Long term culture and characterization of chicken primordial germ cells

Poster (2012, November)

Avian primordial germ cells (PGCs), can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species ... [more ▼]

Avian primordial germ cells (PGCs), can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species. We have developed original methods that allow long term (20 month) expansion of primary cultures of undifferentiated PGCs and their efficient cryopreservation. Blood samples were collected from stage 13-18 embryos, pooled, deposited in cell culture inserts and co-cultivated in the presence of irradiated BRL cells. This physically separated co-culture system along with selective culture medium promoted emergence, selection and proliferation of undifferentiated PGCs lines. Overall, 35% of blood samples gave rise to PGCs cell lines originating from three commercial layer breeds and two Belgian endangered breeds. PGCs lines were first characterised for the expression of the stem cells and PGCs characteristic marker SSEA-1 by FACS (expression rate: 90-99%). RT-PCR confirmed expression of germ-line specific markers (CVH, CDH, DAZL), pluripotency markers (cPouV, cSox2, cNanog), telomerase and CXCR4 receptor. In addition, by means of a quantitative PCR amplification of a chromosome W specific sequence, we demonstrated a drift of all our lines towards the male sex (WL), while they were initially isolated from pooled blood samples with statistically equivalent numbers of male and female embryos (35 females: 29 males). PGCs were subsequently efficiently cryopreserved by slow freezing or by a newly developed vitrification method. Labelled PGCs from 10 lines were injected in recipient embryos. Colonization of the genital ridges confirmed that PGCs retain their gonadal migratory ability, both after long-term culture (min 3, max 20 month) and after cryopreservation. [less ▲]

The three-dimensional reconstruction of the innervation pattern in the lymphoid compartment of the ovine pharyngeal tonsil highlighted a possible way of neuro-invasion by the scrapie agent.

Conference (2012, October 19)

Neuroimmune connections in ovine pharyngeal tonsil: potential site for prion neuroinvasion

in Cell & Tissue Research (2012)

Recent studies have proved the possible implication of nasal associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of this neuroinvasion are still being ... [more ▼]

Recent studies have proved the possible implication of nasal associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of this neuroinvasion are still being debated. To determine the potential sites for prion neuroinvasion inside the ovine pharyngeal tonsil, the topography of neurofilaments heavy (200 kDa) (NFH), neurofilaments light (70 kDa) (NFL) and glial fibrillar acidic protein (GFAP) was semi-quantitatively analysed inside the different compartments of the tonsil. The results showed that the most innervated areas were the interfollicular area and the connective tissue located beneath the respiratory epithelium. Even if the germinal centre of the lymphoid follicles was poorly innervated, the existence of rare follicular dendritic cell-nerve synapses inside the germinal centre indicates that this mechanism of neuroinvasion is possible but unlikely to be unique. The host PRNP genotype did not influence the pattern of innervation in these different tonsil compartments, unlike age: an increase of nerve endings in a zone of high trafficking cells beneath the respiratory epithelium occurred with ageing. A minimal age-related increase of innervation inside the lymphoid follicles was also observed. An increase in nerve fibre density around the lymphoid follicles, in an area rich in mobile cells able to transport PrPd, could ensure a more efficient infectivity, not in the early phase but in the advanced phase of lymphoinvasion after amplification of PrPd, or could act as direct site of entry during neuroinvasion. [less ▲]

Features of follicular dendritic cells in ovine pharyngeal tonsil: An in vivo and in vitro study in the context of scrapie pathogenesis

in Veterinary Immunology and Immunopathology (2011), 141

Although the alimentary tract has been suggested as the most likely portal of entry in natural scrapie, a growing amount of data indicates that the respiratory system and more specifically the pharyngeal ... [more ▼]

Although the alimentary tract has been suggested as the most likely portal of entry in natural scrapie, a growing amount of data indicates that the respiratory system and more specifically the pharyngeal tonsils serve as a natural portal of entry for scrapie. This study describes for the first time the broad cell populations in the lymphoid compartment of pharyngeal tonsils and more specifically inside the lymphoid follicles where the scrapie agent accumulates during the period of latency. Follicular dendritic cells (FDCs), stromal cells located in the light zone of the germinal centre of lymphoid follicles, seem to be the principal causal factor in the accumulation of the infectious agent in transmissible spongiform encephalopathy (TSE) diseases. Knowing that efficient lymphoreticular prion propagation requires PrPc expression, we analysed the expression of PrPc with the mouse monoclonal antibody Pri 909 both in situ and on FDC-cluster-enriched cell suspensions. In situ, a positive staining was observed in the germinal centre of pharyngeal lymph follicles. The germinal centre labelling was due to the presence of a follicular dendritic network as revealed after immunogold staining of isolated FDC clusters. Our results suggest that the pharyngeal lymphoreticular system and more specifically PrPc expressing follicular dendritic cells could serve as a prion “reservoir” during the latency phase, thus playing a key role during the scrapie lymphoinvasion. [less ▲]

Morphology of the digital sheath in horses

in Anatomia, Histologia, Embryologia (2010, August), 39(4), 288

Introduction: The digital sheath is located in the palmar (plantar) face of the distal limb. It consists of a synovium, divided into intimate and vascularised supportive layers, and ligaments that ... [more ▼]

Introduction: The digital sheath is located in the palmar (plantar) face of the distal limb. It consists of a synovium, divided into intimate and vascularised supportive layers, and ligaments that strengthen the tendon sheath in its palmar (plantar) face. The synovium surrounds the digital flexor tendons during their passage within the sheath. The synovium fluid, produced by the filtering of the blood and by the intima cells, allows lubrification within the sheath. Lameness, originating from digital sheath pathology is not rare, and it is important, for any equine practitioner, to know its normal morphology. The aim of this study is to precise the morphology of the digital sheath, particularly its synovium because the literature is not unanimous. Methods: The digitals tips of 7 "sound" horses were collected. Samples were taken within the 3 annular ligaments (Proximal, Digital Proximal, Digital Distal). The digital flexor tendons were sampled with the visceral sheath of the synovial membrane at the level of the proximal sesamoïd bones and just proximal to the medium scutum. All these samples were embedded in tissue-tek and freezed for cryosectioning. The proximal and distal recessus of the synovial membrane were also sampled, fixed in formalin and embedded in paraffin. Sections were stained with haematoxylin/eosin. One sample of the synovial membrane was taken for electronic microscopy and embedded in? Results: Annular ligaments are composed of dense connective tissue but are transversal strengthening of the fascia rather than true ligaments. The digital flexor tendons showed the typical organisation of the collagen network. Type III fibrocytes were observed within the deep digital flexor tendon. The synovial membrane showed two layers: an intima (with visceral and parietal sheath) with fibroblast-like cells and macrophagic cells, and a sub-intima composed of fibrous tissue that was sometime so thickened that it was difficult to cut! The recessus contained large synovial fringe with adipose tissue. Conclusion: This study permitted to precise the morphology of the digital sheath in horse which may help to better understand the pathological changes. [less ▲]

Neuroimmune contact between peripheral nervous system and follicular dendritic cells in ovine lymph organs

Poster (2008, October)

Oral scrapie infection modifies the homeostasis of Peyer's patches' dendritic cells

in Histochemistry & Cell Biology (2007), 128(3), 243-251

In transmitted prion diseases the immune system supports the replication and the propagation of the pathogenic agent (PrPSc). DCs, which are mobile cells present in large numbers within lymph organs, are ... [more ▼]

In transmitted prion diseases the immune system supports the replication and the propagation of the pathogenic agent (PrPSc). DCs, which are mobile cells present in large numbers within lymph organs, are suspected to carry prions through the lymphoid system and to transfer them towards the peripheral nervous system. In this study, C57Bl/6 mice were orally inoculated with PrPSc (scrapie strain 139A) and sacrificed at the preclinical stages of the disease. Immunolabelled cryosections of Peyer's patches were analysed by confocal microscopy. Membrane prion protein expression was studied by flow cytometry. In Peyer's patches (PP), dissected at day one and day 105 after oral exposure to scrapie, we observed an increased population of DCs localised in the follicular-associated epithelium. On day 105, PrPSc was found in the follicles inside the PP of prion-infected mice. A subset of Peyer's patches DCs, which did not express cellular prion protein on their surface in non-infected mice conditions, was prion-positive in scrapie conditions. Within Peyer's patches oral scrapie exposure thus induced modifications of the homeostasis of DCs at the preclinical stages of the disease. These results give new arguments in favour of the implication of DCs in prion diseases. [less ▲]

Distribution of nerve fibres in bovine and human mucosal associated lymphoid tissues

Poster (2006, October)

Prion cell tropism varies significantly among animal species, depending on both the agent strain and host-specific factors. For example, prions show high lymphotropism in scrapie infected sheep and vCJD ... [more ▼]

Prion cell tropism varies significantly among animal species, depending on both the agent strain and host-specific factors. For example, prions show high lymphotropism in scrapie infected sheep and vCJD, but little, if any, in sCJD or BSE. In particular, the BSE strain is associated with significant PrP-res accumulation in tonsils, spleen and appendix in humans, whereas it is largely confined to the nervous system in infected cattle. Therefore, at least in the case of BSE and vCJD, it appears that host properties can influence the accumulation of the infectious agent in lymphoid organs. Mature FDC play an important role in prion pathogenesis, since neuroinvasion following peripheral challenge is significantly impaired in their absence. The proximity between these FDC and sympathetic nerve endings is known to affect the speed of prion neuroinvasion. In this study, we analysed the mucosal innervation and the interface between nerve fibres and FDC in bovine and human tonsils and in ileal and jejunal bovine Peyer’s patches using a panel of antibodies observed by confocal microscopy. Since differences in the innervation of lymphoid organs depending on age have been reported, we analysed three categories of bovine ages (new born calves, calves less than 12 months old and bovines older than 24 months) and two categories of human ages (patients less than 5 years old and patients older than 25 years). In both species, hypothetical ways of innervation by-passing germinal centre could be postulated: nerve fibres are widely distributed in antigens/cells traffic area (the lamina propria, the interfollicular zone, the suprafollicular dome in Peyer’s patches and the lymphoepithelial area in tonsils). We pointed out that, only in ileal and jejunal Peyer’s patches and in tonsils of bovines older than 24 months, nerve fibres are observed to be in contact with FDC. In contrast, in human tonsils, no nerve fibres established contact with FDC, whatever the age. Thus, innervation of germinal centres can be said to be an age-dependent dynamic process in bovines and a weak innervation of the secondary lymphoid organs could thus be a rate-limiting step to neuroinvasion in humans. This variation could influence the way of neuroinvasion and thus, the differences of susceptibility of bovines and humans to the BSE agent. [less ▲]

Implication of Peyer’s patches dendritic cells in prion diseases.

Poster (2005, June)

Cellular and nervous environment of mouse mesenteric lymph node germinal centres

Conference (2005, April)