnzimáticaRESUMO
In this chapter, we describe the methods we have used to show that chicken leukemia inhibitory factor (LIF) maintains chicken embryonic stem (ES) cells in an undifferentiated state in culture. Recombinant chicken LIF (rchLIF) was expressed as a fusion protein linked to glutathione S-transferase (GST) and purified to greater than 90% purity in two chromatography stages, the first an affinity step using the GST tail, which was cleaved before further purification by gel chromatography. Chicken ES cells were obtained by culturing chicken blastodermal cells isolated from stage X embryos of freshly laid chicken eggs. These cells can be maintained in media containing rchLIF for at least 9 d without any other cytokines or feeder cells. Chicken ES cells were characterized by the expression of alkaline phosphatase activity, stage-specific embryonic antigen (SSEA)-1 and embryonal carcinoma cell monoclonal antibody-1. In addition, the phosphorylation of signal transducers and activators of transcription-3 by LIF, which is sufficient to maintain the undifferentiated state of ES cells, was detected by Western blotting analysis.
Assuntos
Embrião de Galinha/citologia , Células-Tronco Pluripotentes/citologia , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Embrião de Galinha/metabolismo , Galinhas/genética , Clonagem Molecular , DNA Complementar/genética , Interleucina-6/genética , Interleucina-6/farmacologia , Fator Inibidor de Leucemia , Antígenos CD15/metabolismo , Camundongos , Dados de Sequência Molecular , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Transformação GenéticaRESUMO
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 SinaisRESUMO
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.