Induction of surface antigen CD69 expression in T-lymphocytes following exposure to actinomycin D.

The expression of surface antigen CD69 in immune response cells is typically associated with the early stage(s) of cell activation, with maximal expression levels within 4 h of appropriate antigenic or mitogenic stimulation, and maintenance of these high expression levels for 18-24 h. The expression profiles of CD69 in human peripheral blood mononuclear cells (PBMC) cultured with actinomycin D prior to mitogenic stimulation were evaluated by direct immunofluorescence using flow cytometry. Pretreatment of PBMC suspensions with low, non-toxic levels of actinomycin D stimulated CD3+ T-lymphocytes to express CD69 in a concentration-dependent manner. Furthermore, CD4+ T-lymphocytes were the primary cells responding in this fashion. Secondary mitogenic stimulation following antibiotic treatment potentiated cellular CD69 expression in these assays. CD69 expression was profoundly suppressed with in vitro actinomycin D concentrations >/=1-2 microg/ml, presumably by interference with cellular transcription/translation mechanisms. Parallel thymidine incorporation assays indicated that actinomycin D effectively inhibited thymidine uptake in a concentration-dependent manner, with complete inhibition at >/=0.1 microg/ml. The evaluation of cell cycling dynamics following antibiotic treatment, with and without secondary mitogen stimulation, indicated no substantial changes in DNA synthesis over controls. The diversity of these responses suggests that expression of CD69 may not solely reflect mitogenic activation status but may, under some conditions, result from induced cellular stress.

Immunophenotypic characterization of tumor infiltrating lymphocytes and peripheral blood lymphocytes isolated from melanomatous and non-melanomatous Sinclair miniature swine.

Selectively-bred Sinclair miniature swine exhibit a high incidence of congenital malignant melanoma which either proves fatal (10-15% of tumor-bearing piglets) or spontaneously regresses with a biphasic immunological phenomenon (85-90%) and no recurrence of malignancy. Mononuclear leukocytes were isolated from cutaneous melanomas and peripheral blood specimens collected from melanomatous (tumor-bearing) Sinclair swine during second-phase regression, and from peripheral blood specimens collected from non-melanomatous (tumor-free) Sinclair swine and control Hanford swine. Leukocyte identities were determined with single- and dual-parameter indirect immunofluorescence assays via flow cytometry. Assays for the specific surface antigens CD45, CD2, CD4, CD8, CD1, MHC class II, and N1 were employed to develop immunophenotypic profiles within the gated lymphocyte clusters from each TIL and PBL suspension. Significantly more CD8+ T-lymphocytes were identified in TIL suspensions than in peripheral blood leukocyte (PBL) suspensions (P < and = 0.05), regardless of breed or tumor status. Conversely, PBL suspensions contained significantly higher percentages of CD4+ T-lymphocytes than the levels found in TIL suspensions (P < and = 0.05). Virtually all TIL were MHC class II+, whereas the percentages of PBL expressing this antigen were markedly lower (P < and = 0.05). The percentages of T-lymphocytes co-expressing CD4 and CD8, a normal subset unique to swine, were generally consistent in all TIL and PBL suspensions examined. The results of this study have firmly established the immunophenotypic identities of cells associated with the second-phase regression phenomenon of this melanoma and have identified specific variations in the leukocyte profiles of the respective TIL and PBL suspensions.

Instrument-dependent fluorochrome sensitivity in flow cytometric analyses.

Flow cytometry has become the preferred technique by which critical clinical evaluations are made such as CD4 counts and aneuploid analyses. Mounting concern has arisen over the numerous techniques, reagents, and different flow cytometric employed to determine these data. Several studies have documented significant differences in results when different flow cytometers are utilized to analyze the same sample. Fluorochrome-dependent instrument sensitivity also has been reported by numerous investigators. As more and more procedures are performed by cytometric analysis, light scatter and fluorescence limitations, which appear to be instrument dependent, demonstrate that not all flow cytometers have the same capabilities. Attempts were made to calculate molecules of equivalent soluble fluorochrome (MESF) values on nine different flow cytometers using fluorescein isothiocyanate (FITC) and R-phycoerythrin (R-PE) labeled microsphere reference standards produced by Flow Cytometry Standards Corporation (FCSC). Dramatic differences were observed in the ability of some cytometers to resolve these microspheres. The diminished resolution appeared to be instrument model and fluorochrome dependent. We propose that diminished fluorescence resolution in certain flow cytometers could be responsible for significant variability in clinical values reported from laboratories utilizing different flow cytometers.

The utilization of monoclonal antibodies in the diagnosis of unusual leukemias.

Development of monoclonal antibodies to lymphoid cells has allowed for simple methods for the classification of leukemias. Using the monoclonal antibodies, B1, B4, T1, T11, Ia, CALLA, My7, My9, and a polyclonal TdT reagent, we report a number of unusual phenotypes analyzed by flow cytometry. Two cases of mixed linkage leukemia are reported, one marked with anti-CALLA and My9, the other marked with T11 and Ia. Three rare cases of pediatric CLL are reported. Both were of B cell origin with chromosome transformation. These studies demonstrate the clinical importance of monoclonal antibodies in leukemia classification. The results also show that the so-called rare leukemias may not be as rare as previously thought.

Enhancement of mixed lymphocyte culture reactions between donor and recipient in B cell chronic leukemia using antibody-coated beads.

Matching of donors and recipients in cases of B cell chronic lymphocytic leukemia (CLL) is complicated by the fact that the cancer clone dominates the blood. A case of CLL was characterized for its antigenic phenotype using monoclonal antibodies. Iron beads were coated with the appropriate antibody and used to remove the cancer clone. A mixed lymphocyte culture (MLC) utilizing the purified cells as well as unpurified cells and sheep cell rosetted purified cells was performed with the donor. Using coated beads, the T cell population rose from 8% to 95% in one treatment versus 8% to 42% with sheep cells. MLC data were statistically better with bead-treated cells, and the results demonstrated the importance of this technique for transplantation matching.

The ethanol fractionation of mouse serum.

A method for the ethanol fractionation of normal mouse serum has been developed which is rapid and simple to perform. The method was shown to be effective in purification of IgG1, IgG2 and gammaM-gammaA globulins. Gel filtration and zone electrophoresis proved to be effective in separating the gammaM-gammaA globulin combination into their respective components. Immunoelectrophoresis was chosen to assess the homogeneity of the fractions and the results agree with those obtained by other workers.

Effect of gold on the immune response of mice.

Separate concurrent injection of organic gold compounds and antigen into mice resulted in immunoenhancement that could be measured by direct and indirect plaque-forming cells, rosette-forming cells, and serum antibody assays. Kinetics of the immune response showed variable effects through day 9 of the experiment. Studies with British anti-lewisite, a gold antagonist, showed that the gold must stay in the system 1 day to obtain immunoenhancement.

Antiglobulin factors in normal mouse serum reacting with enzyme-digested gamma globulin.

The presence of antiglobulin factors in normal mouse serum was studied. Mice were injected with human 0 Rh(+) erythrocytes and the resulting gamma globulins were digested with papain. The reaction was demonstrated by agglutinations of human 0 Rh (+) perythryocytes sensitized with the Fab fragments when treated with normal mouse serum. Studies suggested that there are two factors in mice, a 7S AND A 19S globulin form. Cross-reactivity between strains of mice was demonstrated.