Quantification of retinal pigment epithelial phenotypic variation using laser scanning cytometry.

PURPOSE: Quantifying phenotypic variation at the level of protein expression (variegation) within populations of retinal pigment epithelium (RPE) cells may be important in the study of pathologies associated with this variation. The lack of quantitative methods for examining single cells, however, and the variable presence of pigment and/or lipofuscin complicate this experimental goal. We have applied the technique of laser scanning cytometry (LSC) to paraffin sections of mouse and human eyes to evaluate the utility of LSC for these measurements. METHODS: Mouse eyes were perfusion fixed in 4% paraformaldehyde and embedded in paraffin. Postmortem human eyes were fixed and dissected to obtain a 9-mm punch, which was then embedded in paraffin. A laser scanning cytometer equipped with violet, argon, and helium-neon lasers and the detectors for blue, green, and long red were used to record the fluorescence of each individual cell at all three wavelengths. Raw data were recorded and processed using the WinCyte software. Individual nuclei were identified by the fluorescence of the 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Next, RPE cells were uniquely identified in the green channel using an anti-retinal pigment epithelium-specific protein 65 kDa (anti-RPE65) monoclonal antibody with an Alexa Fluor 488-labeled secondary antibody. Mn-superoxide dismutase (MnSOD) was quantified in the long-red channel using an anti-MnSOD antibody and an Alexa Fluor 647-labeled secondary antibody. MnSOD(+) and RPE65(+) cells exhibited peaks in the plot of fluorescence intensity versus cell number, which could be characterized by the mean fluorescence intensity (MFI), the coefficient of variation (CV), and the percentage of total RPE cells that were also labeled for MnSOD. RESULTS: RPE cells can be uniquely identified in human and mouse paraffin sections by immunolabeling with anti-RPE65 antibody. A second antigen, such as MnSOD, can then be probed only within this set of RPE. Results are plotted primarily with the population frequency diagram, which can be subdivided into multiple regions. The data collected for each region include the MFI, the CV, and the number of cells that are immunolabeled in that region. Background interference from pigment or autofluorescent material can be successfully overcome by elevating the concentrations of fluorescent secondary antibodies. In the human and mouse eyes, age-related changes in MFI, CV, and percent RPE cells immunolabeled for MnSOD were observed. CONCLUSIONS: The extent of the variability of gene expression in RPE cells at the protein level can be quantified by LSC. Relative changes in the MFI, the CV, and/or percentage of RPE cells double labeled for a second antigen quantify the changes observed. The analysis of these data also suggest whether the effects observed are related to local changes in transcription (alterations of CV) or major changes of protein expression (MFI), which are likely to be due to changes in the chromatin structure. The changes of these variables with age suggest that the observed age-related variegation is primarily due to changes in the chromatin structure in individual cells.

Bisdiamine inhibits extracellular matrix formation and cell proliferation of atrioventricular mesenchyme from developing chick heart valves.

Abnormalities of the cushion tissues lead to atrioventricular septal defects (AVSD) and truncus arteriosus (TA). Bisdiamine exposure in the embryo frequently causes AVSD and TA in the newborn chick, mouse, or rat. We studied the effects of bisdiamine on mesenchymal cells grown in aggregate culture isolated from the developing atrioventricular valves of the stage-36 chick embryo. Fibronectin extracellular matrix formation and cell proliferation in the aggregates were assessed in various media. Chick serum stimulated the cells to produce an extracellular matrix and to divide, and the inclusion of bisdiamine inhibited both responses. If we isolated an extracellular matrix from a monolayer of mesenchymal cells and added the sonicated matrix to the medium containing serum and bisdiamine, the matrix incorporated into the aggregates and the cells entered the mitotic cycle. Our previous work established that cells need to attach to an intact extracellular matrix to begin cell division. Thus, we suggest that bisdiamine inhibits the normal formation of the extracellular matrix, leading to reduced cell proliferation, but it does not affect matrix-cell interaction. The lack of cushion growth in situ may be the cause of AVSD or TA.

Fibroblast growth factor-2 stimulates embryonic cardiac mesenchymal cell proliferation.

The proliferation response of stage 36 chick atrioventricular valve mesenchymal cells to fibroblast growth factor-2 (FGF-2) was studied in the tissue-like environment of three-dimensional cell aggregates maintained in organ culture. The mitogenic effects of FGF-2 on mesenchymal tissue depended on the FGF-2-stimulated formation of a fibronectin-containing extracellular matrix. The matrix was absent in unstimulated aggregates, and co-localized with regions of actively proliferating cells in stimulated aggregates. Inhibition of fibronectin matrix formation by the inclusion of Arg-Gly-Asp-containing peptides, which compete with fibronectin for binding to the cell surface alpha 5 beta 1 integrin receptors, abolished the proliferation effects of FGF-2. Inhibition of sulfation of cell surface glycosaminoglycans by treatment with sodium chlorate significantly reduced both the formation of the fibronectin matrix and cell proliferation in response to FGF-2, suggesting an involvement of the low-affinity sulfated glycosaminoglycan FGF receptor system. Thus, the FGF-stimulated growth of embryonic atrioventricular valve mesenchyme in vitro involves the production of a fibronectin matrix. We suggest that the stimulation of the fibronectin matrix represents an essential element in growth factor signaling of mesenchymal tissue, with the matrix serving as an anchorage substratum for the proliferating cells.

Sepracell-MN for mononuclear cell separation from cattle: effect on mitogen-and antigen-induced lymphocyte blastogenesis.

Mononuclear leukocytes (MNC) were separated from heparinized and EDTA-treated whole bovine blood by centrifugation after mixing with a commercial colloidal silica preparation (Sepracell-MN (S-MN]. Cell yields and lymphocyte blast transformation (LBT) to pokeweed mitogen (PWM), phytohaemagglutinin (PHA), concanavalin A (Con A), and Brucella abortus antigens were tested against MNC obtained from heparinized whole blood using Ficoll-Hypaque (FH). Separation with S-MN was more rapid and less labor intensive than separation with FH. There was a higher average total yield of MNC but a lower percentage of monocytes in the FH- than in the S-MN-separated MNC. In mitogen-induced LBT assays, MNC responded comparably to each mitogen regardless of the separation technique or anticoagulant used, and a cell concentration effect was demonstrated. In general, FH-separated MNC responded greater to PWM than did S-MN/EDTA separated MNC, but S-MN/heparin separated MNC had the greatest LBT responses to PWM. Overall, S-MN/EDTA separated MNC had the greatest responses to PHA, and responses to Con A were variable among experiments with respect to the separation technique. In antigen-induced LBT assays, two B. abortus antigens were used: a heat-killed strain S1119 (HKA) and a gamma-irradiated strain 19 (gamma BA). The LBT responses of three steers vaccinated with live B. abortus strain 19 were compared with three nonvaccinated steers in three separate experiments. Using HKA, FH separation resulted in an overall greater LBT response for vaccinates than nonvaccinates and a greater differential between responses of vaccinates and nonvaccinates than did S-MN derived MNC regardless of the anticoagulant used. Using gamma BA, FH produced the most responsive MNC in one experiment and S-MN/heparin produced the most responsive MNC in the other. At the highest cell concentration tested, FH-separated MNC had the greatest LBT responses for vaccinated calves, but differences between S-MN- and FH-separated MNC responses were not significantly different (P greater than 0.05). In conclusion, S-MN is a rapid and simple technique for separation of MNC from bovine blood. The technique produces an adequate cell population for mitogen-induced LBT studies; however, FH-separated MNC were generally more responsive in the B. abortus-induced LBT assay.

Vaccination of cattle with chemically modified and unmodified salt-extractable proteins from Brucella abortus.

Beef heifers were vaccinated on Day 0 with either salt-extractable protein (CSP) or chemically modified CSP (dCSP) from Brucella abortus Strain 19 in Freund's complete adjuvant (FCA). Six weeks later, vaccination was repeated, and heifers received either the homologous or heterologous vaccine. Another group of heifers received only FCA and saline. Vaccinations with CSP or dCSP stimulated marked antibody responses to B. abortus, as detected by standard serologic tests, an enzyme-linked immunosorbent assay, or a quantitative fluorometric immunoassay. Twelve percent of the heifers were seropositive by the CARD test 1 year after vaccination. Vaccination stimulated an increased cell-mediated immune response as measured by lymphocyte blast transformation (LBT) to B. abortus antigens. Fifty-six weeks after the initial vaccination, the heifers were challenged intraconjunctivally with 1.9 X 10(7) colony-forming units of B. abortus strain 2308. Sixty to 83% of the heifers aborted in each group and 70-83% of the heifers were culture positive. There were no significant differences (P greater than 0.05) among groups with respect to the number of abortions or the number of culture-positive heifers. Antibody responses increased rapidly within 4 weeks after challenge. Overall, antibody responses were greater for heifers that aborted than for those that did not abort. These differences were significant (P less than 0.05) only as measured by the fluorometric procedure. The LBT responses appeared to be higher for vaccinates than for the control group, but these differences were not significant (P greater than 0.20). There was a significantly lower (P less than 0.05) LBT response to heat-killed B. abortus in those heifers that aborted compared to those that did not.

Development of the bursa of Fabricius in the partridge and pheasant.

Development and regression of the bursa of Fabricius was measured in chukar partridge (Alectoris chukar) and ring-necked pheasant (Phasianus colchicus) from hatch to 40 and 34 weeks of age, respectively. Testes weights were recorded for 8 to 34-week-old pheasants and 12 to 30-week-old chukars. Approximately 12 partridges and 12 pheasants of mixed sex were killed every other week and their bursae measured and weighed. Results indicated that chukar bursal weight peaked at about 10 weeks of age in both sexes and then immediately regressed. Chukar bursal weights were highly correlated with age and body weight from hatch to 11 weeks of age but not after 11 weeks. Male chukar bursal weights decreased more rapidly than those of females (P less than .05). In ring-necked pheasants, maximum body weights were attained at 26 and 20 weeks of age for males and females, respectively. Maximum pheasant bursal weights were attained at 10 and 12 weeks of age for males and females, respectively. Bursal weight declined immediately after maximum bursal weights were attained. Age appeared to be a more important factor than body weight with regards to bursal regression in both the chukar and pheasant.