Effect of hyperthermia on murine cell surface histocompatibility antigens.

The effect of heat on the density of cell surface histocompatibility antigens was examined. Antigen density and distribution were determined by radioimmunoassay and flow cytometry after the binding of radioiodinated or fluoresceinated monoclonal antibody (anti-H-2Kk and anti-H-2Kb) to murine lymphoma cells in suspension cultures. Antibody binding was unaffected by temperatures between 37 degrees and 41 degrees following a 30-min heat exposure. At 42 degrees, some inhibition of binding was measurable. However, at 43 degrees, antibody binding was reduced by 30 to 50%, and a further 15 to 20% reduction was observed at 45 degrees. Flow cytometry showed that all cells were equally affected. There was no indication of the selection of a specific cell population. The temperature-dependent decrease in antibody binding was due to a decrease in receptor number and not to changes in the affinity. Measurement of the diffusion coefficient of the lipid probe N,N-dioctadecyl indocarbocyanine iodide showed that heat did not affect significantly the fluidity of the membrane lipids. Hyperthermic temperatures, therefore, have a direct effect on these membrane proteins.

A demonstration of human beta-2-microglobulin specific association with the surface of teleostean cells.

Purified human Beta-2 microglobulin (beta 2m) and a human beta 2m fluorochrome conjugate were used in exchange reactions to demonstrate that beta 2m associates with a teleostean cell surface protein. beta 2m exchange among brown bullhead, channel catfish, fathead minnow, and rainbow trout cells lines was detected by using either radioimmunoassay or flow cytometry. Evidence that beta 2m binds specifically with the surface of teleostean cells and possibly associates with an expressed class I MHC homologue is provided. Moreover, following exchange on brown bullhead cells, a coprecipitated protein of 45 kDa was observed following subsequent immunoprecipitation with the human beta 2m specific antibody B1.1G6. Given that beta 2m is a peripheral protein which has been shown to exchange with MHC expressing cells from different species, co-precipitation results suggest that the 45 kDa protein may represent a class I MHC homologue.

Phase equilibria in binary mixtures of phosphatidylcholine and cholesterol.

The paramagnetic resonance spectra of two spin-labels, 2,2,6,6-tetramethylpiperadinyl-1-oxy and a head-group spin-labeled phosphatidylethanolamine (L-alpha-dipalmitoylphosphatidyl-N-ethanolamine), have been used to study solid-liquid and liquid-liquid phase separations in binary mixtures of dimyristoylphosphatidylcholine and cholesterol. A quantitative analysis of these resonance spectra supports the view that at temperatures below theta m, the chain-melting temperature of the phospholipid, and at cholesterol mole fractions Xc less than 0.2, these mixtures consist of two phases, a solid phase of essentially pure dimyristoylphosphatidylcholine and a fluid phase having a mole fraction of cholesterol equal to 0.2. The spin-label data also provide evidence for fluid-fluid immiscibility in the bilayer membrane at temperatures above the chain melting transition temperature of dimyristoylphosphatidylcholine.

Cell analysis for hematopoietic stem/progenitor cell transplantation.

Cytometric analysis has become an important aspect in the quality control of cells in all phases of hematopoietic cell transplantation. In the stage of donor conditioning the counting of stem and progenitor cells is important and several reliable single platform tests for CD34+ cells have become available recently. It has been shown, that the count of certain subsets of CD34 may predict best time for harvesting stem cells better than just CD34. In many cases manipulation of the cell sample after collection from the donor is necessary before the cells are adequate for transplantation. Characterization of the resulting cell preparations requires reliable quantitative analysis of a variety of cell types like the enumeration of T-cells at the level of one in ten thousand for some allogeneic transplantations. It is discussed how these clinical requirements will need a refinement of cytometric procedures to achieve adequate clinical decisions.

The sensitive detection and quantitation of antibody to HCV by using a microsphere-based immunoassay and flow cytometry.

Antibody to HCV core and NS3 was quantified by using a microsphere immunoassay and flow cytometry. Antibody to core and NS3 was elevated in the 85 seropositive blood donors tested. The amount of either antibody varied over two logs although greater variation was seen with the antibody to NS3 than was seen with antibody to core. In three documented acute HCV cases, the microsphere assay detected antibody prior to antibody detection using the reference methods. Twenty donor samples were indeterminate by the reference methods: 45% of these were indeterminate with the microsphere assay while 25% were negative and 30% were positive. As compared to enzyme immunoassay the microsphere assay showed a 5-fold increase in sensitivity. The microsphere assay demonstrated increased sensitivity for the quantification of specific antibody to HCV core and NS3 and was useful in resolving a significant proportion of indeterminate samples.