The potential for archiving and reconstituting valuable strains of turkey (Meleagris gallopavo) using primordial germ cells.

Diseases such as avian influenza can destroy turkey flocks, potentially resulting in the loss of valuable or rare genetic material. Consequently, there is an urgent need to develop a means to archive such germplasm. Germline chimeras produced by intravascular transfer of primordial germ cells (PGC) have been reported in other avian species but not turkeys. This study examined the feasibility of both establishing an archive of frozen PGC, and producing germline chimeras by injecting the thawed PGC into host embryos. To meet these aims, the following experiments were performed: (1) PGC identification within turkey embryos; (2) development of an efficient method for isolation of turkey PGC; (3) demonstration that PGC can be cryopreserved, recovered, and retain viability; (4) reinjection into embryos and detection of injected PGC. Primordial germ cells were identified using periodic acid-Schiff reagent and the immunological marker OLP-1. Bloodstream PGC were isolated using Ficoll density gradient centrifugation with PGC recovery peaking at stages 13, 14, and 15 with 32 ± 4.9, 33 ± 6.4, and 26 ± 5.4 PGC recovered, respectively. Primordial germ cells were frozen using Dulbecco's modified Eagle medium, 20% fetal calf serum, and 10% dimethylsulfoxide and demonstrated 90 ± 1.7% viability after 3 mo frozen in liquid nitrogen. Freshly isolated and frozen thawed DiI- and Q-Tracker-labeled PGC repopulated stage 30 gonads after vascular transfer into ex ovo cultured embryos. The DiI-labeled cells repopulated gonads less frequently, with 36 ± 13.2% of gonads containing the DiI-labeled PGC, and 7 ± 3.8% of reinjected PGC reaching the gonads of positive embryos. The Q-tracker-labeled cells were detected more frequently in embryos, with 67 ± 21.1% having positive signals, and 44 ± 4.9% of reinjected Q-tracker-labeled PGC colonized the gonads of positive embryos. This study demonstrated the feasibility of using turkey PGC to archive turkey germplasm from different strains because frozen PGC reintroduced into host embryos can colonize the host gonads, suggesting the possibility of producing turkey germline chimeras.

Bone marrow-derived endothelial progenitor cells do not contribute significantly to new vessels during incisional wound healing.

OBJECTIVE: To assess the contribution of bone marrow (BM)-derived endothelial progenitor cells (EPCs) to the neovascularisation of cutaneous incisional wounds. METHODS: Lethally irradiated C57Bl/6 mice were transplanted with BM mononuclear cells from Tie2/lacZ mice, which constitutively overexpressed beta-galactosidase (beta-gal) in endothelial cells (ECs). Chimeras were wounded and the number of X-gal-stained (beta-gal(+)) BM-derived EPCs were calculated in histological wound sections. RESULTS: EPCs were measured in skin sections from unwounded BM transplant (BMT) mice, or at day 1 and 3 postwounding, at the level of 0.1 +/- 0.1 (mean +/- SEM) per skin/wound section. In day-5 to day-14 wounds, the number of EPCs increased gradually (1.3 +/- 0.5 at day 5 and 4.8 +/- 0.9 at day 10), peaking at day 14, when there was a significant increase in the number of EPCs per wound section (6.5 +/- 1.7) when compared to unwounded skin. Between days 14 and 18 postwounding, there was a rapid fall-off in the number of beta-gal(+) EPCs (0.8 +/- 0.5 at day 18) and numbers returned to baseline by day 21 (0.1 +/- 0.1). No evidence of vascular structures derived from BM-derived EPCs ("in situ" vasculogenesis) was observed and it was calculated that these cells contributed only 4.4% +/- 1.5% to total wound ECs at their peak. CONCLUSION: These findings indicate that the revascularization of dermal incisional wounds primarily occurs through angiogenesis because the low frequency and temporal expression of EPCs suggests that they do not make a significant contribution to the neovascularization process.

Human peritoneal adhesions show evidence of tissue remodeling and markers of angiogenesis.

PURPOSE: This study was designed to investigate the vascular structure and angiogenic activity of human peritoneal adhesions. METHODS: Adhesions were collected from patients undergoing laparotomy (n=32). Histologic features were documented and the distribution of mature and immature vascular markers were determined by immunolocalization and quantified by image analysis. The three-dimensional organization of blood vessels was investigated by confocal microscopy. Expression of vascular endothelial growth factor A, its receptor flk-1, and proliferating cell nuclear antigen were assessed by immunohistochemistry as indicators of angiogenic activity. RESULTS: Adhesions were found to be vascularized structures comprising bundles of collagen, interspersed with varying amounts of adipose tissue. Functional blood vessels expressed recognized vascular markers (vWF, CD34, alpha-SMA, and CD105) and formed a branching network similar to that of the peritoneum. Those adhesions expressing vascular endothelial growth factor A and its receptor showed significantly higher numbers of immature vessels as defined by expression of CD105. Omental adhesions (n=16) contained significantly more adipose tissue (P<0.05) and displayed a higher microvessel density (P<0.01) but lower cellularity (P<0.05) compared with nonomental adhesions (n=16). CONCLUSIONS: All adhesions contained functional blood vessels and most showed evidence of cell proliferation. The presence of vascular endothelial growth factor A and its receptor in human adhesions suggests ongoing angiogenic activity. This study demonstrates that adhesions are vascular structures with evidence of tissue remodeling and suggests potential for new prevention strategies involving antiangiogenic therapies.

The microcirculation in acute murine cutaneous incisional wounds shows a spatial and temporal variation in the functionality of vessels.

A mouse perfusion model using fluorescently labeled dextran has been developed to investigate the functionality of blood vessels during cutaneous wound healing. By immunostaining cryostat sections of perfused wounds with antibodies that identify vessels, we were able to assess their functionality. There was an increase in the proportion of CD31(+)-perfused vessels in all wound regions with time, although the vessels of the wound margins and superficial granulation tissue (GT) took the longest to become perfused. More than 50% of the latter vessels were not perfused at 10 days postwounding. This is consistent with the growth of functional vessels from the wound base proceeding to the more superficial GT. The CD34 marker was expressed by a subpopulation of CD31(+) vessels. However, in contrast to CD31(+) vessels, the functionality of CD34(+) vessels did not change significantly with time and 50-75% of CD34(+) vessels in the GT and wound margins were nonfunctional. This might be explained either by apoptosis of the CD34(+) vessels or the loss of the marker with time. This study has important implications for assays of wound-healing angiogenesis based on histology and immunohistochemical markers for vessels, because vessel functionality differs both spatially and temporally during wound healing.

A comparative study of the structure of human and murine greater omentum.

In humans, the greater omentum is a fatty peritoneal fold that extends from the greater curvature of the stomach to cover most abdominal organs. It performs many functions, which include acting as a reservoir of resident peritoneal inflammatory cells, a storage site for lipid, and a regulator of fluid exchange in and out of the peritoneal cavity. Most importantly, the omentum readily adheres to areas of inflammation and peritoneal damage, often leading to adhesion formation. Despite its clinical importance, the omentum remains an understudied organ, and discrepancies exist as to its exact morphology. This study uses a combination of phase contrast microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) to elucidate the structure of the greater omentum of both human and mouse and determine whether it possesses a typical surface mesothelial cell lining similar to other serosa. Results indicated that both human and murine omenta were of similar structure and composed of two distinct types of tissue, one adipose-rich and the other translucent and membranous. The adipose-rich regions were well-vascularised and covered by a continuous mesothelial cell layer except at the sites of milky spots. In contrast, translucent areas were poorly vascularised and contained numerous fenestrations of varying size. The possible function and developmental origin of these gaps is unclear; however, their role in promoting omental adhesion formation and in the successful use of omental graft material is discussed.

An in vitro model to evaluate cell adhesion to metals used in implantation shows significant differences between palladium and gold or platinum.

Adhesion of tissue cells to metallic implants is a major factor that is important for proper tissue integration. Adhesion of Swiss mouse 3T3 fibroblasts to gold, platinum and palladium surfaces was investigated. Immunofluorescence staining for the integrin subunits alphav and beta1 and the focal contact protein vinculin revealed that cells growing on gold and platinum expressed many focal contacts. In contrast, cells on palladium surfaces had reduced numbers of focal contacts shown by vinculin staining and failed to demonstrate expression of alphav and beta1 in focal contacts. Spread cell area was also significantly reduced on palladium than on other surfaces suggesting that cells on palladium were more weakly attached. This may be due to either a different molecular composition of focal contacts in cells grown on palladium surfaces or unusual microstructural properties of the palladium surface. This model is useful to evaluate adhesion of cells to different metal surfaces.

Latent TGF-beta1 activation by platelets.

Platelets are a major source of transforming growth factor-beta1 (TGF-beta1) in the circulation as they release latent growth factor in response to activation. We report here that human platelets, when stimulated with thrombin, activated a significant proportion of the latent TGF-beta released. Latent TGF-beta activation was independent of cytokine release, since activation was delayed compared to platelet degranulation. Activation occured in releasates and did not require the continuous presence of platelets. Classical mechanisms of latent TGF-beta activation were not involved, since activation was not affected by gene deletion and/or inhibitors of the known TGF-beta activators/co-factors, thrombospondin-1 (TSP-1), mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR), plasminogen/plasmin, or several other candidate proteases. In contrast, latent TGF-beta activation was significantly inhibited by the furin inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone and L-hexaarginine. We show that platelets contain a furin-like enzyme which is released upon platelet activation. We conclude that, following activation, platelets release and activate latent TGF-beta1 via mechanisms involving the release and activity of a furin-like proprotein convertase. This novel mechanism of latent TGF-beta activation might represent an important mediator and therapeutic target of platelet TGF-beta1 functions, for example, in early wound repair, fibrosis, or arteriosclerosis.