RESUMEN

Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.

Asunto(s)

Virus de la Leucosis Aviar , Leucosis Aviar , Pollos , Animales , Virus de la Leucosis Aviar/genética , Virus de la Leucosis Aviar/fisiología , Pollos/virología , Leucosis Aviar/virología , Leucosis Aviar/genética , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/genética , Resistencia a la Enfermedad/genética , Sistemas CRISPR-Cas , Edición Génica , Embrión de Pollo , Evolución Molecular , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Fibroblastos/virología , Fibroblastos/metabolismo

RESUMEN

The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva -/- chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/- siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.

Asunto(s)

Virus de la Leucosis Aviar/fisiología , Proteínas Aviares/fisiología , Pollos/virología , Receptores Virales/fisiología , Vitamina B 12/metabolismo , Animales , Virus de la Leucosis Aviar/clasificación , Proteínas Aviares/genética , Embrión de Pollo , Femenino , Mutación del Sistema de Lectura , Edición Génica , Técnicas de Inactivación de Genes , Masculino , Ácido Metilmalónico/sangre , Receptores Virales/genética

RESUMEN

Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na+/H+ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.

Asunto(s)

Virus de la Leucosis Aviar/genética , Leucosis Aviar/inmunología , Proteínas Aviares/genética , Enfermedades de las Aves de Corral/inmunología , Intercambiador 1 de Sodio-Hidrógeno/genética , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/inmunología , Animales Modificados Genéticamente/virología , Leucosis Aviar/genética , Leucosis Aviar/virología , Virus de la Leucosis Aviar/clasificación , Virus de la Leucosis Aviar/fisiología , Proteínas Aviares/inmunología , Sistemas CRISPR-Cas , Pollos , Resistencia a la Enfermedad , Femenino , Edición Génica , Masculino , Enfermedades de las Aves de Corral/genética , Enfermedades de las Aves de Corral/virología , Intercambiador 1 de Sodio-Hidrógeno/inmunología

RESUMEN

Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A.IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus.

Asunto(s)

Virus de la Leucosis Aviar/patogenicidad , Leucosis Aviar/genética , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , Animales , Leucosis Aviar/metabolismo , Leucosis Aviar/virología , Virus de la Leucosis Aviar/clasificación , Virus de la Leucosis Aviar/fisiología , Línea Celular , Pollos , Susceptibilidad a Enfermedades , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/virología , Mesocricetus , Especificidad de la Especie , Internalización del Virus

RESUMEN

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine playing critical roles in host defense and acute and chronic inflammation. It has been described in fish, amphibians, and mammals but was considered to be absent in the avian genomes. Here, we report on the identification and functional characterization of the avian ortholog. The chicken TNF-α (chTNF-α) is encoded by a highly GC-rich gene, whose product shares with its mammalian counterpart 45% homology in the extracellular part displaying the characteristic TNF homology domain. Orthologs of chTNF-α were identified in the genomes of 12 additional avian species including Palaeognathae and Neognathae, and the synteny of the closely adjacent loci with mammalian TNF-α orthologs was demonstrated in the crow (Corvus cornix) genome. In addition to chTNF-α, we obtained full sequences for homologs of TNF-α receptors 1 and 2 (TNFR1, TNFR2). chTNF-α mRNA is strongly induced by lipopolysaccharide (LPS) stimulation of monocyte derived, splenic and bone marrow macrophages, and significantly upregulated in splenic tissue in response to i.v. LPS treatment. Activation of T-lymphocytes by TCR crosslinking induces chTNF-α expression in CD4+ but not in CD8+ cells. To gain insights into its biological activity, we generated recombinant chTNF-α in eukaryotic and prokaryotic expression systems. Both, the full-length cytokine and the extracellular domain rapidly induced an NFκB-luciferase reporter in stably transfected CEC-32 reporter cells. Collectively, these data provide strong evidence for the existence of a fully functional TNF-α/TNF-α receptor system in birds thus filling a gap in our understanding of the evolution of cytokine systems.

Asunto(s)

Proteínas Aviares/genética , Linfocitos T CD4-Positivos/inmunología , Pollos/inmunología , Macrófagos/inmunología , Receptores del Factor de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/genética , Animales , Proteínas Aviares/metabolismo , Células Cultivadas , Clonación Molecular , Cuervos/inmunología , Secuencia Rica en GC/genética , Humanos , Mamíferos/inmunología , FN-kappa B/metabolismo , Paleognatos/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Alineación de Secuencia

RESUMEN

A chicken multiplex cytokine assay (Bio-Plex) to detect four different cytokines (IL-2, IL-12, IL-10, and interferon gamma) simultaneously in plasma samples was designed. Most standard curves range between 1 to 5 pg/mL and 5,000 pg/mL, except for IFNγ with the range of 50 to 25,000 pg/mL. Such a chicken multiplex assay proved to be fast and reliable, and comparable in sensitivity, accuracy, and reproducibility to conventional enzyme-linked immunosorbent assays. Comparison of the multiplex assay with the ELISA technique using the same clones of detection and capture antibodies resulted in correlation coefficients for all cytokines ranging from 0.95 to 0.99. Lower limit of detection and limit of quantification values were obtained for all tested cytokines by the Bio-Plex assay compared with ELISA. To reduce the risk of cross-reaction with other proteins, the Bio-Plex system was used, combining the principle of sandwich immunoassay with the Luminex bead-based technology. The cytokine standard recoveries for each cytokine varied between 86 and 118% in dynamic concentration ranges. A chicken multiplex cytokine assay (Bio-Plex) provided a more complete picture of differences between the Th1/Th2 cytokine profiles of the immunized via a new system of antigen delivery into chicken antigen-presenting cells and control groups. This multiplexed fluorescent-bead-based detection assay can be used as a quantitative or comparative tool for the study of the chicken ex vivo cellular immune response.

Asunto(s)

Proteínas Aviares/sangre , Pollos/sangre , Inmunoensayo/veterinaria , Interferón gamma/sangre , Interleucinas/sangre , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Inmunoensayo/métodos , Reproducibilidad de los Resultados

RESUMEN

Systems of antigen delivery into antigen-presenting cells represent an important novel strategy in chicken vaccine development. In this study, we verified the ability of Rous sarcoma virus (RSV) antigens fused with streptavidin to be targeted by specific biotinylated monoclonal antibody (anti-CD205) into dendritic cells and induce virus-specific protective immunity. The method was tested in four congenic lines of chickens that are either resistant or susceptible to the progressive growth of RSV-induced tumors. Our analyses confirmed that the biot-anti-CD205-SA-FITC complex was internalized by chicken splenocytes. In the cytokine expression profile, several significant differences were evident between RSV-challenged progressor and regressor chicken lines. A significant up-regulation of IL-2, IL-12, IL-15, and IL-18 expression was detected in immunized chickens of both regressor and progressor groups. Of these cytokines, IL-2 and IL-12 were most up-regulated 14 days post-challenge (dpc), while IL-15 and IL-18 were most up-regulated at 28 dpc. On the contrary, IL-10 expression was significantly down-regulated in all immunized groups of progressor chickens at 14 dpc. We detected significant up-regulation of IL-17 in the group of immunized progressors. LITAF down-regulation with iNOS up-regulation was especially observed in the progressor group of immunized chickens that developed large tumors. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that our system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity.

Asunto(s)

Antígenos Virales/inmunología , Pollos/virología , Citocinas/fisiología , Células Dendríticas/inmunología , Virus del Sarcoma de Rous/inmunología , Sarcoma Aviar/prevención & control , Vacunas Virales/inmunología , Animales , Animales Congénicos/inmunología , Animales Congénicos/virología , Anticuerpos Biespecíficos/inmunología , Antígenos CD/inmunología , Pollos/inmunología , Células Dendríticas/virología , Inmunidad Celular/inmunología , Lectinas Tipo C/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , Receptores de Superficie Celular/inmunología , Sarcoma Aviar/inmunología

RESUMEN

The J subgroup of avian leukosis virus (ALV-J) infects domestic chickens, jungle fowl, and turkeys. This virus enters the host cell through a receptor encoded by the tvj locus and identified as Na+/H+ exchanger 1. The resistance to avian leukosis virus subgroup J in a great majority of galliform species has been explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of Na+/H+ exchanger 1. Because there are concerns of transspecies virus transmission, we studied natural polymorphisms and susceptibility/resistance in wild galliforms and found the presence of tryptophan 38 in four species of New World quails. The embryo fibroblasts of New World quails are susceptible to infection with avian leukosis virus subgroup J, and the cloned Na+/H+ exchanger 1 confers susceptibility on the otherwise resistant host. New World quails are also susceptible to new avian leukosis virus subgroup J variants but resistant to subgroups A and B and weakly susceptible to subgroups C and D of avian sarcoma/leukosis virus due to obvious defects of the respective receptors. Our results suggest that the avian leukosis virus subgroup J could be transmitted to New World quails and establish a natural reservoir of circulating virus with a potential for further evolution. IMPORTANCE: Since its spread in broiler chickens in China and Southeast Asia in 2000, ALV-J remains a major enzootic challenge for the poultry industry. Although the virus diversifies rapidly in the poultry, its spillover and circulation in wild bird species has been prevented by the resistance of most species to ALV-J. It is, nevertheless, important to understand the evolution of the virus and its potential host range in wild birds. Because resistance to avian retroviruses is due particularly to receptor incompatibility, we studied Na+/H+ exchanger 1, the receptor for ALV-J. In New World quails, we found a receptor compatible with virus entry, and we confirmed the susceptibilities of four New World quail species in vitro We propose that a prospective molecular epidemiology study be conducted to identify species with the potential to become reservoirs for ALV-J.

Asunto(s)

Virus de la Leucosis Aviar/fisiología , Leucosis Aviar/genética , Leucosis Aviar/virología , Susceptibilidad a Enfermedades , Codorniz , Secuencia de Aminoácidos , Aminoácidos , Animales , Leucosis Aviar/metabolismo , Virus de la Leucosis Aviar/clasificación , Células Cultivadas , Resistencia a la Enfermedad/genética , Evolución Molecular , Expresión Génica , Sitios Genéticos , Especificidad del Huésped , Interacciones Huésped-Patógeno , Filogenia , Polimorfismo Genético , Dominios y Motivos de Interacción de Proteínas , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Replicación Viral

RESUMEN

J subgroup avian leukosis virus (ALV-J) infects domestic chicken, jungle fowl, and turkey and enters the host cell through a receptor encoded by tvj locus and identified as Na+/H+ exchanger 1 (NHE1). The resistance to ALV-J in a great majority of examined galliform species was explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of NHE1, and genetic polymorphisms around this site predict the susceptibility or resistance of a given species or individual. In this study, we examined the NHE1 polymorphism in domestic chicken breeds and documented quantitative differences in their susceptibility to ALV-J in vitro. In a panel of chicken breeds assembled with the aim to cover the maximum variability encountered in domestic chickens, we found a completely uniform sequence of NHE1 extracellular loop 1 (ECL1) without any source of genetic variation for the selection of ALV-J-resistant poultry. In parallel, we studied the natural polymorphisms of NHE1 in wild ducks and geese because of recent reports on ALV-J positivity in feral Asian species. In anseriform species, we demonstrate a specific and highly conserved critical ECL1 sequence without any homologue of tryptophan 38 in accordance with the resistance of duck cells to prototype ALV-J. Last, we demonstrated that the new Asian strains of ALV-J have not evolved their envelope glycoprotein to the entry the duck cells. Our results contribute substantially to the current discussion of possible heterotransmission of ALV-J and its spill-over into the wild ducks and geese.

Asunto(s)

Animales Domésticos , Animales Salvajes , Variación Genética , Intercambiadores de Sodio-Hidrógeno/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Embrión de Pollo , Pollos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Intercambiadores de Sodio-Hidrógeno/química

RESUMEN

Depletion of B cells with the anti-CD20 antibody rituximab is an established therapy for rheumatoid arthritis. However, rituximab has only moderate efficacy, most likely due to insufficient depletion of B cells in lymphoid organs and expansion of pathogenic B cells. We found that an antibody against mouse CD79b profoundly blocks B-cell proliferation induced via the B-cell receptor, CD40, CD180, and chondroitin sulfate, but not via TLR4 or TLR9. Treatment with anti-CD79b also induces death in resting and activated B cells. B-cell inhibition is mediated by cross-linkage of CD79b, but independent of Fc-receptor engagement. In the model of collagen-induced arthritis, an antibody against mouse CD20 depletes B cells very efficiently but fails to suppress the humoral immune response against collagen and the development of arthritis. In contrast, the antibody against CD79b, and a deglycosylated variant of this antibody, almost completely inhibits the increase in anti-collagen antibodies and the development of arthritis. In mice with established arthritis only the fully glycosylated antibody against CD79b is effective. Our data show that targeting B cells via CD79b is much more effective than B-cell depletion with anti-CD20 antibodies for therapy of arthritis. These findings may have important implications for treatment of B-cell-mediated autoimmune diseases.

Asunto(s)

Anticuerpos Monoclonales de Origen Murino/farmacología , Antirreumáticos/farmacología , Artritis Experimental/tratamiento farmacológico , Linfocitos B/inmunología , Antígenos CD79/antagonistas & inhibidores , Depleción Linfocítica , Animales , Antígenos CD/inmunología , Artritis Experimental/inmunología , Artritis Experimental/patología , Linfocitos B/patología , Antígenos CD40/inmunología , Antígenos CD79/inmunología , Proliferación Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Recubrimiento Inmunológico/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos DBA , Receptores de Antígenos de Linfocitos B/inmunología , Rituximab

RESUMEN

BACKGROUND: Infection with the protozoan Trypanosoma cruzi manifests in mammals as Chagas heart disease. The treatment available for chagasic cardiomyopathy is unsatisfactory. METHODS/PRINCIPAL FINDINGS: To study the disease pathology and its inhibition, we employed a syngeneic chicken model refractory to T. cruzi in which chickens hatched from T. cruzi inoculated eggs retained parasite kDNA (1.4 kb) minicircles. Southern blotting with EcoRI genomic DNA digests revealed main 18 and 20 kb bands by hybridization with a radiolabeled minicircle sequence. Breeding these chickens generated kDNA-mutated F1, F2, and F3 progeny. A targeted-primer TAIL-PCR (tpTAIL-PCR) technique was employed to detect the kDNA integrations. Histocompatible reporter heart grafts were used to detect ongoing inflammatory cardiomyopathy in kDNA-mutated chickens. Fluorochromes were used to label bone marrow CD3+, CD28+, and CD45+ precursors of the thymus-dependent CD8α+ and CD8ß+ effector cells that expressed TCRγδ, vß1 and vß2 receptors, which infiltrated the adult hearts and the reporter heart grafts. CONCLUSIONS/SIGNIFICANCE: Genome modifications in kDNA-mutated chickens can be associated with disruption of immune tolerance to compatible heart grafts and with rejection of the adult host's heart and reporter graft, as well as tissue destruction by effector lymphocytes. Autoimmune heart rejection was largely observed in chickens with kDNA mutations in retrotransposons and in coding genes with roles in cell structure, metabolism, growth, and differentiation. Moreover, killing the sick kDNA-mutated bone marrow cells with cytostatic and anti-folate drugs and transplanting healthy marrow cells inhibited heart rejection. We report here for the first time that healthy bone marrow cells inhibited heart pathology in kDNA+ chickens and thus prevented the genetically driven clinical manifestations of the disease.

Asunto(s)

Enfermedades Autoinmunes/prevención & control , Trasplante de Médula Ósea , Cardiomiopatía Chagásica/prevención & control , Enfermedad de Chagas/terapia , Animales , Apoptosis , Pollos/genética , ADN de Cinetoplasto/genética , Rechazo de Injerto , Inmunización , Mutación , Miocardio/patología , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología

RESUMEN

Glycosaminoglycans have anti-inflammatory properties and interact with a variety of soluble and membrane-bound molecules. Little is known about their effects on B cells and humoral immune responses. We show that CS but not dextran or other glycosaminoglycans induces a pronounced proliferation of B cells in vitro compared with TLR4 or TLR9 ligands. With the use of inhibitors and KO mice, we demonstrate that this proliferation is mediated by the tyrosine kinases BTK and Syk but independent of CD44. Antibodies against Ig-α or Ig-ß completely block CS-induced B cell proliferation. Injection of CS in mice for 4-5 days expands B cells in the spleen and results in a marked increase of CD138(+) cells in the spleen that is dependent on BTK but independent of CD4(+) T cells. Long-term treatment with CS for 14 days also increases CD138(+) cells in the bone marrow. When mice were immunized with APC or collagen and treated with CS for up to 14 days during primary or after secondary immune responses, antigen-specific humoral immune responses and antigen-specific CD138(+) plasma cells in the bone marrow were reduced significantly. These data show that CD138(+) cells, induced by treatment with CS, migrate into the bone marrow and may displace other antigen-specific plasma cells. Overall, CS is able to interfere markedly with primary and fully established humoral immune responses in mice.

Asunto(s)

Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sulfatos de Condroitina/farmacología , Inmunidad Humoral/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Células Plasmáticas/inmunología , Sindecano-1/inmunología , Agammaglobulinemia Tirosina Quinasa , Animales , Células de la Médula Ósea/inmunología , Movimiento Celular/inmunología , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Inmunidad Humoral/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/inmunología , Quinasa Syk , Sindecano-1/genética , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunología

RESUMEN

BACKGROUND: Although there is extensive evidence for the amoeboid invasiveness of cancer cells in vitro, much less is known about the role of amoeboid invasiveness in metastasis and the importance of Rho/ROCK/MLC signaling in this process. RESULTS: We analyzed the dependence of amoeboid invasiveness of rat and chicken sarcoma cells and the metastatic activity of chicken cells on individual elements of the Rho/ROCK/MLC pathway. In both animal models, inhibition of Rho, ROCK or MLC resulted in greatly decreased cell invasiveness in vitro, while inhibition of extracellular proteases using a broad spectrum inhibitor did not have a significant effect. The inhibition of both Rho activity and MLC phosphorylation by dominant negative mutants led to a decreased capability of chicken sarcoma cells to metastasize. Moreover, the overexpression of RhoA in non-metastatic chicken cells resulted in the rescue of both invasiveness and metastatic capability. Rho and ROCK, unlike MLC, appeared to be directly involved in the maintenance of the amoeboid phenotype, as their inhibition resulted in the amoeboid-mesenchymal transition in analyzed cell lines. CONCLUSION: Taken together, these results suggest that protease-independent invasion controlled by elements of the Rho/ROCK/MLC pathway can be frequently exploited by metastatic sarcoma cells.

Asunto(s)

Cadenas Ligeras de Miosina/metabolismo , Sarcoma/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Pollos , Invasividad Neoplásica , Ratas , Sarcoma/patología , Transducción de Señal

RESUMEN

UNLABELLED: Comparing the gene expression profiles of metastatic and nonmetastatic cells has the power to reveal candidate metastasis-associated genes, whose involvement in metastasis can be experimentally tested. In this study, differentially expressed genes were explored in the v-src-transformed metastatic cell line PR9692 and its nonmetastatic subclone PR9692-E9. First, the contribution of homeodomain only protein X (HOPX) in metastasis formation and development was assessed. HOPX-specific knockdown decreased HOPX expression in the nonmetastatic subclone and displayed reduced cell motility in vitro. Critically, HOPX knockdown decreased the in vivo metastatic capacity in a syngeneic animal model system. Genomic analyses identified a cadre of genes affected by HOPX knockdown that intersected significantly with genes previously found to be differentially expressed in metastatic versus nonmetastatic cells. Furthermore, 232 genes were found in both screens with at least a two-fold change in gene expression, and a number of high-confidence targets were validated for differential expression. Importantly, significant changes were demonstrated in the protein expression level of three metastatic-associated genes (NCAM, FOXG1, and ITGA4), and knockdown of one of the identified HOPX-regulated metastatic genes, ITGA4, showed marked inhibition of cell motility and metastasis formation. These data demonstrate that HOPX is a metastasis-associated gene and that its knockdown decreases the metastatic activity of v-src-transformed cells through altered gene expression patterns. IMPLICATIONS: This study provides new mechanistic insight into a HOPX-regulated metastatic dissemination signature.

Asunto(s)

Proteínas Aviares/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/genética , Metástasis de la Neoplasia/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Sarcoma Experimental/genética , Animales , Proteínas Aviares/metabolismo , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Transformación Celular Neoplásica/genética , Pollos , Regulación hacia Abajo , Factores de Transcripción Forkhead/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes src , Proteínas de Homeodominio/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Sarcoma Experimental/patología , Sarcoma Experimental/secundario

RESUMEN

Subgroup J avian leukosis virus (ALV-J) is unique among the avian sarcoma and leukosis viruses in using the multimembrane-spanning cell surface protein Na(+)/H(+) exchanger type 1 (NHE1) as a receptor. The precise localization of amino acids critical for NHE1 receptor activity is key in understanding the virus-receptor interaction and potential interference with virus entry. Because no resistant chicken lines have been described until now, we compared the NHE1 amino acid sequences from permissive and resistant galliform species. In all resistant species, the deletion or substitution of W38 within the first extracellular loop was observed either alone or in the presence of other incidental amino acid changes. Using the ectopic expression of wild-type or mutated chicken NHE1 in resistant cells and infection with a reporter recombinant retrovirus of subgroup J specificity, we studied the effect of individual mutations on the NHE1 receptor capacity. We suggest that the absence of W38 abrogates binding of the subgroup J envelope glycoprotein to ALV-J-resistant cells. Altogether, we describe the functional importance of W38 for virus entry and conclude that natural polymorphisms in NHE1 can be a source of host resistance to ALV-J.

Asunto(s)

Virus de la Leucosis Aviar/fisiología , Receptores Virales/genética , Receptores Virales/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Tropismo Viral , Internalización del Virus , Animales , Aves , Análisis Mutacional de ADN , Triptófano/genética , Triptófano/metabolismo

RESUMEN

The group of closely related avian sarcoma and leukosis viruses (ASLVs) evolved from a common ancestor into multiple subgroups, A to J, with differential host range among galliform species and chicken lines. These subgroups differ in variable parts of their envelope glycoproteins, the major determinants of virus interaction with specific receptor molecules. Three genetic loci, tva, tvb, and tvc, code for single membrane-spanning receptors from diverse protein families that confer susceptibility to the ASLV subgroups. The host range expansion of the ancestral virus might have been driven by gradual evolution of resistance in host cells, and the resistance alleles in all three receptor loci have been identified. Here, we characterized two alleles of the tva receptor gene with similar intronic deletions comprising the deduced branch-point signal within the first intron and leading to inefficient splicing of tva mRNA. As a result, we observed decreased susceptibility to subgroup A ASLV in vitro and in vivo. These alleles were independently found in a close-bred line of domestic chicken and Indian red jungle fowl (Gallus gallus murghi), suggesting that their prevalence might be much wider in outbred chicken breeds. We identified defective splicing to be a mechanism of resistance to ASLV and conclude that such a type of mutation could play an important role in virus-host coevolution.

Asunto(s)

Alpharetrovirus/fisiología , Proteínas Aviares/genética , Pollos/genética , Predisposición Genética a la Enfermedad , Enfermedades de las Aves de Corral/genética , Empalme del ARN , Receptores Virales/genética , Sarcoma Aviar/genética , Eliminación de Secuencia , Alpharetrovirus/genética , Secuencia de Aminoácidos , Animales , Proteínas Aviares/metabolismo , Secuencia de Bases , Pollos/metabolismo , Pollos/virología , Intrones , Datos de Secuencia Molecular , Enfermedades de las Aves de Corral/metabolismo , Enfermedades de las Aves de Corral/virología , Receptores Virales/metabolismo , Sarcoma Aviar/metabolismo , Sarcoma Aviar/virología

RESUMEN

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located on chromosome 17p13.3, a region frequently hypermethylated or deleted in human neoplasias. In mouse, Hic1 is essential for embryonic development and exerts an antitumor role in adult animals. Since Hic1-deficient mice die perinatally, we generated a conditional Hic1 null allele by flanking the Hic1-coding region by loxP sites. When crossed to animals expressing Cre recombinase in a cell-specific manner, the Hic1 conditional mice will provide new insights into the function of Hic1 in developing and mature tissues. Additionally, we used gene targeting to replace sequence-encoding amino acids 186-893 of Hic1 by citrine fluorescent protein cDNA. We demonstrate that the distribution of Hic1-citrine fusion polypeptide corresponds to the expression pattern of wild-type Hic1. Consequently, Hic1-citrine "reporter" mice can be used to monitor the activity of the Hic1 locus using citrine fluorescence.

Asunto(s)

Alelos , Regulación del Desarrollo de la Expresión Génica , Genes Supresores de Tumor , Factores de Transcripción de Tipo Kruppel/genética , Animales , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/metabolismo , Femenino , Eliminación de Gen , Marcación de Gen , Genes Reporteros , Integrasas/genética , Integrasas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Factores de Transcripción/genética

RESUMEN

The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.

Asunto(s)

Cisteína/metabolismo , Serina/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt1/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Desarrollo Embrionario , Humanos , Lipoilación , Ratones , Datos de Secuencia Molecular , Mutación , Ratas , Proteínas Wnt/genética , Proteína Wnt1/genética , Proteína Wnt3 , Proteína Wnt3A , Xenopus/embriología , Xenopus/metabolismo , Proteínas de Xenopus

RESUMEN

Metastatic spreading of cancer cells is a highly complex process directed primarily by the interplay between tumor microenvironment, cell surface receptors, and actin cytoskeleton dynamics. To advance our understanding of metastatic cancer dissemination, we have developed a model system that is based on two v-src transformed chicken sarcoma cell lines-the highly metastatic parental PR9692 and a non-metastasizing but fully tumorigenic clonal derivative PR9692-E9. Oligonucleotide microarray analysis of both cell lines revealed that the gene encoding the transcription factor EGR1 was downregulated in the non-metastatic PR9692-E9 cells. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from parental PR9692 cells. Microarray analysis of EGR1 reconstituted cells revealed the activation of genes that are crucial for actin cytoskeleton contractility (MYL9), filopodia formation (MYO10), the production of specific extracellular matrix components (HAS2, COL6A1-3) and other essential pro-metastatic abilities.

Asunto(s)

Transformación Celular Neoplásica/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Metástasis de la Neoplasia/genética , Proteína Oncogénica pp60(v-src)/metabolismo , Sarcoma/genética , Sarcoma/patología , Animales , Adhesión Celular , Línea Celular , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/patología , Pollos , Citoesqueleto/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Cinética , Proteína Oncogénica pp60(v-src)/genética , Fenotipo

RESUMEN

Retroviruses and retrovirus-derived vectors integrate nonrandomly into the genomes of host cells with specific preferences for transcribed genes, gene-rich regions, and CpG islands. However, the genomic features that influence the transcriptional activities of integrated retroviruses or retroviral vectors are poorly understood. We report here the cloning and characterization of avian sarcoma virus integration sites from chicken tumors. Growing progressively, dependent on high and stable expression of the transduced v-src oncogene, these tumors represent clonal expansions of cells bearing transcriptionally active replication-defective proviruses. Therefore, integration sites in our study distinguished genomic loci favorable for the expression of integrated retroviruses and gene transfer vectors. Analysis of integration sites from avian sarcoma virus-induced tumors showed strikingly nonrandom distribution, with proviruses found prevalently within or close to transcription units, particularly in genes broadly expressed in multiple tissues but not in tissue-specifically expressed genes. We infer that proviruses integrated in these genomic areas efficiently avoid transcriptional silencing and remain active for a long time during the growth of tumors. Defining the differences between unselected retroviral integration sites and sites selected for long-terminal-repeat-driven gene expression is relevant for retrovirus-mediated gene transfer and has ramifications for gene therapy.

Asunto(s)

Virus del Sarcoma Aviar/fisiología , Cromosomas/virología , Provirus/fisiología , Sarcoma Aviar/virología , Integración Viral , Animales , Virus del Sarcoma Aviar/genética , Pollos , Expresión Génica , Terapia Genética/métodos , Vectores Genéticos , Provirus/genética