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.

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.

[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.

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.

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 characterization and expression of porcine Siglec-5.

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.

Antigen targeting to APC: from mice to veterinary species.

Antigen delivery to receptors expressed on antigen presenting cells (APC) has shown to improve immunogenicity of vaccines in mice. An enhancement of cytotoxic T lymphocyte (CTL), helper T cell or humoral responses was obtained depending on the type of APC and the surface molecule targeted. Although this strategy is being also evaluated in livestock animals with promising results, some discrepancies have been found between species and pathogens. The genetic diversity of livestock animals, the different pattern of expression of some receptors among species, the use of different markers to characterize APC in large animals and sometimes the lack of reagents make difficult to compare results obtained in different species. In this review, we summarize the data available regarding antigen targeting to APC receptors in cattle, sheep and pig and discuss the results found in these animals in the context of what has been obtained in mice.

Expression of TLR4 in swine as assessed by a newly developed monoclonal antibody.

Toll-like receptors (TLRs) constitute an ancient family of pattern recognition receptors for conserved microbial structures that allow rapid detection of invading pathogens, triggering immune responses. TLR4 binds lipopolysaccharides (LPS) being involved in the recognition of Gram-negative bacteria. Herein we describe the generation and characterisation of a monoclonal antibody, named 3H3, against porcine TLR4. Its specificity was confirmed by reactivity with TLR4 expressing CHO cell transfectants. On peripheral blood leukocytes TLR4 was preferentially expressed on myelomonocytic cells, with monocytes expressing higher levels than granulocytes. Staining of lung tissue sections showed that TLR4 is also expressed on epithelial cells lining the bronchial tract, a distribution consistent with a surveillance function of bacterial invasion.

Analysis of chemokine receptor CCR7 expression on porcine blood T lymphocytes using a CCL19-Fc fusion protein.

The chemokine receptor CCR7 has been a useful marker for the characterization of human and mouse T cell subsets. We have produced the porcine CCR7 ligand CCL19 fused to the human IgG1 Fc fragment, and used it to analyse CCR7 expression in swine. CCL19-Fc bound to and induced the migration of cells expressing porcine CCR7 but not of untransfected cells, corroborating its specificity. On blood lymphocytes, CCL19-Fc labelled the majority of CD4(+) T cells expressing the 2E3 marker, associated with a naïve phenotype, whereas the 2E3(-) cells were mostly negative. Among CD8(+) T cells CCL19-Fc labelled two subsets: one, CD8ß(hi) CD11a(lo) CD45RA(+), perforin(-/lo) , which produced low amounts of IFN-γ after stimulation, which might correspond to naïve cells; and a second small population of CD8ß(lo) cells which expressed high levels of CD11a, and were mostly CD45RA(-), a phenotype which resembles that of human central memory T cells.

Porcine myelomonocytic markers and cell populations.

This review focuses in what is currently known about swine myeloid markers, the expression and function of these receptors in the biology of porcine myelomonocytic cells, the regulation of their expression along the different developmental stages of these cells and their utility to investigate the heterogeneity of monocyte and macrophage populations. Although the number of monoclonal antibodies recognizing surface antigens expressed on either swine granulocytes or monocytes is low compared with those available for human or mouse, they have contributed significantly to study the members of myeloid lineages in this species, allowing to discriminate different maturation stages of these cells in bone marrow and to reveal the heterogeneity of blood monocytes and tissue macrophages. Porcine myeloid cells share many similarities with humans, highlighting the relevance of the pig as a biomedical model.

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.

Change of mechanical properties during short-term natural weathering of MSWI bottom ash.

The present work describes the change of mechanical properties during the natural weathering of freshly quenched processed bottom ash. An unconfined uniaxial compression to failure test of the unbound material was used to determine compressive strength and modulus of elasticity. Three main stages of mechanical behavior were determined. In the first stage, during a period lower than 30 days, mechanical properties suddenly increase, with a compressive strength and elastic modulus 7 times greater than the initial parameters. During the second stage, compressive strength and modulus of elasticity lightly increase until approximately 90 days of curing time. Starting from this period both mechanical properties remain steady and independent of the curing time. The neoformed phases, the elevated water content, and the improvement of particle contact after compaction act as a binder layer among particles, increasing the mechanical parameters during the short-term natural weathering process. Because of this, the freshly compacted bottom ash progresses from behaving as an unbound material into a bound pavement material. These mechanical properties obtained from freshly quenched bottom ash are 6-7 times greater than those obtained from previously weathered bottom ash. The bottom ash expansion and leaching of metals were also evaluated.

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.

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.

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.

Expression of porcine CD163 on monocytes/macrophages correlates with permissiveness to African swine fever infection.

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.

A new epitope on swine CD5 molecule detected by monoclonal antibody 5F12/9.

This paper describes the production and characterization of a monoclonal antibody (MAb), 5F12/9, that recognizes a new epitope on porcine CD5. Conformation of its CD5 specificity was obtained by means of sequential immunoprecipitation and Western blot experiments in combination with anti-CD5 MAb 1H6/8, whereas cross-blocking experiments with both MAbs showed that they reacted with different epitopes.

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.