David J. Hurley

Numbers and percent of T lymphocytes in bovine peripheral blood during the periparturient period

To determine if periparturient immunosuppression in dairy cattle might be due to an alteration in total numbers of percent of T lymphocytes, we examined the numbers and percent of T lymphocyte subsets in peripheral blood from periparturient dairy cows, some of which received recombinant bovine granulocyte colony stimulating factor (rbG-CSF) during the study. Beginning 2 weeks preparatum through 4 weeks postpartum, peripheral blood mononuclear cells (PBMC) were collected and labeled with monoclonal antibodies to BoCD5, BoCD4, and BoCD8, and the percent of cells positive for each marker measured by flow cytometry. The percent of PBMC expressing BoCD5 (total T cells), and BoCD8 (T suppressor/cytotoxic cells) was not significantly different between the groups, or at different times before and after calving. The percent of PBMC expressing BoCD4 (T helper cells) was not significantly different between the groups, however, within both groups there was a higher percent of BoCD4+ cells after calving than during the prepartum period. In cows receiving rbG-CSF, total numbers of PBMC were significantly increased compared to controls during the postpartum treatment period.

An evaluation of the mononuclear cells eerived from bovine mammary gland dry secretions using leukocyte antigen specific monoclonal antibodies, light scattering properties and non-specific esterase staining

The distribution of mononuclear cells isolated from the bovine mammary gland during the nonlactating (dry) period was examined using monoclonal antibodies against leukocyte cell surface antigens, cellular light scattering properties, and the presence of nonspecific esterase. Most of the mononuclear cells isolated during the dry period were lymphocytes. T cells predominated until about 1 week prior to parturition. During the week prior to calving, the percentage of B cells increased until it approximated T cells. The ratio of CD4:CD8 cells was 2-3:1 for mammary gland T cells. This was similar to the ratio found in peripheral blood. At dry-off, about 12% of mammary mononuclear cells were macrophages. The macrophage percentage increased (to about 30%) at mid-dry and remained at this levels until parturition. PMNs were isolated with the mononuclear cells during the first 2 weeks dry and the week prior to calving. Three methods were used to identify mammary macrophages. Esterase staining (as an enzymatic method), forward angle/90 degrees light scatter (based on size and internal complexity), and MHC class II/forward angle light scatter (based on size and surface markers) were compared. Each method yielded similar specificity for macrophage identification. Non-adherent cell fractions, obtained by passage of the cells over Sephadex G-10 columns, were enriched in CD4 positive T cells, somewhat depleted of B cells, and depleted of macrophages and PMNs. Cells eluted from G-10 columns, with lidocaine, were mostly lymphocytes, but reflected the cells loaded onto the column.

Mitogenic activity of snake venom lectins.

Abstract. Five lactose‐inhibitable lectins have been isolated from snake venoms. These five share certain biochemical properties but are not identical (Gartner, Stocker & Williams, 1980; Gartner & Ogilvie, 1984). In this study the lectins were tested for their ability to stimulate lymphocytes to undergo DNA synthesis. We found that three of the lectins were comparable in mitogenic activity to the T cell lectin, concanavalin A (Con A). the mitogenic activity was blocked by lactose, a sugar which also blocks the haemagglutination activity of these lectins. Although mitogenic response appeared to be due to T cells, it depended on the presence of accessory cells in the culture. This requirement for macrophages could be replaced by the phorbol ester tumour promoter, 12‐o‐tetradecanoylphorbol‐13‐acetate (TPA).

Characterization of resting and phorbol ester or concanavalin A activated bovine lymph node cells with leukocyte specific monoclonal antibodies

Bovine lymph node cells (LNC) have been used as a model to study cell activation and proliferation. Because monoclonal antibodies to bovine lymphoid-specific surface antigens have only recently become available, these cells have not been previously characterized in regard to subpopulations. Furthermore, it was not known how expression of lymphoid differentiation antigens and subset proportionalities might change following different modes of activation of LNC. Therefore, the distribution of cell-surface differentiation antigens in unstimulated LNC as well as in LNC incubated with the mitogen concanavalin A (Con A) or the phorbol ester, phorbol dibutyrate (PDBU), was measured using a series of leukocyte-specific monoclonal antibodies and flow cytometry. Unstimulated LNC were found to have similar proportions of T cells, B cells (sIgM positive), and MHC Class II positive cells similar to bovine peripheral blood mononuclear leukocytes (reviewed by Baldwin et al., 1988a). Treatment of the LNC with PDBU or mitogenic doses of Con A induced changes in the expression of surface antigens consistent with the changes observed with human and mouse cells after similar activation. However, these two compounds did not cause identical effects. After treatment with PDBU, the percentage of cells expressing CD4 as well as the density of surface expression decreased. An increase in the percentage of cells expressing and/or density of surface expression of the pan T cell antigens CD2, CD5, CD6, MHC Class II and J5, a T cell activation antigen, also occurred. PDBU treatment also increased the percentage of CD8 positive cells. The change in CD6 following PDBU treatment has not been reported previously. Con A treatment led to a significant increase in the percentage of cells bearing CD8, CD6, MHC Class II and J5, but it had no effect on the percentages of cells positive for the other T cell markers CD5, CD4, or CD2. Because Con A is a complete mitogen and PDBU is not, the changes observed following Con A stimulation probably reflected an expansion of a particular subpopulation. In contrast, PDBU most likely modifies surface antigen expression directly. Neither treatment affected the B cell subpopulation.

Diffusion of a Small Molecule in the Aqueous Compartment of Mammalian Cells

Cell biologists, biochemists, biophysicists, and other life scientists who study the mammalian cell are in general concerned with the basic questions, What is the cell made of? How does it work? In order to answer these questions, scientists are continuing to develop new techniques and approaches which are revealing a variety of information. It is interesting that some of this information remains buried, only to be rediscovered a decade or so later as new or even old models of cell structure and function are proposed (see Conklin, 1940, for an historical review).

Culture conditions for blastogenic responses of bovine mammary mononuclear cells.

Several experimental parameters were examined to determine optimal conditions for proliferative responses of mammary mononuclear cells (MMC) obtained from six nonlactating dairy cows. These parameters were: pre-incubation of cells in medium prior to assay, mitogen concentration, assay incubation time, and type of culture medium. Response variables included viability of cells and the rate of proliferation as assessed by tritiated thymidine incorporation. Pre-incubation of cells in medium had no effect on the proliferative response of MMC. Whereas Concanavalin A (ConA; 3.3 or 6.6 micrograms/ml) and phytohemagglutinin (PHA; 1, 5, 10 micrograms/ml) did stimulate proliferation of MMC, the higher doses did not stimulate greater proliferation than the lower doses of mitogens. The greatest mitogenic response was obtained on days 2 and 3 of incubation. Proliferative responses were significantly higher at all mitogen levels tested in a 50-50 mixture of Rosewell Park Memorial Institute medium 1640 and Liebovitz-15 medium (RPMI/L-15) than in RPMI alone. Viability of MMC was also significantly higher in the RPMI/L-15 medium. To test whether the significant effect of media on blastogenesis was specific for mononuclear cells from the bovine mammary gland, peripheral blood lymphocytes (PBL) from four dairy cows were cultured with ConA and PHA in a mitogen assay in both RPMI and RPMI/L-15. Viability was measured on day of collection and on all culture days. PBL were stimulated equally in both media. PBL viability decreased significantly on day 1 in both RPMI and RPMI/L-15. These results suggest that the optimal culture conditions for blastogenic responses of mammary mononuclear cells and peripheral blood lymphocytes may differ.