Expression of two distinct T cell receptor alpha/beta heterodimers by an antigen-specific T cell clone.

A cytochrome c-specific, MHC-restricted T cell clone with two complete rearrangements of the same V beta 1 gene element was shown to express two different TCR alpha/beta heterodimers. Antipeptide antisera specific for TCR C beta 1 and C beta 2 peptides each immunoprecipitated distinct disulfide-linked cell surface heterodimers. The clone was derived from immunized allogeneic chimeric mice, and displayed multiple Ia specificities, including the ability to recognize antigen in association with both I-Ek and I-Es Ia molecules, as well as alloreactivity to the I-Eb molecule. It will be important to determine whether each receptor contributes independently to the overall specificity of the clone.

Expression of Siglec-1, -3, -5 and -10 in porcine cDC1 and cDC2 subsets from blood, spleen and lymph nodes and functional capabilities of these cells.

Dendritic cells are professional antigen-presenting cells that play a critical role in the development of immune responses. DCs express a variety of Siglecs on their surface, which play a regulatory role modulating their activation through interaction with sialylated structures expressed by cells or pathogens. Here, we characterized the phenotype of porcine conventional dendritic cells subsets from blood, spleen and lymph nodes, emphasizing the analysis of the expression of Siglecs. Siglec-1 was detected in type 1 cDC and, at lower levels, in type 2 cDC in the spleen, being low to negative in blood and lymph node cDC. Siglec-3 and Siglec-5 were expressed in cDC1 at lower levels than in cDC2. Porcine cDCs did not express Siglec-10. cDC2 showed a higher capacity to phagocytose microspheres and to process DQ™-OVA than cDC1, but none of these functions was affected by engagement of Siglec-3 and -5 with antibodies on blood cDC.

Analysis of the expression of porcine CD200R1 and CD200R1L by using newly developed monoclonal antibodies.

CD200R1 and CD200R1-like are paired receptors which modulate activation of immune cells. Here, we describe the characterisation of their porcine homologues. Analysis of database porcine sequences shows an exceptionally high homology between the extracellular Ig-like domains of these receptors, being the rest more dissimilar. We have obtained two mAbs, PCT1 and PCT3, against a CD200R1-Fc recombinant protein, that bind on CHO cells expressing GFP-tagged CD200R1. The specificity of these mAbs was analysed on CD200R1 L, and also on a CD200R1 splicing variant that lacks the V-type Ig domain. PCT1 bound to both CD200R1 and CD200R1L, but not to the splicing variant, what suggests that recognises an epitope in the V-type Ig domain. PCT3 reacted with both CD200R1 variants, but not CD200R1L, probably binding to an epitope in the N-terminal sequence of CD200R1. Analysis of porcine cells with these mAbs showed expression of CD200R1/CD200R1L on B cells, monocytes and alveolar macrophages.

Characterisation of porcine bone marrow progenitor cells identified by the anti-c-kit (CD117) monoclonal antibody 2B8/BM.

c-kit (CD117) plays an important role in the early stages of haematopoiesis. Previous studies of porcine haematopoietic stem cells have relied for their identification on the use of the c-kit ligand stem cell factor. Here, we describe a new mAb, 2B8/BM, that recognizes a 155-kDa protein expressed on a small subset (2-8%) of bone marrow haematopoietic cells. 2B8/BM(+) cells have a blast appearance, and are mostly negative for lineage-specific markers or express low levels of CD172a or SLA-II. In in vitro colony-forming unit assays these cells were able to give rise to erythroid and myeloid colonies. Altogether these data suggested that the 2B8/BM antigen might be the porcine orthologue of the human c-kit. This specificity was confirmed by the binding of mAb 2B8/BM to CHO cells transfected with a plasmid encoding the porcine c-kit ectodomain. This antibody can facilitate the isolation and enrichment of porcine stem cells to be used in procedures aimed to induce xenograft tolerance or to test their potential to repair damaged tissues and organs.

Phenotypic and functional characterization of porcine granulocyte developmental stages using two new markers.

Here, we describe two new surface antigens, named 6D10 and 2B2, whose expression is restricted to porcine granulocytes. 6D10 is only detected in neutrophils and its expression decreases from promyelocytes to mature cells. By contrast, 2B2 antigen is selectively expressed in mature neutrophils, eosinophils and basophils. The expression of these antigens along granulocyte maturation allows the discrimination of several developmental stages of granulocytes based on phenotypic, morphological and functional characteristics previously established. Moreover, these new markers are useful tools to easily characterize the different granulocytes lineages (neutrophils, eosinophils and basophils). By using multiparameter flow cytometric analysis, we have performed a phenotypic and functional characterization of the granulocyte subsets identified by the combination of 6D10 and 2B2 antigens.

The SCL gene is formed from a transcriptionally complex locus.

We describe the structural organization of the human SCL gene, a helix-loop-helix family member which we believe plays a fundamental role in hematopoietic differentiation. The SCL locus is composed of eight exons distributed over 16 kb. SCL shows a pattern of expression quite restricted to early hematopoietic tissues, although in malignant states expression of the gene may be somewhat extended into later developmental stages. A detailed analysis of the transcript(s) arising from the SCL locus revealed that (i) the 5' noncoding portion of the SCL transcript, which resides within a CpG island, has a complex pattern of alternative exon utilization as well as two distinct transcription initiation sites; (ii) the 5' portions of the SCL transcript contain features that suggest a possible regulatory role for these segments; (iii) the pattern of utilization of the 5' exons is cell lineage dependent; and (iv) all of the currently studied chromosomal aberrations that affect the SCL locus either structurally or functionally eliminate the normal 5' transcription initiation sites. These data suggest that the SCL gene, and specifically its 5' region, may be a target for regulatory interactions during early hematopoietic development.

[Influence of preoperative bone mass density in periprosthetic bone remodeling after implantation of ABG-II prosthesis: A 10-year follow-up]. / Influencia de la masa ósea preoperatoria en la remodelación femoral periprotésica tras la implantación de una prótesis ABG-II: resultados tras 10 años de seguimiento.

INTRODUCTION: Preoperative bone mass index has shown to be an important factor in peri-prosthetic bone remodelling in short follow-up studies. MATERIAL AND METHODS: Bone density scans (DXA) were used to perform a 10-year follow-up study of 39 patients with a unilateral, uncemented hip replacement. Bone mass index measurements were made at 6 months, one year, 3 years, 5 years, and 10 years after surgery. Pearson coefficient was used to quantify correlations between preoperative bone mass density (BMD) and peri-prosthetic BMD in the 7 Gruen zones at 6 months, one year, 3 years, 5 years, and 10 years. RESULTS: Pre-operative BMD was a good predictor of peri-prosthetic BMD one year after surgery in zones 1, 2, 4, 5 and 6 (Pearson index from 0.61 to 0.75). Three years after surgery it has good predictive power in zones 1, 4 and 5 (0.71-0.61), although in zones 3 and 7 low correlation was observed one year after surgery (0.51 and 0.57, respectively). At the end of the follow-up low correlation was observed in the 7 Gruen zones. Sex and BMI were found to not have a statistically significant influence on peri-prosthetic bone remodelling. CONCLUSION: Although preoperative BMD seems to be an important factor in peri-prosthetic remodelling one year after hip replacement, it loses its predictive power progressively, until not being a major factor in peri-prosthetic remodelling ten years after surgery.

The respiratory DC/macrophage network at steady-state and upon influenza infection in the swine biomedical model.

Human and mouse respiratory tracts show anatomical and physiological differences, which will benefit from alternative experimental models for studying many respiratory diseases. Pig has been recognized as a valuable biomedical model, in particular for lung transplantation or pathologies such as cystic fibrosis and influenza infection. However, there is a lack of knowledge about the porcine respiratory immune system. Here we segregated and studied six populations of pig lung dendritic cells (DCs)/macrophages (Mθs) as follows: conventional DCs (cDC) 1 and cDC2, inflammatory monocyte-derived DCs (moDCs), monocyte-derived Mθs, and interstitial and alveolar Mθs. The three DC subsets present migratory and naive T-cell stimulation capacities. As observed in human and mice, porcine cDC1 and cDC2 were able to induce T-helper (Th)1 and Th2 responses, respectively. Interestingly, porcine moDCs increased in the lung upon influenza infection, as observed in the mouse model. Pig cDC2 shared some characteristics observed in human but not in mice, such as the expression of FCɛRIα and Langerin, and an intra-epithelial localization. This work, by unraveling the extended similarities of the porcine and human lung DC/Mθ networks, highlights the relevance of pig, both as an exploratory model of DC/Mθ functions and as a model for human inflammatory lung pathologies.

Analysis of functional heterogeneity of porcine memory CD4+ T cells.

Previous studies have identified swine helper memory T cells as CD4+CD8alpha+SLADR+. We have recently described a new porcine surface antigen (2E3) selectively expressed on CD4+ T cells that allows to divide these cells into naive (2E3+) and effector/memory (2E3-). However, although the majority of CD4+2E3- cells are CD8alpha+SLADR+, a minor proportion do not express SLADR and/or CD8alpha. Here, we have analyzed the functional capacity of these CD4+2E3- subsets to proliferate to a recall antigen. Both SLADR- and CD8alpha- cells proliferated in response to lysozyme, but at lower levels compared to the whole population CD4+2E3-. Besides, after activation with PMA plus ionomycin, CD4+2E3-SLADR- T cells produced IFNgamma and TNFalpha, although they did also in lower proportion than the whole CD4+2E3- population. Most of the IFNgamma-TNFalpha+, IFNgamma+TNFalpha+, IFNgamma+TNFalpha- cells were CD8alpha+ and CD45RA-, while IFNgamma-TNFalpha- cells showed a less differentiate phenotype.

Differential expression of chemokine receptors and CD95 in porcine CD4(+) T cell subsets.

Among other differences, naïve and memory T cells show distinct migratory patterns and susceptibility to CD95-mediated cell death. We have recently characterised in the pig two subsets of CD4(+) T cells, based on the expression of the 2E3 marker, that display phenotypic and functional features of naïve (CD4(+)2E3(+)) and effector/memory (CD4(+)2E3(-)) T cells. In this study, we have analysed the expression of several chemokine receptors, as well as the distribution of CD95 antigen (APO-1/Fas) in these CD4(+) T cell subsets. CD4(+)2E3(-) T cells express high levels of CXCR3 and CCR4 transcripts but not of CCR7. On the contrary, CCR7 is clearly detected in CD4(+)2E3(+) T cells, whereas CXCR3 and CCR4 are negative or present at trace levels. These subsets also differ in the expression of CD95 antigen, being CD95 positive cells significantly more abundant in the CD4(+)2E3(-) cell subset. These findings, although based on a small number of animals, fit well with those reported for naïve and memory CD4(+) T cells in humans.

Molecular characterization of porcine Siglec-10 and analysis of its expression in blood and tissues.

Siglecs are sialic acid binding Ig-like proteins involved in the control of leukocyte responses. In this study we describe the characterization of a porcine orthologue of Siglec-10. A cDNA clone was obtained from a porcine library which encodes a protein with sequence homology to human Siglec-10. This cDNA codes for a type I transmembrane protein containing four Ig-like domains, a transmembrane region, and a cytoplasmic tail with three tyrosine-based motifs, including a membrane-proximal Grb2-binding motif, and two ITIM motifs. When expressed on transfected cells, porcine Siglec-10 was able to bind red blood cells in a sialic acid-dependent manner. Monoclonal antibodies were developed against this protein and used to examine its cell and tissue distribution in the pig. Siglec-10 was found to be expressed on blood B cells and B cell areas of the spleen and lymph nodes. A weak expression was also detected on monocytes.

Molecular and functional characterization of porcine Siglec-3/CD33 and analysis of its expression in blood and tissues.

A cDNA clone encoding a 380 a-a type 1 transmembrane protein with homology to human Siglec-3/CD33 was obtained from a swine small intestine library. An analysis of protein sequence identified two immunoglobulin-like domains, a transmembrane region, and a carboxi-terminal tail with two tyrosine-based signalling motifs. Binding assays of Siglec-3 transfected CHO cells to polyacrylamide glycoconjugates showed a preference for α2-6-linked sialic acids. Using mAbs raised against a fragment containing the two Ig-like domains, porcine Siglec-3 was found to be expressed on monocytes and granulocytes, and their bone marrow precursors. It was also detected in lymph node, splenic and alveolar macrophages. MAbs immunoprecipitated, from granulocyte lysates, a protein of 51-60 kDa under both non-reducing and reducing conditions. MAbs were also used to analyse functional activity of Siglec-3 on bone marrow and blood cells. Engagement of Siglec-3 by mAb had no apparent effect on cell proliferation or cytokine production.

Monoclonal antibodies 2F6/8 and 2A10/8 recognize a porcine antigen (SWC7) expressed on B cells and activated T cells.

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.

Phenotypic characterization of porcine IFN-gamma-producing lymphocytes by flow cytometry.

We have developed a three-colour flow cytometric assay for phenotypic characterization of porcine IFN-gamma-producing lymphocytes. Analyses of activated swine peripheral blood mononuclear cells (PBMC) showed a significant difference in the proportion of IFN-gamma producing cells between young and adult animals (13.2+/-5.8% versus 34.2+/-5.7%). The majority of IFN-gamma producing cells were alphabeta T lymphocytes, although there was also an important proportion of gammadelta T cells particularly in young animals. Within the alphabeta T lymphocytes, the double positive CD4(+)CD8(lo) subset, that contains memory T cells, produced high levels of IFN-gamma, whereas the CD8(hi) T cells ranged from low to high levels of IFN-gamma. Also, consistent with a higher production by memory T cells, the CD45RA(-) subset of both CD4(+) and CD8(+) cells contained higher numbers of IFN-gamma producing cells than the CD45RA(+) subset. Finally, no production of IFN-gamma by either B cells (CD21(+)) or monocytes (SWC3(+)) was detected. This assay may be useful for the assessment of cell-mediated immunity in vaccine trials and may contribute to our understanding of the role of IFN-gamma in protective immunity against important viral diseases of the pig.

Monoclonal antibody 2F4/11 recognizes the alpha chain of a porcine beta 2 integrin involved in adhesion and complement mediated phagocytosis.

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).

Analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection.

The cellular immune response to a European isolate of porcine reproductive and respiratory syndrome (PRRS) virus in animals recovered from the experimental infection has been studied in vitro. Peripheral blood mononuclear cells (PBMC) from these pigs proliferated specifically when they were stimulated with PRRS virus. This response was not detectable until 4 weeks after inoculation and remained for more than 3 months. Addition of blocking monoclonal antibodies to the cultures showed that this proliferation was mainly dependent on CD4(+) cells with the participation of SLA-class II molecules. T-cell cultures established by stimulating responding cells with PRRS virus and maintained in culture for up to 3 weeks showed an increase of CD8(+) CD4(+) and CD4(-) CD8(+) subsets within activated cells, gated according to their light scatter parameters, whereas CD4(+) CD8(-) cells declined along the time in culture. Within the activated cells, those expressing the TcR gammadelta receptor also increased, being most of them also positive for the CD8 marker. By RT-PCR, T-cells responding to the virus showed a Th1 type cytokine production pattern. During the culture period the cytotoxic activity against K-562 cells increased from 15 to 35% of specific lysis. This cellular immune response may play a relevant role in the clearance of PRRS virus and the recovery of the infection.

Porcine reproductive and respiratory syndrome (PRRS) virus down-modulates TNF-alpha production in infected macrophages.

The effect of porcine reproductive and respiratory syndrome (PRRS) virus infection on the synthesis and secretion of TNF-alpha and other pro-inflammatory cytokines by porcine alveolar macrophages (PAM) was investigated as well as the effect that TNF-alpha has on the replication of this virus. A clear reduction of phorbol myristate acetate (PMA)-induced expression of TNF-alpha mRNA was observed in cells incubated with PRRS virus. Moreover, the presence of PRRS virus also induced a decrease in IL-1 alpha and MIP-1 beta mRNAs expression with respect to PMA-stimulated uninfected cells. According to these results, exposure to the PRRS virus led to a reduction of the TNF-alpha protein in supernatants of PMA-stimulated PAM. On the other hand, addition of recombinant porcine TNF-alpha to cultures clearly reduced virus replication; however the addition of TNF-alpha to cultures containing IFN-alpha did not result in a further reduction of the produced by IFN-alpha alone. This indicates the lack of synergy in the effect of these cytokines on viral replication.

Characterization of five monoclonal antibodies specific for swine class II major histocompatibility antigens and crossreactivity studies with leukocytes of domestic animals.

A set of five monoclonal antibodies (mAb) against porcine major histocompatibility complex (MHC), or swine leukocyte antigens (SLA), class II molecules has been characterized. These mAbs appear to recognize monomorphic determinants on SLA-DR (2F4, 1F12 and 2E9/13) and SLA-DQ (BL2H5 and BL4H2) molecules, as assessed by flow cytometry and immunoprecipitation. By Western blot, the 2F4, 1F12, BL2H5 and BL4H2 epitopes were located on the beta-chains of these molecules. mAbs 2F4 and 1F12 crossreact with leucocytes of dog, cattle, horse and human; mAbs 2E9/13, BL2H5 and BL4H2 bind leucocytes of cattle but not those of human, dog and horse. These mAbs effectively blocked the mixed lymphocyte reaction and the proliferative response to viral antigens (African swine fever virus) and to staphylococcal enterotoxin B. Therefore, these mAbs can be useful reagents for studying MHC class II molecules of pig and crossreactive species, and the immunological processes where they are involved.

2E3, a new marker that selectively identifies porcine CD4+ naive T cells.

We describe a novel antigen recognized by mAb 2E3 selectively expressed in the periphery by a subset of porcine CD4+ T cells. Both, CD4+CD8alpha- and CD4+CD8alphalow T cell subpopulations express this antigen. CD4+2E3+ T cells show phenotypical and functional characteristics of nai;ve cells. The majority of them are CD29low, CD45RAhigh, CD49dlow, CD11alow, CD18low, and SLA-II-. After mitogen activation CD4+2E3+ T cells express high levels of IL-2 mRNA, but only traces of IFN-gamma or IL-4 mRNA. Indeed a minor percentage of cells stained positive for IFN-gamma when assessed by flow cytometry. Moreover, CD4+2E3+ T cells did not proliferate in response to the recall antigen lysozyme, although they did efficiently to the mitogen ConA. By contrast, CD4+2E3- T cells show phenotypical and functional characteristics of primed cells. They express markers associated to a memory phenotype, respond to the recall antigen lysozyme, and produce high amounts of IFN-gamma and IL-4.

Phenotypic characterization of monocyte subpopulations in the pig.

We have recently described the existence of two subsets of porcine monocytes based on the expression of CD163. In this study we compare the expression of a number of cell surface antigens in CD163+ and CD163- monocyte subsets using three-color flow cytometry. These monocyte subsets show differences with respect to the expression of MHC class II antigens (SLA-DR and DQ) and a variety of adhesion molecules (CD11a, wCD11c, wCD29, CD49d) that are expressed at higher levels on CD163+ monocytes, and of CD14 that is higher expressed by CD 163- cells. These differences on phenotype could reflect differences in the ability of these two subsets to migrate to tissues and may account for the higher allostimulatory capacity of CD163+ cells. In some aspects, the phenotype of CD163+ monocytes resembles that of mature macrophages. In vitro serum-induced maturation of monocytes into macrophages lead to the expression of SWC9 together with an increase in the expression of CD163 and a reduction in that of CD14. These results delineate a maturation pathway where CD14hiCD163-SWC9- monocytes develop into CD14loCD163+SWC9- monocytes and these cells into CD14loCD163+SWC9+ macrophages.