The molecular organisation of bimolecular lipid membranes. The dielectric structure of the hydrophilic/hydrophobic interface.

Improvements to a previously described very low-frequency impedance-measuring technique have now allowed the characterisation of a third, electrically distinct, type of substructural region in phosphatidylcholine biomolecular lipid membranes. This region was found to have properties intermediate to those of the hydrophobic (hydrocarbon) layer and the regions containing the polar heads of the phosphatidylcholine molecules. Its properties are consistent with it being associated with the oxygen-rich carboxyl ester portions of the phosphatidylcholine molecules which lie at the hydrophilic/hydrophobic interface. We will refer to these regions in the membrane as the acetyl regions. The individual properties of the three distinct types of region in the phosphatidylcholine membranes were determined at KCl electrolyte concentrations of 1, 10, 100 and 1000 mM. It was found that with increasing KCl concentration: (a) The capacitance, CH, of the hydrophobic region increased slightly, indicating a decrease in the thickness of this region. (b) The conductance, GH, of this hydrophobic region increased by a factor of 20 in going from 1 to 1000 mM electrolyte. (c) The capacitance of the acetyl region was independent of KCl concentration although its conductance increased 5-fold over the range 1-1000 mM KCl. (d) The volume-specific electrical properties of the region containing the polar heads appeared to be essentially independent of KCl concentration. However, a change in thickness of these regions was observed which was consistent with the cholinephosphate dipole being oriented normal to the bilayer surface in 1 mM KCl and parallel to the surface in 1000 mM KCl external solutions.

Perturbations to lipid bilayers by spectroscopic probes as determined by dielectric measurements.

Dielectric measurements on lecithin/cholesterol bimolecular lipid membranes have indicated that the series of extrinsic fluorescent probe molecules, the n-(9-anthroyloxy) fatty acids, cause significant perturbation to the bilayer structure at concentrations equivalent to those used in fluorescence experiments (0.1 mol%). Perturbations were observed in the capacitance and conductance of the electrically distinct substructural regions of the bilayer that were consistent with the putative location of the probe molecules. Inclusion of stearic acid decreased the thickness of the hydrocarbon region of the membrane, presumably by expanding the average surface area per unit membrane mass, and also significantly disrupted the surface regions. The attachment of the anthroyloxy moiety to position 2 of a fatty acid accentuated both these effects. Attachment at position 12 had the reverse effect by increasing the volume of the hydrocarbon region without further disturbance of the surface organisation. The 9-positioned probe had an intermediate effect. The degree of perturbation by the 2-positioned probe was dependent on the probe concentration within the range (probe:lipid) 1:1000 to 1:10 000. The technique therefore detects perturbation of structure at probe levels which are lower than those commonly used in fluorescence-labelling experiments.

Reovirus infection enhances expression of class I MHC proteins on human beta-cell and rat RINm5F cell.

Viruses are implicated in the pathogenesis of beta-cell destruction in type I (insulin-dependent) diabetes. The aim of our study was to investigate whether reovirus 1 or reovirus 3, which are known to infect beta-cells and induce autoimmunity in susceptible mice, could alter the expression of the major histocompatibility complex (MHC) proteins by human beta-cells and rat insulinoma RINm5F cells. Forty-eight hours after infection of either human beta-cells or RINm5F cells with reovirus 1 or reovirus 3, cytopathic effects were noted. By flow-cytofluorometric analysis, infected RINm5F cells exhibited a seven- to eightfold increase in the surface expression of class I MHC proteins. Upregulation of class I MHC proteins on reovirus 3-infected RINm5F cells was inhibited by 80% after preexposure of the virus to reovirus 3 antiserum. When analyzed by double-indirect immunofluorescence microscopy, human beta-cells infected with reoviruses 1 or 3 also exhibited markedly increased levels of class I MHC proteins. Reovirus infection of human beta-cells or RINm5F cells was not accompanied by the induction of class II MHC proteins. These findings suggest that 1) in addition to direct cytopathic effects, reovirus infection may contribute to beta-cell destruction by increasing expression of class I MHC proteins and therefore reactivity with cytotoxic T-lymphocytes; and 2) some viruses may increase MHC protein expression independent of and before the action of cytokines (e.g., interferon-gamma and tumor necrosis factor) released by immunoinflammatory cells.

Expression of class I MHC proteins on RIN-m5F cells is increased by interferon-gamma and lymphokine-conditioned medium.

To examine whether products of the immune system interact with the pancreatic beta-cell, rat insulinoma cells (RIN-m5F line) were cultured in the presence of conditioned medium from concanavalin A-activated mouse spleen cells (CAS medium). Indirect immunofluorescence and flow cytometry revealed that after culture in CAS medium, RIN-m5F cells had an 8- to 10-fold increase in class I major histocompatibility complex (MHC) proteins, whereas class II MHC proteins remained undetectable, and the level of insulin and/or insulin-like growth factor 1 receptors was unchanged. The stimulation of class I MHC expression on RIN-m5F cells by CAS medium could be mimicked by recombinant interferon-gamma. Analysis of 125I-surface-labeled cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that in the presence of CAS medium, there was a major increase in the expression of proteins of 48,000, 32,000, and 12,000 Mr and a minor increase in proteins of 17,000 and 9,000 Mr. Precipitation with monoclonal antibody identified the 48,000- and 12,000-Mr proteins as the class I MHC protein and beta 2-microglobulin, respectively. The ability of lymphokine-conditioned medium to increase the expression of RIN-m5F cell surface proteins, including the class I MHC proteins, provides a potential mechanism for enhancing the immune-mediated destruction of the beta-cell.

Commercial high speed machines open new opportunities in high throughput flow cytometry (HTFC).

Two recent events have opened a new domain of flow cytometry applications which we term high throughput flow cytometry (HTFC). The release of a commercial high speed sorter in 1994 placed HTFC within the reach of anyone who could buy one of the new machines and not just the handful of advanced laboratories worldwide that had custom built their own high speed sorters. The advent in 1999 of HTFC analysis capabilities of 100000 cells/s marks the second stage in this enabling of HTFC. We describe the technical basis of HTFC. The commercial high speed sorters measure cells in dead-times three to six times shorter than conventional machines. They can sort with high yield and high purity at rates from 25000 to 60000 cells/s, depending on their settings, mainly by virtue of their use of high drop creation rates 100000 drops/s or more. Finally, one series can analyse the measured cells at rates exceeding these sort-rates and at least six times faster than conventional sorters could. The performance of the systems made by the three manufacturers can be readily assessed for single laser systems. Comparison becomes difficult for multiple beam machines, due to requirements for multi-beam sampling for each cell and due to the demands of fluorescence compensation between signals from one laser and between signals from two or three lasers. Applications are described in the field of rare cell analysis and isolation as well as from sorting of abundant cell populations.

Flow-cytometric determination of fluorescence ratios between differently stained particles is dependent on excitation intensity.

By use of a flow cytometer, the fluorescence of cells stained with hematoporphyrin derivative and the fluorescence of plastic beads stained with different dyes were analysed as a function of the intensity of the exciting laser light. The ratios of the fluorescence values of stained and unstained cells as well as of stained cells and beads were sensitively dependent on excitation intensities. As a consequence of this finding, the normalization of cellular fluorescence by use of reference particles needs to be made on a well-defined and reproduced intensity of the exciting laser light.

Uptake and retention of hematoporphyrin derivative in an in vivo/in vitro model of cerebral glioma.

Photoirradiation treatment depends on exposing tumors to a photosensitizer and light to achieve selective tumor kill. We evaluated the kinetics of uptake of a photosensitizer, hematoporphyrin derivative (HpD), in an animal model of cerebral glioma to ascertain the optimal time for photoirradiation therapy. Animal models of cerebral glioma were established by implanting cells from the rat C6 glioma cell line into rat brains or as xenografts in adult mouse brains. C6 cells (10(7] injected into the frontal lobe of adult Wistar rats produced intracranial tumors greater than 5 mm in diameter in 90% of the animals at 21 days. Tumors greater than 4 mm in diameter developed in adult mouse brains within 14 days after 10(6) cells were implanted into the frontal lobe. These two tumor models were used to investigate the localization of HpD. After HpD administration, its presence was detected in fresh, unfixed specimens by fluorescence emission after excitation with an ultraviolet lamp. Fluorescence was determined quantitatively by an image analysis method using an optical data digitometer. The fluorescence, which was highly localized selectively to the intracerebral tumor, was just detectable 5 minutes after an intravenous injection of HpD. Patchy, bright fluorescence was evident 4 hours after injection, and the tumor was uniformly fluorescent after 6 hours. A minimal dose of 0.5 mg of HpD per kg of body weight was necessary to produce detectable fluorescence, and the dose of HpD necessary to produce detectable fluorescence was 4 mg/kg of body weight. The intracarotid route of administration was unsatisfactory because seizures were induced, and intrathecal injection did not produce significant fluorescence in the tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Future clinical role for flow cytometry.

It appears that there is an essential, not just supportive, role for flow cytometry in the clinical context, particularly in providing early information in clinical oncology. High flow rate enumeration and sorting of rare cells, combined with microscopy, offer immediate benefits in the clinical processing of at least some cancers. These benefits would be in diagnosis (perhaps very early detection of metastatic cells in the present prediagnostic phase of solid tumor growth), monitoring, prognosis, and therapy. Importantly, flow cytometric measures can be implemented immediately, and measurement times are short. The value of high flow rate operation of existing facilities in clinical, "supportive" flow cytometry should be better appreciated, if only because shorter measurement times and on-line analysis would make the existing facilities more cost effective: higher throughput for the same overheads. Finally, the wisdom of employing nonsorting cytometers for clinical use should be strongly questioned. Thus, what future impact will the application of flow sorting have in clinical fields old and new, e.g., in bacterial infection measurements in peripheral blood? In particular, nonsorting machines will be unable to adopt the "essential" clinical role I have proposed here.

Calibration of flow cytometric fluorescence standards using the isoparametric analysis of ligand binding.

To extract ligand-cell binding parameters from flow cytometric fluorescence data, one needs a method of converting the measured cellular fluorescence to the actual number of bound molecules producing that fluorescence. Bead standards containing a known number of fluorophores do not necessarily provide the correct conversion factor. We therefore present a method for calibrating flow cytometric bead standards. The technique uses the isoparametric analysis (Chatelier et al: EMBO J 5:1181-1186, 1986) to construct a plot of fluorescence per cell versus the number of bound ligands per cell, thus allowing a direct comparison of the quantum yields of the bead-associated fluorophore with that of the cell-bound fluorophore. The potential of this analysis is demonstrated by contrasting the fluorescent brightness of fluorescein isothiocyanate associated with thymocyte nuclei and synthetic polymer beads with that of fluoresceinated epidermal growth factor bound to A431 cells. When the standards are improperly calibrated, then the number of ligand-binding sites is incorrectly reported, and the derived Scatchard plot may exhibit an apparent positive or negative cooperativity as well as a strong dependence on cell concentration.

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry.