New tools in cytometry.

Cytometry aims to analyze cells, of any type, using dedicated instruments. The quantitative aspect makes flow cytometry (FCM) a good complementary tool for morphology. Most of the identification tools are based on immunostaining of cell structure details and more and more tools are available in terms of specificities and labels. FCM is under exponential development thanks to technical, immunological and data analysis progresses. Actual generations are now routinely using 6 to 10 simultaneous immuno-labeling on 20 to 100,000 cells, at high speed and short sample preparation and can easily detect rare events at frequency below 10-4 cells. Data interpretation is complex and requires expertise. Mathematical tools are available to support analysis and classification of cells based. Cells from tissues can also be analyzed by FCM after mechanical and or enzymatic separation, but in situ cells can also be analyzed with the help of cytometry. Very new instruments bring spectral analysis, image in flow and mass spectrometry. Medical applications are very broad, notably in hemopathies, immunology, solid tumors, but also microbiology, toxicology, drug discovery, food and environmental industry. But, the limit of FCM is its dependence on operator from sample preparation, instrument settings up to data analysis and a strong effort is now under progress for standardization and constitution of international data bank for references and education.

Interleukin-1 receptor antagonist in normal and psoriatic epidermis.

The objective of these studies was to characterize the IL-1 inhibitory activity present in normal and psoriatic epidermis from clinically stable lesions. Fractionation of normal epidermal cytosol on a molecular sizing column failed to reveal the presence of IL-1 inhibitory bioactivity. However, specific ELISAs indicated that both the IL-1 receptor antagonist (IL-1ra) and IL-1 alpha were present in overlapping peaks. Further fractionation of the normal epidermal cytosol by anion exchange chromatography separated these two molecules, revealing the IL-1 inhibitory bioactivity of the IL-1ra molecule. Similar studies on psoriatic epidermal cytosol indicated the presence of IL-1 inhibitory bioactivity and IL-1ra protein. The IL-1 inhibitory bioactivity of both normal and psoriatic cytosol was neutralized by a mAb specific for IL-1ra. The ratio of IL-1ra to IL-1 alpha proteins was significantly increased in involved psoriatic skin compared with normal skin. By Western blot analysis this IL-1ra was approximately 20 kD, slightly larger than monocyte-derived IL-1ra and equivalent to an intracellular variant of IL-1ra expressed by keratinocytes. Polymerase chain reaction indicated the presence of mRNA for both forms of IL-1ra in normal epidermis, with both forms increased in psoriatic-involved skin. Immunofluorescence studies revealed the IL-1ra protein to be concentrated in the stratum granulosum of normal skin and in the basal-midbasal layers of psoriatic epidermis. These results suggest that the balance between intracellular IL-1ra and IL-1 alpha may be an important influence on keratinocyte growth and/or differentiation, as well as on the inflammatory potential of IL-1 in injured skin.

Microbicidal activity measured by flow cytometry: Optimization and standardization for detection of primary and functional deficiencies.

Microbicidal activity is related to the production of reactive oxygen species (ROS) that can be measured by flow-cytometry using rhodamine 123 (R123). Few assays have been proposed to measure ROS production, usually on heparinized samples but none of them is standardized. Here we propose to improve the test by selecting polymorphonuclears (PMN) and monocytes, labelled and activated in one step to keep the test short, and to standardize the process even between different systems (i.e. Navios™ and FACSCanto™) using fluorescence intensity target setting ("FITS"). We applied this test on 15 patients without inflammation, 19 patients from an intensive care unit (ICU) and 11 healthy volunteers. RESULTS: Provided calcium restitution, we show that the test can be performed on EDTA that is a better sample preservative. The results were highly correlated between instruments (r2=0.898). PMN CD16 (and not CD14) expression was altered under stimulation with E. coli (MdFI=239.3±93.5) or PMA (139.7±76.8) as compared to resting sample (307.6±145.1). RH123 was strongly and homogeneously induced by PMA (14.2±6.6) and more heterogeneously by E. coli (MdFI 21.9±23.4) as compared to unstimulated PNN (0.9±1.3, p<0.0001). The test is useful not only for genetic disorders but also for secondary deficiencies as observed in ICU (E. coli RH123 MFI=10.5±11.1 patients vs 30.1±26.5 in healthy donors). In ICU, CD16 expression was already altered on unstimulated samples (MdFI=197.4±131.2 vs 418, 2±81.3 in healthy donors; p≤0.0001). Bacterial stimulation was dependent of the complement that partly explains deficiency to bacterial stimulus in ICU patients.

Combination chemotherapy of L1210 leukemia with 1-beta-D-arabinofuranosylcytosine and 5-azacytidine.

1-beta-D-Arabinofuranosylcytosine (cytarabine; ara-C) and 5-azacytidine (5-azaCR), cytosine nucleoside antimetabolites with different mechanisms of action, are both effective in the treatment of human leukemia, and the clinical use of these two agents in combination has been suggested. We have studied the therapeutic effect in L1210 leukemic mice of single i.p. doses of ara-C and 5-azaCR in combination. Therapeutic effects observed depended markedly on the sequence and time interval between the doses of each agent. Antagonism was observed when both agents were administered simultaneously. The optimal therapeutic effect was observed when 5-azaCR was administered after ara-C at a time when tumor DNA synthesis had maximally recovered after the ara-C dose. The dose-interval effect and correlation with recovery of DNA synthesis capacity were also observed in studies in vitro in which the survival of L1210 cells in culture was examined. ara-C was shown to inhibit the incorporation of [4-14C]-5-azaCR-derived radioactivity into DNA of L1210 cells in culture, and the therapeutic effects observed are interpreted in terms of these latter results and the mechanisms of action of the two agents.

Etodolac, aspirin, and gastrointestinal microbleeding.

The effects of etodolac, a new nonsteroidal anti-inflammatory drug, on gastrointestinal (GI) microbleeding were quantitatively assessed in two studies in healthy adult men. The first was a two-group, open-label, parallel comparison of etodolac, 600 mg/day, aspirin, 2600 mg/day, and placebo in 20 subjects; the second was a four-group, double-blind, parallel comparison of etodolac, 600, 800, and 1200 mg/day, aspirin, 2600 mg/day, and placebo in 41 subjects. Subjects in both studies received a single-blind placebo on days 1 through 7, either etodolac or aspirin on days 8 through 14, and a single-blind placebo on days 15 through 19. GI blood loss (milliliters per day) was estimated by the radiolabeled (51Cr) erythrocyte method and was based on daily radioactivity counts of stool specimens and regression-estimated daily blood radioactivity. Etodolac, 600 mg/day, induced no significant GI blood loss at any time during the experiments, nor was there significant blood loss after 800 and 1200 mg/day in experiment 2. Blood loss was noted after aspirin in both.

Sandwich ELISA formats designed to detect 17 kDa IL-1 beta significantly underestimate 35 kDa IL-1 beta.

The IL-1 beta precursor (proIL-1 beta) represents a significant component of total IL-1 beta production in certain cell types such as keratinocytes, fibroblasts and alveolar macrophages. It has been presumed that immunodetection systems for the mature 17 kDa IL-1 beta can be used interchangeably for the 35 kDa intracellular proIL-1 beta. However, during attempts to purify alveolar macrophage proIL-1 beta, we found that conventional enzyme-linked immunoassays (ELISAs) (using antibodies directed against the 17 kDa mature IL-1 beta) underestimated the amounts of 35 kDa proIL-1 beta by at least ten-fold compared to detection by Western blot techniques. This difference was due to the fact that ELISAs, with an antigen capture format (i.e., that use more than one epitope), can more readily see these distinct epitopes on mature or partially processed IL-1 beta than on the proIL-1 beta molecule. This problem does not occur with the Western blot technique, either because only one antibody is needed and hence there is no stearic blockade of a second epitope or because it denatures 35 kDa proIL-1 beta during the immobilization step, presumably better exposing epitopes as expressed on mature 17 kDa IL-1 beta. The problem with the ELISA can be partially corrected by proteolytic removal of the aminoterminus of 35 kDa proIL-1 beta with neutrophil elastase. More accurate determinations of proIL-1 beta by ELISA can be made by using 35 kDa proIL-1 beta as the reference standard (when the 35 kDa proIL-1 beta is free of molecular weight IL-1 beta). These data suggest that there are conformational differences between the carboxyterminus of 35 kDa proIL-1 beta and mature 17 kDa IL-1 beta which may affect immunodetection when using antibodies directed against mature 17 kDa IL-1 beta.

ara-Cytidine acylates. Use of drug design predictors in structure-activity relationship correlation.

This manuscript if one of a series of investigations into modifying the pharmacologic properties of the antitumor, antiviral, and immunosuppressive nucleoside ara-cytidine (cytarabine, Cytosar). The present paper summarizes our studies on depot ester derivatives of the nucleoside. We are able to predict with reasonable accuracy the biological activity as measured by increased life span in the L1210 leukemic mouse from a combination of two predictor variables: (1) the solubility of the ester in water and (2) its rate of hydrolysis by the mixed esterase system of animal plasma. We have tried unsuccessfully to correlate enzymatic hydrolysis rates with an alkaline hydrolysis model. Calculated Hansch partition (p) values had a correlation of r equal to 0.86 with water solubility. These p values had no additional predictive value. Based on our results, two esters were selected for clinical trial in cancer and rheumatoid arthritis.

A human leukocyte antigen identified by a monoclonal antibody.

Leukocyte antigens of approximate molecular weight 200,000 daltons have been described in mouse, rat, and man. We describe here the reactivities of a monoclonal antibody, GAP 8.3, which identified such an antigen on human leukocytes. We found the leukocyte antigen H-T200 on T and B lymphocytes, granulocytes, monocytes, and platelets, but not on erythrocytes or nonhematopoetically derived cells. Resting and activated T cells had more antigen on their surfaces than did resting B lymphocytes and EBV-transformed B cells, respectively. The leukocyte antigen was detected on approximately 75% of bone marrow cells; cells of the erythroid series comprised the negative population. The GAP 8.3 antibody and its F(ab')2 fragments had no effect on in vitro stimulation of peripheral blood cells by mitogens or allogeneic cells. Antigen isolated from T cell lines had a higher electrophoretic mobility than did antigen from B cell lines; antigen from the myeloid line U937 comigrated with that from B cell lines. In addition, we detected very small but reproducible differences in the electrophoretic mobility of the antigen on two T cell lines.

Ferrioxamine as a magnetic resonance contrast agent. Preclinical studies and phase I and II human clinical trials.

The preclinical and clinical trial experience with ferrioxamine (S-FDF; Salutar, Inc.) as a contrast agent for magnetic resonance imaging (MRI) is summarized. The results in 44 patients or subjects show that the drug is safe and well tolerated when given intravenously. In certain conditions, early results show that the use of this contrast agent provides more information than can be obtained with MRI alone.

Dual processing of two-dimensional exchange data in magic angle spinning NMR of solids.

We discuss procedures for processing data in rotor-synchronized two-dimensional magic angle spinning (2D MAS) NMR exchange measurements for both structural and dynamical studies. We show, both mathematically and experimentally, that there are two distinct data processing procedures that lead to 2D MAS exchange spectra with purely absorptive crosspeaks. One procedure is that described previously by Hagemeyer, Schmidt-Rohr, and Spiess (HSS). The other procedure is related, but different, and leads to crosspeak intensities given by the formulae of Herzfeld, Roberts, and Griffin (HRG). In 2D MAS exchange experiments on doubly (13)C-labeled l-alanylglycylglycine, we demonstrate that the HSS and HRG crosspeak intensities can be extracted separately from the same data set and contain independent information. Processing and analysis of 2D MAS exchange data with both the HSS and the HRG procedures may enhance utilization of the information content of 2D MAS exchange measurements.

Ethylnitrosourea treatment increases lectin binding to mouse germ cells.

Germ cell toxicity was assessed by investigating the binding of FITC-labeled lectins to mouse testis cells before and 18 days after treatment with ethylnitrosourea (ENU). Flow cytometry of testis cells dual-labeled with FITC-lectin plus the DNA stain, propidium iodide, allowed analysis of haploid (1C), diploid (2C), and dividing (4C) cell populations. Soybean agglutinin, wheat germ agglutinin, concanavalin A and Limax flavus agglutinin bound to normal mouse testis cells containing 1C, 2C or 4C DNA. Asparagus pea lectin and Bandeireae simplicifolia I isolectin B4 did not. ENU treatment reduced the number of testis cells and increased lectin binding, particularly of those lectins which bound to untreated cells.

Monoclonal antibody to HLA-A3.

The monoclonal antibody GAP A3 detects the HLA allospecificity A3. Reactivity of the monoclonal was in exact concordance with the presence of the A3 antigen as defined by conventional alloantisera on a panel of 59 cells from individuals with well-characterized HLA antigens. Reactivity with GAP A3 segregated with HLA-A3 in a family where three of eight siblings inherited the paternal A3 antigen. GAP A3 precipitated appropriate 44,000- and 12,000-dalton bands on SDS-polyacrylamide gels under reducing conditions from an HLA-A3-positive, but not an HLA-A3-negative B lymphoblastoid cell line. Thus, by serological, familial, and biochemical criteria, GAP A3 defines the allospecificity HLA-A3.

Comparison of fecal blood loss after use of aspirin and suprofen.

The effects of aspirin and suprofen on gastrointestinal blood loss were compared in a double-blind, crossover study of 20 healthy male subjects. Fecal blood loss was measured by 51Cr-labeled red cells. Subjects treated with aspirin (2,600 mg/day) experienced a mean fecal blood loss of 4.2 ml/day, compared with subjects treated with suprofen (800 mg/day) whose mean fecal blood loss was 1.8 ml/day. There was significantly greater (p less than 0.01) blood loss in the aspirin group than in the suprofen group. Mean fecal blood loss in the suprofen group did not differ appreciably from the fecal blood loss observed during the placebo period (0.4 ml/day).

Phosphorylation of a Mr 70,000 protein is associated with interleukin 2 receptor expression.

The human T cell hybrid II23 was isolated from fusions between human peripheral blood lymphocytes which had been stimulated with phytohemagglutinin (PHA) and a subline of the human T cell line CEM called CEM.TET1. This hybrid does not constitutively express detectable levels of interleukin 2 (IL 2) receptors but can be induced to express receptors by stimuli shown to activate T cells. Antibody to CD3 (a component of the T cell receptor) coupled to agarose or PHA (greater than 3 micrograms/ml) induced both IL 2 production and receptor expression on II23 cells. Phorbol 12-myristate 13-acetate (PMA) induced IL 2 receptor expression on II23 cells but not IL 2 secretion. Because PMA is a known activator of the Ca2+/phospholipid-dependent enzyme protein kinase C, proteins of stimulated and unstimulated cells were analyzed by two-dimensional gel electrophoresis for changes in phosphoprotein patterns. A Mr 70,000 protein with a pI of 6.2 was phosphorylated in hybrids stimulated by PMA, anti-CD3 antibody coupled to agarose or PHA, i.e. by the same stimuli which induce IL 2 receptors on these cells. The immunosuppressive drug cyclosporin A inhibited IL 2 release without altering induction of IL 2 receptors or phosphorylation of the Mr 70,000 protein. The 70-kDa protein was located in the cytosol, where it remained phosphorylated for at least 4 h after stimulation. A protein with the same migratory properties on two-dimensional gels was similarly phosphorylated after stimulation of normal peripheral blood T lymphocytes, indicating that the phenomenon was not due to hybridization or transformation. This 70-kDA protein may therefore be involved in the pathway which leads to the transcription and expression of IL 2 receptors.