RESUMO
Development of agonistic monoclonal antibodies (mAbs) against the pro-apoptotic molecule death receptor 4 (DR4) [or tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor 1] is an attractive anti-cancer strategy because of their potential for inducing tumor-specific cell death. In this study, we humanized an agonistic anti-DR4 AY4 scFv raised in mice (mAY4) by grafting the complementarity-determining regions (CDRs) onto a fixed human framework, while preserving the so-called Vernier zone residues, a group of framework (FR) residues directly underneath the CDRs, with the murine residues in the humanized antibody, hAY4. The humanized hAY4 scFv maintained the antigen binding affinity and epitope specificity of mAY4. To investigate how the valence of hAY4 scFv affects DR4-mediated cell death, bivalent and trivalent forms of hAY4 scFv were generated by linking a hinge region to the coiled-coil domain of a dimerizing leucine zipper and trimerizing isoleucine zipper, respectively. Compared to the monovalent and bivalent forms, the trivalent hAY4 scFv induced more potent caspase-dependent apoptotic cell death as evidenced by increased activation of caspase-8 and downstream pro-apoptotic molecules. Our results suggest that like other TNF family receptors, avidity-mediated oligomerization of DR4 augments the receptor-mediated apoptotic cell death by promoting intracellular cell death signaling.
Assuntos
Anticorpos/imunologia , Afinidade de Anticorpos/imunologia , Citotoxicidade Imunológica , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/química , Células HCT116 , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Fragmentos de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Cinética , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de SequênciaRESUMO
The chicken interleukin-17D was cloned from a testis cDNA library prepared from the Korean native chicken. The full-length chicken IL-17D (chIL-17D) cDNA consisted of a 348 nucleotide sequence encoding an open reading frame of 116 amino acids with a predicted molecular mass of 13.3kDa. Comparison of the deduced amino acid sequence of chIL-17D with homologous proteins from human, mouse and opossum revealed 64%, 53% and 76% identity, respectively, including six conserved cysteine residues present in the mammalian polypeptides. The chIL-17D gene transcript was expressed in a wide range of tissues, and highest levels were in pancreas, thymus and lung. Following Eimeria maxima infection, levels of the chIL-17D mRNA were up-regulated in the intestinal jejunum, bursa, lung, and spleen but decreased in the thymus. Infected chickens also expressed greater levels of chIL-17D mRNA in CD4(+), CD8(+) and TCR1(+) intestinal intraepithelial lymphocytes while decreased expression was seen in TCR2(+) cells. Treatment of CHCC-OU2 fibroblasts with chIL-17D recombinant protein induced the expression of IL-6 and IL-8. Collectively, these results suggest that chL-17D has structural and functional similarities to mammalian IL-17Ds and that it plays an important role in local gut innate immune responses during experimental coccidiosis.
Assuntos
Galinhas/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas/imunologia , Clonagem Molecular , Coccidiose/imunologia , Coccidiose/veterinária , DNA Complementar/genética , DNA de Protozoário/genética , Eimeria , Dados de Sequência Molecular , Doenças das Aves Domésticas/imunologia , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismoRESUMO
Intestinal intraepithelial lymphocytes (IELs) are the primary immune effector cells in the gut and play a critical role in eliciting protective immunity to enteric pathogens such as Eimeria, the etiologic agent of avian coccidiosis. In this study, a microarray of genes expressed by intestinal IELs from Eimeria-infected chickens was constructed using the expressed sequence tag (EST) strategy. The avian intestinal IEL cDNA microarray (AVIELA) contained duplicates of 9,668 individual ESTs (6,654 known genes and 3,014 unique singletons of unknown identity) and was used to analyze gene expression profiles during primary and secondary Eimeria maxima infections. Following primary inoculation with E. maxima, the expression levels of 74 genes were significantly altered more than two-fold over the 3-day infection period (51 up-regulated, 23 down-regulated). Following secondary infection, the expression levels of 308 genes were significantly altered (62 up-regulated, 246 down-regulated). Pathway gene analysis indicated that many of the modulated genes were related to apoptosis, JAK/STAT, MAPK, interleukin, and TLR signaling pathways, and involving innate and adaptive immune responses. This chicken IEL microarray will provide a valuable resource for future transcriptional profiling of the genes involved in protective immunity to chicken enteric pathogens.
Assuntos
Galinhas , Coccidiose/veterinária , Eimeria/imunologia , Enteropatias Parasitárias/veterinária , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Doenças das Aves Domésticas/parasitologia , Animais , Coccidiose/imunologia , Coccidiose/parasitologia , Perfilação da Expressão Gênica , Imunidade Celular/imunologia , Enteropatias Parasitárias/imunologia , Enteropatias Parasitárias/parasitologia , Linfócitos/imunologia , Linfócitos/parasitologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Doenças das Aves Domésticas/imunologia , Organismos Livres de Patógenos EspecíficosRESUMO
The incidence of necrotic enteritis (NE) due to Clostridium perfringens (CP) infection in commercial poultry has been increasing at an alarming rate. Although pre-exposure of chickens to coccidia infections is believed to be one of the major risk factors leading to NE, the underlying mechanisms of CP virulence remain undefined. The objectives of this study were to utilize an experimental model of NE produced by Eimeria maxima (EM) and CP coinfection to investigate the pathologic and immunologic parameters of the disease. Broilers coinfected with EM plus CP exhibited more severe gut pathology compared with animals given EM or CP alone. Additionally, EM/CP coinfection increased the numbers of intestinal CP bacteria compared with chickens exposed to an identical challenge of CP alone. Coinfection with EM and CP repressed nitric oxide synthase gene expression that was induced by EM alone, leading to lower plasma NO levels. Intestinal expression of a panel of cytokine and chemokine genes following EM/CP coinfection showed a mixed response depending on the transcript analyzed and the time following infection. In general, IFN-alpha, IFN-gamma, IL-1beta, IL-2, IL-12, IL-13, IL-17, and TGF-beta4 were repressed, whereas IL-8, IL-10, IL-15, and LITAF were increased during coinfection compared with challenge by EM or CP alone. These results are discussed in the context of EM and CP to act synergistically to create a more severe disease phenotype leading to an altered cytokine/chemokine response than that produced by infection with the individual pathogens.
Assuntos
Galinhas , Infecções por Clostridium/veterinária , Clostridium perfringens , Coccidiose/veterinária , Eimeria , Doenças das Aves Domésticas , Animais , Galinhas/microbiologia , Galinhas/parasitologia , Infecções por Clostridium/complicações , Infecções por Clostridium/imunologia , Infecções por Clostridium/patologia , Coccidiose/complicações , Coccidiose/imunologia , Coccidiose/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Enterocolite Necrosante/microbiologia , Enterocolite Necrosante/parasitologia , Enterocolite Necrosante/veterinária , Feminino , Regulação da Expressão Gênica , Intestinos/microbiologia , Intestinos/patologia , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/parasitologia , Doenças das Aves Domésticas/patologiaRESUMO
The current study was conducted to evaluate the effect of dietary supplementation with a lyophilized powder made from plums (P) on host protective immune responses against avian coccidiosis, the most economically important parasitic disease of poultry. One-day-old White Leghorn chickens were fed from the time of hatch with a standard diet either without P (control and P 0 groups) or supplemented with P at 0.5% (P 0.5) or 1.0% (P 1.0) of the diet. Animals in the P 0, P 0.5, and P 1.0 groups were orally challenged with 5000 sporulated oocysts of Eimeria acervulina at day 12 post-hatch, while control animals were uninfected. Dietary supplementation of P increased body weight gain, reduced fecal oocyst shedding, and increased the levels of mRNAs for interferon-gamma and interleukin-15 in the P 1.0 group at 10 days post-infection compared with the P 0 group. Furthermore, chickens fed either the P 0.5 or P 1.0 diets exhibited significantly greater spleen cell proliferation compared with the non-plum P 0 group. These results indicate that plum possesses immune enhancing properties, and that feeding chickens a plum-supplemented diet augments protective immunity against coccidiosis.
Assuntos
Galinhas/imunologia , Coccidiose/veterinária , Dieta/veterinária , Eimeria/imunologia , Doenças das Aves Domésticas/prevenção & controle , Prunus/imunologia , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Coccidiose/imunologia , Coccidiose/prevenção & controle , Fezes/parasitologia , Feminino , Regulação da Expressão Gênica/fisiologia , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-15/genética , Interleucina-15/metabolismo , Linfócitos/imunologia , Masculino , Oocistos , Doenças das Aves Domésticas/imunologia , Organismos Livres de Patógenos Específicos , Baço/citologia , Aumento de PesoRESUMO
Coccidiosis is recognized as the major parasitic disease of poultry and is caused by the apicomplexan protozoa Eimeria. Increasing evidence shows the complexity of the host immune response to Eimeria and microarray technology presents a powerful tool for the study of such an intricate biological process. Using an avian macrophage microarray containing 4906 unique gene elements, we identified important host genes whose expression changed following infection of macrophages with sporozoites of Eimeria tenella (ET), Eimeria acervulina (EA), and Eimeria maxima (EM). This approach enabled us to identify a common core of 25 genetic elements whose transcriptional expression is induced or repressed by exposure to Eimeria sporozoites and to identify additional transcription patterns unique to each individual Eimeria species. Besides inducing the expression of IL-1beta, IL-6, and IL-18 and repressing the expression of IL-16, Eimeria treated macrophages were commonly found to induce the expression of the CCL chemokine family members macrophage inflammatory protein (MIP)-1beta (CCLi1), K203 (CCLi3), and ah221 (CCLi7). However, the CXCL chemokine K60 (CXCLi1) was found to be induced by macrophage exposure to E. tenella but was repressed upon macrophage exposure to E. maxima and E. acervulina. Fundamental analysis of avian chemokine and cytokine expression patterns offers insight into the unique avian immunological responses to these related but biologically unique pathogens.
Assuntos
Coccidiose/veterinária , Eimeria , Regulação da Expressão Gênica , Macrófagos/parasitologia , Doenças das Aves Domésticas/imunologia , Animais , Aves , Linhagem Celular , Quimiocinas/biossíntese , Quimiocinas/genética , Coccidiose/genética , Coccidiose/imunologia , Regulação da Expressão Gênica/imunologia , Interleucinas/biossíntese , Interleucinas/genética , Macrófagos/imunologia , Doenças das Aves Domésticas/genética , Especificidade da EspécieRESUMO
The inflammatory response to parasites, bacteria, and viruses is mediated by multiple host factors. TNF-alpha is one of the most pleiotropic cytokines in mammals, but has yet to be identified in avian species. In the current study, we isolated a full-length cDNA encoding the chicken homologue of LPS-induced TNF-alpha factor (LITAF), transcription factor, with an open reading frame of 148 amino acids and a predicted molecular mass of 16.0kDa. Quantitative RT-PCR analysis showed that chicken LITAF mRNA was predominantly expressed in spleen and intestinal intraepithelial lymphocytes (IELs). LITAF mRNA levels were up-regulated following in vitro stimulation of macrophages for 4 h with Escherichia coli or Salmonella typhimurium endotoxin, and 18-48 h after treatment with Eimeria acervulina, E. maxima, or E. tenella, three causative agents of avian coccidiosis. LITAF mRNA was up-regulated by more than 700-fold in IELs isolated from E. maxima-infected birds. Purified recombinant LITAF protein expressed in E. coli or COS7 cells exhibited cytotoxic activity against chicken tumor cell lines in vitro, presumably through autocrine activation of TNF-alpha or its chicken homologue. This supposition was strengthened by the fact that treatment of chicken macrophages with recombinant LITAF induced the expression of TL1A (TNFSF 15), a member of the TNF ligand super family. We conclude that chicken LITAF may play an important role in the regulation of TNF-alpha gene expression during the course of coccidiosis or tumorigenesis.
Assuntos
Proteínas Aviárias/química , Proteínas Aviárias/genética , Galinhas/genética , Clonagem Molecular , Proteínas Nucleares/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Proteínas Aviárias/isolamento & purificação , Galinhas/metabolismo , Coccidiose/metabolismo , DNA Complementar , Eimeria/fisiologia , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genéticaRESUMO
Chicken monoclonal antibody (mAb), 8C3, which is reactive with a sporozoite antigen of Eimeria acervulina, is a potential therapeutic agent against avian coccidiosis caused by Eimeria spp. However, production of large amounts of 8C3 mAb in cell culture system is labor intensive and not cost-effective. Accordingly, recombinant single chain variable fragment (ScFv) antibody was constructed by amplification of the V(H) and V(L) genes from chicken hybridoma, 8C3 and when expressed in E. coli gave 5 mg l(-1). The expressed protein showed antigen binding activity equivalent to that of the parental mAb. In addition, nucleotide sequence comparison of 8C3 gene to the germline chicken V(L) genes suggested that the gene conversion with (V)lambda pseudogenes might contribute to the diversification of V(L) genes in chickens.
Assuntos
Biotecnologia/métodos , Fragmentos de Imunoglobulinas/química , Proteínas Recombinantes/química , Animais , Anticorpos Monoclonais/química , Sequência de Bases , Galinhas , Coccidiose/prevenção & controle , Primers do DNA/química , DNA Complementar/metabolismo , Relação Dose-Resposta Imunológica , Eimeria/metabolismo , Ensaio de Imunoadsorção Enzimática , Hibridomas/metabolismo , Região Variável de Imunoglobulina/química , Imunoterapia/métodos , Modelos Genéticos , Dados de Sequência Molecular , Doenças das Aves Domésticas/prevenção & controle , Homologia de Sequência do Ácido Nucleico , Esporozoítos/metabolismoRESUMO
Korean mugwort herb is a preparation of dried leaves from Artemisia species and has been used as a traditional medicine in Asia. An oligosaccharide fraction, AVF3, purified from the preparation promoted survival of the mouse thymocytes in culture. A mouse gene array study suggests that the AVF3 may modulate Fas/FasL dependent apoptotic cell death and thus has influence on the survival of the thymocytes in culture. RT-PCR analysis confirmed the down-regulation of the Fas gene by the AVF3 treatment, supporting that the AVF3 modulated thymocyte death by suppressing the Fas gene expression.
Assuntos
Apoptose/efeitos dos fármacos , Artemisia/química , Oligossacarídeos/isolamento & purificação , Oligossacarídeos/farmacologia , Timo/efeitos dos fármacos , Timo/metabolismo , Receptor fas/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Receptor fas/genéticaRESUMO
A polysaccharide fraction, AIP1, purified from Artemisia iwayomogi was shown to have immunomodulating and anti-tumor activities in mice. In order to determine how the AIP1 fraction exhibits the immunomodulating activity, the effect of the fraction on the apoptosis of mouse spleen cells was investigated. Treatment of the mouse spleen cells with the AIP1 fraction resulted in the suppression of apoptotic death and an extension of cell survival in culture, indicating that the fraction might modulate the death of spleen cells. Treatment of the mice with the AIP1 fraction in vivo also resulted in less apoptosis of the spleen cells, which indicates the physiological relevance of the anti-apoptosis effect of the fraction in vitro. A mouse gene array was used to determine the profile of the gene expression change showing a pattern of up- and down-regulated genes by the AIP1 treatment. This study provides preliminary information regarding the immunomodulatory mechanism of the AIP1 fraction.