RESUMEN
In this study we describe the characterization of the porcine orthologue of Siglec-5. A cDNa clone was obtained from a porcine cDNa library derived from swine small intestine which encodes a 555 a-a type 1 transmembrane protein with sequence homology to human Siglec-5. This protein consists of four Ig-like domains, a transmembrane region, and a cytoplasmic tail with two tyrosine-based signalling motifs. When expressed as a recombinant protein fused to the Fc region of human IgG1, porcine Siglec-5 was able to bind porcine red blood cells in a sialic acid-dependent manner. Monoclonal antibodies (mAb) were developed against porcine Siglec-5 and used to analyse its expression in bone marrow and blood cells, and lymphoid tissues. Porcine Siglec-5 expression was mainly restricted to myelomonocytic cells and their precursors, being detected also, although at low levels, on plasmacytoid dendritic cells and B lymphocytes. In lymphoid tissues, ellipsoids of the spleen and subcapsular and medullar sinuses of lymph nodes were positive for Siglec-5. These mAbs were able to precipitate, from granulocyte lysates, a protein of approximately 85 kDa under non-reducing conditions, indicating that porcine Siglec-5 is expressed as a monomer in the plasma membrane.
Asunto(s)
Células Dendríticas/inmunología , Eritrocitos/metabolismo , Células Mieloides/inmunología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Porcinos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Clonación Molecular , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , TranscriptomaRESUMEN
The mesoporous silicon microparticles (MSMPs) are excellent vehicles for releasing molecules inside the cell. The aim of this work was to use MSMPs to deliver viral specific MHC class I restricted epitopes into human antigen presenting cells (monocyte derived dendritic cells, MDDCs) to facilitate their capture, processing, and presentation to CD8+ (cytotoxic) T lymphocytes. We show for the first time that MSMPs vehiculation of antigenic peptides enhances their MHC class I presentation by human MDDCs to CD8 T lymphocytes.
Asunto(s)
Presentación de Antígeno/inmunología , Compuestos de Calcio , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Nanopartículas , Silicatos , Células Presentadoras de Antígenos/inmunología , Antígenos Virales/química , Antígenos Virales/inmunología , Compuestos de Calcio/química , Células Cultivadas , Células Dendríticas/metabolismo , Epítopos/administración & dosificación , Epítopos/inmunología , Humanos , Nanopartículas/química , Nanopartículas/toxicidad , Nanopartículas/ultraestructura , Tamaño de la Partícula , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Silicatos/química , Propiedades de SuperficieRESUMEN
Monocytes-macrophages, the target cells of African swine fever virus (ASFV) are highly heterogeneous in phenotype and function. In this study, we have investigated the correlation between the phenotype of specific populations of porcine macrophages and their permissiveness to ASFV infection. Bone marrow cells and fresh blood monocytes were less susceptible to in vitro infection by ASFV than more mature cells, such as alveolar macrophages. FACS analyses of monocytes using a panel of mAbs specific for porcine monocyte/macrophages showed that infected cells had a more mature phenotype, expressing higher levels of several macrophage specific markers and SLA II antigens. Maturation of monocytes led to an increase in the percentage of infected cells, which correlated with an enhanced expression of CD163. Separation of CD163+ and CD163- monocytes demonstrated the specific sensitivity of the CD163+ subset to ASFV infection. In vivo experiments also showed a close correlation between CD163 expression and virus infection. Finally, mAb 2A10 and, in a lower extent, mAb 4E9 were able to inhibit, in a dose-dependent manner, both ASFV infection and viral particle binding to alveolar macrophages. Altogether, these results strongly suggest a role of CD163 in the process of infection of porcine monocytes/macrophages by ASFV.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación Mielomonocítica/fisiología , Asfarviridae/fisiología , Macrófagos/virología , Monocitos/virología , Receptores de Superficie Celular/fisiología , Animales , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Células Cultivadas , Macrófagos/química , Monocitos/química , Fenotipo , Receptores de Superficie Celular/análisis , PorcinosRESUMEN
CD1 molecules are specialized in presenting lipidic antigens to T lymphocytes. They are structurally and evolutionary related to MHC molecules and show very limited polymorphism. We have previously described and partially characterized a new human CD1A allele differing from the wild type CD1A by a substitution of Cysteine by Tryptophan at position 52 in the alpha1 domain of the CD1A molecule. The frequency of this allele varies from 10% in individuals of Caucasian origin to 56% in Chinese people. The aim of the present work was to structurally characterize this CD1A allele. To do this we have cloned and sequenced the full-length cDNA encoding the new CD1A allele. The cDNA sequence of this allele encodes a protein differing the wild type in two amino acids at positions 14 (Threonine versus Isoleucine) and 52 (Cysteine versus Tryptophan). The cDNAs encoding both wild type and mutant CD1A were cloned in the expression vector pSRalphaNeo and transfected into C1R and L721.221 cells. Cell surface expression of the protein products in transfected cell lines were analyzed by flow cytometry and immunoprecipitation using CD1a-specific monoclonal antibodies. Our results indicate that both allelic products are efficiently expressed on the cell surface.
Asunto(s)
Alelos , Antígenos CD1/química , Antígenos CD1/genética , Variación Genética/inmunología , Anticuerpos Monoclonales/análisis , Antígenos CD1/biosíntesis , Antígenos CD1/inmunología , Línea Celular Transformada , Clonación Molecular/métodos , ADN Complementario/aislamiento & purificación , Citometría de Flujo , Vectores Genéticos/biosíntesis , Vectores Genéticos/inmunología , Humanos , Pruebas de Precipitina , Transfección , Células Tumorales CultivadasRESUMEN
En los últimos años se han descubierto diversos genes relacionados con los que codifican las moléculas HLA de clase I a los que se ha denominado en conjunto genes HLA "no clásicos". Hasta el momento se han descrito 9 familias "no clásicas" diferentes que contabilizan un total de 17 genes funcionales. Solamente para una de estas familias, MR1, la función sigue siendo completamente desconocida. Además, la estructura tridimensional de al menos una molécula representativa ha sido descifrada en 6 familias. Aunque muestran importantes diferencias en términos de secuencia aminoacídica las distintas moléculas HLA de clase I no clásicas presentan una estructura tridimensional muy parecida. Sorprendentemente, estas moléculas tan parecidas en su forma llevan a cabo funciones muy heterogéneas que van desde la presentación de antígenos lipídicos a los linfocitos T hasta la regulación del metabolismo del hierro (AU)
Asunto(s)
Animales , Humanos , Genes MHC Clase I/fisiología , Genes MHC Clase I/genética , Secuencia de Aminoácidos , Polimorfismo GenéticoRESUMEN
We characterized the MHC-related 1 ( MR1) locus in two nonhuman primates species, Pongo pygmaeus and Pan troglodytes. MR1 cDNA sequences encoding several isoforms generated through alternative splicing were observed in both species. Amino acid alignment between the five species in which MR1 has been characterized to date - human, chimpanzee, orangutan, mouse, and rat - reveals a very high degree of conservation specially in the alpha1 and alpha2 domains of the molecule. The main differences concentrate in the transmembrane and cytoplasmic domains. In the three primates species there is a lysine residue inside the putative transmembrane domain which is not present in rodents. Furthermore, the MR1 cytoplasmic region is longer in rodents, with a conserved serine-containing motif that could be involved in endocytosis; remarkably, this motif is absent in the three primate species. We also describe the presence in the chimpanzee of a sequence homologous to the MR1P1 pseudogene previously found in humans.
Asunto(s)
Genes MHC Clase I/genética , Antígenos de Histocompatibilidad Clase I/genética , Pan troglodytes/genética , Pongo pygmaeus/genética , Secuencia de Aminoácidos , Animales , Línea Celular Transformada , Clonación Molecular , Secuencia Conservada , Humanos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
We describe in this report the production and characterization of monoclonal antibodies (mAb) to the swine homologues of CD11a and CD18 antigens, and their use for phenotypic and functional analysis of porcine leukocytes. Monoclonal antibodies BL1H8 and BL2F1 precipitated two bands of approximately 170 and 95 kDa, whereas mAb BA3H2 brought down three bands of 170, 155 and 95 kDa, from alveolar macrophage lysates. Clearance of macrophage lysates with mAbs BL1H8 and BL2F1 resulted in complete removal of the 170-kDa band. The cell distribution of the molecules recognized by these mAbs was similar to that of human LFA-1. It was found on all leukocytes, although its expression varied among the different leukocyte subpopulations, with monocytes, granulocytes and a subset of CD8+ cells expressing the highest levels. Cross-blocking studies showed that these antibodies recognize different epitopes on porcine LFA-1. Both anti-LFA-1 mAbs strongly inhibited the mitogenic response of PBMC to ConA, whereas the anti-CD18 mAb had no effect. These anti-LFA-1 mAbs also inhibited the mixed lymphocyte reaction (MLR) and the NK cell-mediated lysis of K-562 cells.
Asunto(s)
Anticuerpos Monoclonales , Antígenos CD/inmunología , Antígenos CD18/inmunología , Leucocitos/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Macrófagos Alveolares/inmunología , Animales , Reacciones Cruzadas , Epítopos/inmunología , Citometría de Flujo , Granulocitos/inmunología , Humanos , Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Monocitos/inmunología , PorcinosRESUMEN
This report describes the production and characterization of two monoclonal antibodies (mAbs), 2F6/8 and 2A10/8, that recognize a porcine antigen (SWC7) expressed on B cells in lymphoid tissues. The antigen was not detectable on resting PBMC but its expression could be induced after treatment with phorbol esters (PMA) but not by ConA, PWM, LPS or Ca ionophore. Kinetic studies showed that the antigen was expressed 24 h after PMA treatment, peaked at day 2 or 3 and slightly declined by day 6. Interestingly, the antigen was also found on a subset of CD3 + T cells, with levels of expression similar to those of B cells. By immunohistochemistry, the antigen was detected on follicular dendritic cells of germinal centers in tonsils, spleen, lymph nodes and Peyer's patches. MAb 2F6/8 precipitates a molecule of approximately 40 kDa under non-reducing conditions, and 24 kDa under reducing conditions. The restricted and tightly regulated expression of this antigen may reflect an important role in B cell differentiation within the germinal center. These mAbs will be useful reagents for phenotypic analysis of porcine lymphoid cell populations by flow cytometry and immunohistochemistry.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos/inmunología , Linfocitos B/metabolismo , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Antígenos/biosíntesis , Antígenos/metabolismo , Concanavalina A/farmacología , Epítopos/análisis , Citometría de Flujo , Inmunohistoquímica , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Pruebas de Precipitina , Porcinos , Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Distribución TisularRESUMEN
We have analyzed the production of tumor necrosis factor alpha (TNF-alpha) induced by in vitro infection with African swine fever (ASF) virus (ASFV) and the systemic and local release of this inflammatory cytokine upon in vivo infection. An early increase in TNF-alpha mRNA expression was detected in ASFV-infected alveolar macrophages, and high levels of TNF-alpha protein were detected by ELISA in culture supernatants from these cells. When animals were experimentally infected with a virulent isolate (E-75), enhanced TNF-alpha expression in mainly affected organs correlated with viral protein expression. Finally, elevated levels of TNF-alpha were detected in serum, corresponding to the onset of clinical signs. TNF-alpha has been reported to be critically involved in the pathogenesis of major clinical events in ASF, such as intravascular coagulation, tissue injury, apoptosis, and shock. In the present study, TNF-alpha containing supernatants from ASFV-infected cultures induced apoptosis in uninfected lymphocytes; this effect was partially abrogated by preincubation with an anti-TNF-alpha specific antibody. These results suggest a relevant role for TNF-alpha in the pathogenesis of ASF.
Asunto(s)
Fiebre Porcina Africana/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Fiebre Porcina Africana/complicaciones , Fiebre Porcina Africana/patología , Virus de la Fiebre Porcina Africana/patogenicidad , Animales , Apoptosis , Macrófagos/metabolismo , ARN Mensajero/análisis , Porcinos , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: We have analyzed by electron microscopy, immunohistochemistry, and flow cytometry the development of chicken caecal tonsil, the largest lymphoid organ of avian gut-associated lymphoid tissue (GALT). METHODS: White Leghorn chickens of different ages obtained from a local supplier were routinely processed by transmission electron microscopy. For both immunohistochemistry and flow cytometry, we tested a battery of specific monoclonal antibodies (mAbs) to chicken cell markers on caecal cryosections or cell suspensions, respectively. RESULTS: A rudimentary caecal tonsil occurs at the end of incubation. The organ grows just after birth, reaching the adult condition 4 days later. Firstly (4 days to 2 weeks), it contains predominantly T lymphocytes, principally TcR alphabeta+ and CD4+ cells, which occupy largely the named caecal diffuse lymphoid tissue. In adult tonsils (6-week-old chickens) however, B lymphocytes, mainly expressing either IgM or IgA, predominate. They occur in both the subepithelial zone and the germinal centers, in which there are also a few T cells. After 2 weeks the CD8+ lymphocytes gradually become more numerous than CD4+ cells. In the tonsillar epithelium CD8+TcRgammadelta+ T cells, CD8+TcRgammadelta-alphabeta-, presumably NK cells, and a few B lymphocytes are the main cell subpopulations. CONCLUSIONS: Chicken caecum grows fast after hatching. The diffuse lymphoid tissue largely contains TcR alphabeta CD4+ or CD8+ cells. CD8+ cells of caecal epithelium represent gammadelta T cells or NK cells. B lymphocytes which occur in the subepithelial zone, germinal centers, and, in few numbers, the caecal epithelium predominantly express either IgM or IgA.
Asunto(s)
Linfocitos B/citología , Ciego/crecimiento & desarrollo , Pollos/crecimiento & desarrollo , Tejido Linfoide/crecimiento & desarrollo , Subgrupos de Linfocitos T/citología , Animales , Linfocitos B/inmunología , Ciego/citología , Ciego/embriología , Ciego/inmunología , Embrión de Pollo/citología , Pollos/inmunología , Citometría de Flujo , Técnicas para Inmunoenzimas , Inmunoglobulinas/análisis , Tejido Linfoide/citología , Tejido Linfoide/embriología , Tejido Linfoide/inmunología , Microscopía Electrónica , Receptores de Antígenos de Linfocitos T , Subgrupos de Linfocitos T/inmunologíaRESUMEN
This report describes the obtention and characterization of two monoclonal antibodies (mAbs), 6E3/7 [mAb 6E3/7 was submitted to the Second International Swine CD Workshop, where it has been assigned to CD45R] and 3C3/9, which recognize the isoform of highest molecular weight of porcine CD45. This conclusion is based on their cell reactivity and tissue distribution, identical to that reported for the human high molecular weight isoform of CD45, and on data from immunoprecipitation and immunoblotting analyses which show that these mAbs react with the largest polypeptide of those precipitated by mAb 2A5, that recognizes an epitope shared by all CD45 isoforms. These mAbs react with 60% of peripheral blood mononuclear cells (PBMC) but not with alveolar macrophages, granulocytes, platelets or erythrocytes. Antigen expression on PBMC is heterogeneous and is reduced after in vitro activation with mitogens. B cells and CD8+ T cells express more antigen than CD4+ T cells. Using immunoperoxidase techniques, the antigen was detected on B cell areas of lymph nodes and Peyer's patches, and on a subpopulation of medullary thymocytes. These mAbs will be useful reagents for functional and phenotypic analysis of porcine lymphoid cell populations by flow cytometry and immunohistochemistry.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos Comunes de Leucocito/química , Antígenos Comunes de Leucocito/inmunología , Animales , Bovinos , Reacciones Cruzadas , Perros , Mapeo Epitopo/veterinaria , Caballos , Humanos , Isomerismo , Antígenos Comunes de Leucocito/biosíntesis , Activación de Linfocitos , Peso Molecular , Especificidad de Órganos/inmunología , Especificidad de la Especie , PorcinosRESUMEN
Macrophages are widely distributed in most tissues of the body, where they play important roles in host defense and repair of tissue damage. In this report we describe the production and characterization of a panel of six monoclonal antibodies (mAb) against porcine macrophages and their use for phenotyping tissue macrophages. All mAbs were produced by immunizing mice with porcine alveolar macrophages. Three of them (2A10/11, 3B11/11 and 3F7/11) react mainly with macrophages and, at a lower extent, blood monocytes, whereas the others (1E12/11, 2C12/10 and 4E9/11) also recognize granulocytes. Antigens recognized by these antibodies could be characterized by Western blot and/or immunoprecipitation, with the exception of that one recognized by 2C12/10. By their behavior in SDS-PAGE under reducing and nonreducing conditions, all seem to be single polypeptides, whose apparent molecular weight under reducing conditions are: 1E12/11 and 3B11/11 larger than 204 kDa; 2A10/11, 150 kDa; 4E9/11, 125-170 kDa; and 3F7/11, 135 kDa. Immunohistochemical analyses of both lymphoid and non-lymphoid organs using these mAbs reveal important antigenic heterogeneity among tissue macrophages. These mAbs are, therefore, useful tools for the study of porcine macrophage maturation and differentiation and for determining their heterogeneity both in normal and pathological conditions.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos , Diferenciación Celular , Citometría de Flujo , Inmunohistoquímica , Macrófagos/citología , Ratones , Ratones Endogámicos BALB C , Monocitos/citología , Porcinos , Distribución TisularRESUMEN
The characterization of a new mAb, named 2F4/11, specific for porcine myelomonocytic cells is described. This mAb immunoprecipitates a non-covalently linked heterodimer of 155,000/95,000, which is expressed by granulocytes, monocytes and tissue macrophages but not by lymphocytes, erythrocytes or platelets. Immunoblot analysis localizes the 2F4/11 epitope on the largest subunit of the heterodimer. Mab 2F4/11 is able to block phagocytosis of complement-opsonized zymosan particles by PMN granulocytes and alveolar macrophages, as well as adherence to plastic surfaces of PMA-activated PMN. Together, these results suggest that mAb 2F4/11 recognizes the CD11b or alpha chain of the porcine complement type 3 receptor (CR3).
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD18/inmunología , Monocitos/inmunología , Animales , Adhesión Celular/inmunología , Activación de Complemento , Monocitos/citología , Especificidad de Órganos , Fagocitosis/inmunología , PorcinosRESUMEN
Although numerous authors have correlated high levels of circulating estrogens with thymic involution, a systematic analysis to date on the histological changes affecting the thymus gland in that situation is lacking. In the present study we report both histological and ultrastructural changes occurring in the thymus of adult Wistar rats which received a single dose either of 100 micrograms or 500 micrograms of estradiol benzoate. Both doses induced thymic involution which correlated well with histological changes observed in the lymphoid populations but also with profound modifications in the thymic epithelial component. Moreover, intrathymic erythro-and granulopoiesis, increased numbers of both macrophages and plasma cells, and important variations in the thymic vascular permeability occurred in estradiol benzoate treated rats. These results are discussed from the perspective that changes in both the non-lymphoid cell components of thymic microenvironments and vascular permeability are essential to understand the general effects of sex steroids on the immune system.
Asunto(s)
Estradiol/farmacología , Timo/efectos de los fármacos , Timo/ultraestructura , Animales , Estradiol/sangre , Femenino , Inyecciones Intraperitoneales , Ratas , Ratas Wistar , Maduración Sexual , Timo/patologíaRESUMEN
In the present study we have evaluated morphometrically the contribution of thymocytes to the thymic involution induced by a single injection either of 100 micrograms or 500 micrograms of estradiol benzoate. Our results demonstrate that changes in the numbers of both cortical and medullary thymocytes contribute to thymic involution although the importance of the first is quantitatively higher. On the other hand, while cortical pyknosis and a decreased mitotic index could be important for explaining the estrogen-dependent thymic changes, the release of lymphocytes from thymus seems to be the main factor inducing the thymic involution as well as the lack of recovery observed at the end of the experimental period.
Asunto(s)
Estradiol/farmacología , Linfocitos/ultraestructura , Timo/citología , Animales , Permeabilidad Capilar/efectos de los fármacos , Estradiol/administración & dosificación , Femenino , Inyecciones Intraperitoneales , Linfocitos/efectos de los fármacos , Índice Mitótico/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Wistar , Linfocitos T/efectos de los fármacos , Linfocitos T/ultraestructura , Timo/efectos de los fármacos , Timo/ultraestructuraRESUMEN
In the present study we confirm and extend previous reports about the existence of a T-dependent area in the bursa of Fabricius of some birds, analyzing ultrastructurally the cell content of the so-called diffusely-infiltrated lymphoid tissue of the bursa of the spotless starling, Sturnus unicolor. It consists of lymphocytes, plasma cells, macrophages (Mphis) and interdigitating cells (IDCs) in a supporting reticular stroma which, apart from blood capillaries, contains postcapillary high-endothelial venules (HEVs). The presence of both IDCs and HEVs confirms the T-cell-dependent nature of this area, as previously claimed for other avian species, and emphasizes other functions, apart from its role in B-cell maturation, for the bursa of Fabricius specially in adult birds.
Asunto(s)
Aves/anatomía & histología , Bolsa de Fabricio/ultraestructura , Tejido Linfoide/ultraestructura , Envejecimiento/fisiología , Animales , Aves/fisiología , Bolsa de Fabricio/crecimiento & desarrollo , Bolsa de Fabricio/fisiología , Tejido Linfoide/crecimiento & desarrollo , Tejido Linfoide/fisiología , Microscopía Electrónica , Linfocitos T/ultraestructuraRESUMEN
An in vitro assay was used to study the involvement of gill cells in the trapping and processing of particulate antigens. Gills were routinely processed for light microscopy after being placed in medium containing either Yersinia ruckeri O-antigen-labelled fluorescent beads, unlabelled fluorescent beads, Y, ruckeri O-antigen or formalin-killed Y. ruckeri, for 0, 30 s, 1, 5 and 30 min. Y. ruckeri formalin-killed cells, Y. ruckeri O-antigen and fluorescent beads labelled with Y. ruckeri O-antigen were taken in by gill epithelial cells as soon as 30 s after administration. In contrast, unlabelled fluorescent beads adhered to the epithelial cell membranes, but did not occur inside the gill cells. These results are discussed principally in relationship with the specificity of antigen trapping.