Analysis of chromosome damage for biodosimetry using imaging flow cytometry.

The dicentric chromosome assay (DCA), which involves counting the frequency of dicentric chromosomes in mitotic lymphocytes and converting it to a dose-estimation for ionizing radiation exposure, is considered to be the gold standard for radiation biodosimetry. Furthermore, for emergency response, the DCA has been adapted for triage by simplifying the scoring method [1]. With the development of new technologies such as the imaging flow cytometer, it may now be possible to adapt this microscope-based method to an automated cytometry method. This technology allows the sensitivity of microscopy to be maintained while adding the increased throughput of flow cytometry. A new protocol is being developed to adapt the DCA to the imaging cytometer in order to further increase the rapid determination of a biological dose. Peripheral blood mononuclear cells (PBMC) were isolated from ex vivo irradiated whole blood samples using a density gradient separation method and cultured with PHA and Colcemid. After 48h incubation, the chromosomes were isolated, stained for DNA content with propidium iodide (PI) and labelled with a centromere marker. Stained chromosomes were then analyzed on the ImageStream(×) (EMD-Millipore, Billerica, MA). Preliminary results indicate that individual chromosomes can be identified and mono- and dicentric chromosomes can be differentiated by imaging cytometry. A dose response curve was generated using this technology. The details of the method and the dose response curve are presented and compared to traditional microscope scoring. Imaging cytometry is a new technology which enables the rapid, automated analysis of fluorescently labelled chromosomes. Adapting the dicentric assay to this technology has the potential for high throughput analysis for mass casualty events.

Immunolocalization of the cellular adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule (PECAM), in human endometrium throughout the menstrual cycle.

In the present study the presence and distribution of cellular adhesion molecules involved in leukocyte binding were investigated in human endometrium. Endometrial biopsies (n = 45) were collected from women at all stages of normal menstrual cycles. Consecutive cryostat sections of endometrium were immunostained with monoclonal antibodies to intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule (PECAM) and haematoxylin and eosin. Primary antibody binding was visualized using a streptavidin-biotin system. Strong staining for PECAM was observed in endothelial cells of all vessel types and in focal areas of stroma including single cells, small clusters and larger aggregates of cells. At menstruation, however, almost the entire stroma stained for PECAM which was temporally related to a massive influx of leukocytes. ICAM-1 staining, which was consistently less intense than PECAM staining, was detected in vascular endothelial cells during the cycle, reaching a peak at menstruation. Unlike PECAM, ICAM-1 staining did not occur consistently across all vessel types. Stromal staining for ICAM-1 was rare except at menstruation, when almost the entire stroma showed positive staining for ICAM-1. No glandular or luminal epithelial staining was detected for either PECAM or ICAM-1. This study demonstrates that PECAM and ICAM-1 are expressed on endothelial cells of veins, arterioles and capillaries, and stromal cells within human endometrium.

Vascular response in a non-uterine site to implantation-stage embryos following interspecies transfers between the rat, mouse, and guinea-pig.

Pre-implantation-stage embryos from rats, mice, and guinea-pigs were transferred to a non-uterine site--the anterior chamber of the eye--of female recipients. All 9 combinations of transfers were performed: 3 allogeneic (intraspecies) transfers as controls, and 6 xenogeneic (interspecies) transfers. Implantation, as judged by extravasation from blood vessels of the iris or ciliary body occurred with success rates of 90.4% per transfer in the control rat group. 76.9% in the control mouse group, and 81.8% in the control guinea-pig group. Significantly reduced implantation rates occurred in the rat to guinea-pig (0%), mouse to rat (46.9%), mouse to guinea-pig (6.7%), and guinea-pig to rat (0%) groups compared to controls. Reductions, although not significant, also occurred in the other 2 groups: rat to mouse (77.8%), and guinea-pig to mouse (44.4%). These results together with some ultrastructural and light-microscopical observations suggest a degree of species specificity involved in the vascular response to the implanting embryo. We propose that the peri-implantation embryo produces a signal(s) which is to some extent species specific and which in the normal allogeneic situation is responsible for the early vascular effects seen at implantation in most eutherian mammals.

Vascular response in a non-uterine site to implantation-stage embryos in the rat and guinea-pig: in vivo and ultrastructural studies.

Preimplantation-stage embryos were transferred to the anterior eye chamber of recipient rats and guinea-pigs. After implantation had occurred the influence of the embryo on the iris vasculature was examined ultrastructurally. In both species, the earliest effect of embryonic implantation was an increased stromal oedema. Under increasing embryonic influence the vascular endothelial cells showed an increased number of projections into the vascular lumen, while in the rat, endothelial projections were also found pushing back into the basement membrane. In the rat, the endothelium became very irregular in thickness prior to complete disintegration and loss during more advanced stages of implantation. Rat embryonic trophoblast was found invading iris vasculature, particularly in areas where the iridial endothelium was partially or completely missing. Other cells in the iris, including the stroma, appeared to be less affected. In the guinea-pig, however, trophoblast cells appeared to be capable of invading the vasculature by displacing endothelial cells that still appeared morphologically normal.

Embryo implantation in the anterior chamber of the eye. Effects on uterine allografts and the microvasculature.

A model suitable for the in vivo study of embryo implantation in the presence or absence of uterine tissue has been developed. After transplantation of a uterine allograft to the anterior chamber of rat eyes, embryos were transferred to control, normally cycling, and pseudopregnant recipients. Implantation rates were 86%, 46% and 49%, respectively, for the three groups. Despite embryo growth rates being delayed by 3-4 days, histologic studies showed relatively normal egg cylinder stage embryos equivalent to those of day 8 of normal development. Uterine allografts varied greatly in appearance, from inert-looking pieces of connective tissue to recognizable uterine structures with an epithelium and a degree of decidual response. Reduced implantation rates in the presence of a uterine allograft may be due either to the uterine production of blastotoxic substances or the presence of a chronic inflammatory reaction resulting from the rejection of the allograft by the recipient. This model provides a unique system for continuous, noninvasive in vivo observations of the implantation process.