Selective tonicity-induced expression of the neutral amino-acid transporter SNAT2 in oligodendrocytes in rat brain following systemic hypertonicity.

Sodium-coupled neutral amino-acid transporter member 2 (SNAT2) belongs to the family of neutral amino-acid transporters. SNAT2 is encoded by the gene Slc38a2, whose expression was reported to increase in vitro in fibroblasts, endothelial and renal cells exposed to a hypertonic medium. SNAT2 tonicity-induced expression brings about cellular accumulation of amino-acid, which contributes to osmoadaptation to hypertonicity. Since brain osmoadaptation is observed in relationship to neurological disorders resulting from pathological osmotic imbalances in blood plasma, we have investigated, through immunocytochemistry, SNAT2 expression in brain of rats subjected to systemic hypertonicity. Following prolonged systemic hypertonicity (24 h), small, strongly immunolabeled elements were observed that were not present in sham-treated animals. They were evenly distributed in the gray matter, with a lower density in the forebrain and a higher density in the brain stem. However the highest density by far was observed in white matter, where they were frequently aligned in chain-like rows. These observations suggested an oligodendrocyte location that was further established by double immunofluorescent labeling, using the oligodendrocyte phenotypic markers 2'-3'-cyclic nucleotide 3'phosphodiesterase and carbonic anhydrase II. SNAT2-positive elements were found associated with oligodendrocyte cell bodies, while oligodendrocyte processes were devoid of labeling. A quantitative analysis performed in the cerebral cortex indicated that virtually all SNAT2-positive elements were associated with oligodendrocyte cell bodies and conversely that the overwhelming majority of oligodendrocytes showed SNAT2 immunolabeling. The tonicity-induced expression of SNAT2 was not observed following acute systemic hypertonicity (6 h). Our results suggest that the osmoadaptation of brain oligodendrocytes to hypertonicity relies upon amino-acid accumulation through the tonicity-induced expression of SNAT2. The possible significance of these findings in relationship to the selective loss of oligodendrocytes observed in osmotic demyelination syndrome is discussed.

Large discrepancies in cellular distribution of the tonicity-induced expression of osmoprotective genes and their regulatory transcription factor TonEBP in rat brain.

Osmoprotective genes are tonicity-activated genes involved in cellular osmoadaptation to hypertonicity and considered to be regulated by a specific transcription factor called tonicity-responsive enhancer-binding protein (TonEBP). In the brain we had previously established that TonEBP was expressed and tonicity-induced in neurons only. Here we have compared in various brain regions of rats subjected to systemic hypertonicity, the cellular expression of TonEBP through immunocytochemistry and the cellular expression of osmoprotective genes, namely aldose reductase (AR), sodium-dependent myo-inositol transporter (SMIT), betaine/GABA transporter (BGT1) and taurine transporter (TauT), by in situ hybridization using non-radioactive digoxigenin-labeled riboprobes. In neurons where TonEBP was strongly tonicity-induced, AR-mRNA labeling was strongly increased in some subsets (e.g. hippocampus pyramidal cells, cerebellar Purkinje cells and neurons of the hypothalamic magnocellular nuclei) but remained undetectable in some other subsets (e.g. neurons in cerebral cortex). Tonicity-induced AR-mRNA labeling was observed only several hours after the tonicity-induced expression of TonEBP. SMIT-mRNA labeling was tonicity-induced as densely and evenly distributed dots in neuron poor regions (e.g. cerebral cortex layer I and hippocampus stratum lacunosum-moleculare). The tonicity-induced expression of SMIT-mRNA may thus occur in non-neuronal cells, presumably astrocytes, where TonEBP is neither significantly expressed, nor tonicity-induced. In neurons showing a strong tonicity-induced expression of TonEBP, no SMIT-mRNA labeling was observed. BGT1-mRNA and TauT-mRNA labeling could not be detected, even after systemic hypertonicity. The present work reveals large discrepancies between the cellular distribution of the tonicity-induced expression of osmoprotective genes and that of their regulatory transactivator TonEBP. Depending on the cell subsets and the osmoprotective genes, TonEBP may appear insufficient or conversely unnecessary for the tonicity-induced activation of an osmoprotective gene. Altogether our results show that brain cells, even from the same class, activate distinct osmoprotective genes through distinct activation processes to adapt to hypertonicity.

Differential cellular distribution of tonicity-induced expression of transcription factor TonEBP in the rat brain following prolonged systemic hypertonicity.

In a previous work performed on cerebral cortex and hippocampus we reported that tonicity-responsive enhancer binding protein (TonEBP), originally identified as a transactivator of osmoprotective genes involved in osmoadaptation of renal cells, was induced in neurons only, but to varying levels, following acute systemic hypertonicity. Whether or not this cellular specificity reflected a unique ability of neurons or a differential time course among brain cells for tonicity-induction of TonEBP was investigated throughout the brain in this study by subjecting the animals to prolonged systemic hypertonicity. In normal rats, TonEBP immunolabeling and TonEBP-mRNA in situ hybridization labeling showed a widespread, uneven and parallel distribution. TonEBP was expressed primarily in the cell nuclei of neurons, where it was heterogeneously distributed in a nucleoplasmic and a granular pool. In rats subjected to prolonged systemic hypertonicity, TonEBP labeling increased in the cell nuclei of neurons only. The tonicity-induced expression of TonEBP for a given cell group of neurons was rather uniform but varied greatly among neuronal cell groups and was positively correlated with the average size of the cell nuclei, as determined by quantitative analysis of digitized images. The detailed distribution of tonicity-induced expression of TonEBP is reported throughout the brain. In normal rats, a very minor proportion of non-neuronal cells, identified as a subset of astrocytes and possibly oligodendrocytes, showed faint nuclear immunolabeling, which however did not increase in hypertonic animals. Ependymocytes, capillary endothelial cells, and microglial cells showed no TonEBP labeling, even in hypertonic animals. Altogether our data indicate that neurons, albeit possibly to a varying extent, are the only brain cells able to use TonEBP-mediated processes for adaptation to a systemic hyperosmotic unbalance.

[The choroid plexuses: a dynamic interface between the blood and the cerebrospinal fluid]. / Les plexus choroïdes: une interface dynamique entre sang et liquide cephalo-rachidien.

The choroid plexuses form one of the interfaces that control the brain microenvironment by regulating the exchanges between the blood and the central nervous system. They appear early during brain development. Originating from four different areas of the neural tube, they protrude into the ventricular system of the brain. The choroidal mechanisms involved in the control of brain homeostasis include the structural properties of the epithelial cells that restrict diffusional processes, as well as specific exchange and secretion mechanisms. In addition to the anatomical and histological organization of the choroidal tissue, this review describes the mechanism of cerebrospinal fluid secretion which is the most studied function of the choroid plexus. Experimental evidence for an implication of the choroid plexuses in neuroprotective mechanisms and in the supply of biologically active polypeptides to the brain are also reviewed.

Transcription factor tonicity-responsive enhancer-binding protein (TonEBP) which transactivates osmoprotective genes is expressed and upregulated following acute systemic hypertonicity in neurons in brain.

Tonicity-responsive enhancer-binding protein (TonEBP) was initially identified as a transcription factor involved in adaptation of renal cells to hypertonicity by activation of osmoprotective genes encoding proteins for accumulation of compatible osmolytes. Since brain osmoadaptation is observed in relationship to neurological disorders resulting from pathological osmotic disbalances of blood plasma we have investigated through immunocytochemistry TonEBP expression in cerebral cortex and hippocampus of normal rat and rats submitted to an acute systemic hypertonicity or to a prolonged systemic hypotonicity. TonEBP-expressing cells were identified using double immunofluorescence and appropriate cell type markers. Their relative proportion was determined by quantitative image analysis. In normal rats TonEBP expressed primarily in neurons where it was strictly located in the cell nucleus but heterogeneously distributed into a nucleoplasmic pool and a granular pool. In animals made acutely hypertonic TonEBP labeling increased dramatically exclusively in the nuclei of neurons and reached a maximum within 1 h. In hypertonic animals TonEBP labeling covered the whole cell nucleus of virtually all neurons, appeared finely punctuated but was no more granular. Optical density of the labeling as determined by image analysis correlated linearly with the increased plasma osmolality. In animals made hypotonic for several days no conspicuous decrease of TonEBP labeling was observed. In normal animals a very minor proportion of non-neuronal cells showed a faint TonEBP nuclear labeling. This proportion increased slightly in hypertonic animals. Nevertheless these non-neuronal TonEBP-positive nuclei which belonged to oligodendrocytes and to a small subpopulation of astrocytes remained always very weakly labeled when compared with neuron nuclei. Brain capillary endothelial cells as well as microglial cells showed no TonEBP-labeling even in hypertonic animals. Our data demonstrate that in brain TonEBP is significantly expressed and tonicity-overexpressed in neurons and accordingly suggest that neurons only among brain cells accumulate compatible osmolytes through TonEBP-mediated activation of osmoprotective genes to adapt to acute systemic hypertonicity.

Individuality of Ag-selected and preimmune TCR repertoires.

We have combined flow cytometry and single-cell PCR to characterize the TCRBV repertoires selected by individual mice in a model CD8 response against a defined peptide/MHC complex (CW3 170-1 79/Kd). Ourresults established thatdifferent mice select individually distinct yet structurally similar CW3-specific repertoires. Repertoire selection appears to be flexible depending on the immunizing cell dose. Using a single-donor, matched-pair-recipient adoptive transfer strategy, we demonstrated that the CW3-specific TCR repertoires of normal mice are already distinct at the preimmune level. We combine our data with computer simulations to test models for the composition of an Ag-specific preimmune repertoire and its selection during an immune response.

Maternal endothelial soluble cell adhesion molecules with isolated small for gestational age fetuses: comparison with pre-eclampsia.

OBJECTIVE: 1. To evaluate the activation profile of the endothelium in pregnancies complicated by small for gestational age fetuses compared with pre-eclampsia and normal pregnancy, by measuring the plasma levels of soluble adhesion molecules soluble E-selectin, intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1. 2. To determine whether soluble adhesion molecules were related to the severity of small for gestational age fetuses and pre-eclampsia. DESIGN: Observational study. PARTICIPANTS: Sixteen women with small for gestational age fetuses; 15 women with pre-eclampsia and 15 healthy primigravidae were recruited as controls. METHODS: Plasma levels of soluble E-selectin, intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 were measured by ELISA. RESULTS: Compared with the healthy controls, soluble E-selectin was significantly increased in both small for gestational age fetuses and pre-eclampsia, whereas intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 were increased only in pre-eclampsia. In the small for gestational age fetuses group, soluble E-selectin correlated inversely with the ratio between birthweight and the expected normal birthweight (r = -0.4, P = 0.007). In the pre-eclampsia group, a significant correlation was observed between vascular cell adhesion molecule-1 and blood pressure (r = 0.54, P = 0.039). CONCLUSIONS: Endothelial activation, reflected by raised levels of soluble E-selectin, is a feature of small for gestational age fetuses and is correlated with the severity of the disease. Differences in the profile of soluble cell adhesion molecules suggest variations in the degrees of endothelial activation between pre-eclampsia and small for gestational age fetuses.

Specific alteration in the expression of glial fibrillary acidic protein, glutamate dehydrogenase, and glutamine synthetase in rats with genetic absence epilepsy.

Astrocytes play a predominant role in energy metabolism and in the catabolism of gamma-aminobutyric acid (GABA) and glutamate, neurotransmitters critically involved in epileptic processes. We show specific astrocytic alterations in the genetic absence epilepsy rats from Strasbourg (GAERS). Spontaneous absence seizures appear in this strain in the cortex and thalamus after the age of 1 month. In these brain structures, we demonstrate increased GFAP expression in both adult and young GAERS, suggesting that reactive astrocytes are already present before the onset of seizures. Glutamate dehydrogenase (GDH) and glutamine synthetase (GS), which are localized mainly in astrocytes and involved in glutamate catabolism, are shown to be differentially altered. GDH expression was increased in the thalamus of both young and adult GAERS and in the cortex of young GAERS. GS expression was slightly decreased in the thalamus of young GAERS. These astrocytic modifications are not adaptive responses to seizures, as the modifications appear before the development of absence seizures. Thus, astrocytes might be involved in the neuronal processes giving rise to epileptic seizures in this strain.

Comparative T cell receptor repertoire selection by antigen after adoptive transfer: a glimpse at an antigen-specific preimmune repertoire.

The low frequency of precursor cells specific for any particular antigen (Ag) makes it difficult to characterize preimmune T cell receptor (TCR) repertoires and to understand repertoire selection during an immune response. We have undertaken a combined adoptive transfer single-cell PCR approach to probe the Ag-specific preimmune repertoires of individual mice. Our strategy was to inject paired irradiated recipient mice with normal spleen cells prepared from individual donors and to compare the TCR repertoires subsequently selected during a CD8 response to a defined model Ag. We found that although some TCRs were shared, the TCR repertoires selected by mice receiving splenocytes from the same donor were not identical in terms of the TCRs selected and their relative frequencies. Our results together with computer simulations imply that individual mice express distinct Ag-specific preimmune TCR repertoires composed of expanded clones and that selection by Ag is a random process.

In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Direct evidence of endothelial injury in acute myocardial infarction and unstable angina by demonstration of circulating endothelial cells.

Circulating endothelial cells (CECs) have been detected in association with endothelial injury and therefore represent proof of serious damage to the vascular tree. Our aim was to investigate, using the technique of immunomagnetic separation, whether the pathological events in unstable angina (UA) or acute myocardial infarction (AMI) could cause desquamation of endothelial cells in circulating blood compared with effort angina (EA) and noncoronary chest pain. A high CEC count was found in AMI (median, 7.5 cells/mL; interquartile range, 4.1 to 43.5, P <.01 analysis of variance [ANOVA]) and UA (4.5; 0.75 to 13.25 cells/mL, P <.01) within 12 hours after chest pain as compared with controls (0; 0 to 0 cells/mL) and stable angina (0; 0 to 0 cells/mL). CEC levels in serial samples peaked at 15.5 (2.7 to 39) cells/mL 18 to 24 hours after AMI (P <.05 repeated measures ANOVA), but fell steadily after UA. Regardless of acute coronary events, the isolated cells displayed morphologic and immunologic features of vascular endothelium. The CECs were predominantly of macrovascular origin. They did not express the activation markers intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin, although some were positive for tissue factor. CECs failed to exhibit characteristics of apoptosis (TUNEL assay) excluding this event as a possible mechanism of cell detachment. The presence of CECs provides direct evidence of endothelial injury in AMI and UA, but not in stable angina, confirming that these diseases have different etiopathogenic mechanisms.

Immunologic phenotype of cultured endothelial cells: quantitative analysis of cell surface molecules.

Endothelial cells express a wide spectrum of surface molecules involved in multiple vascular functions. We quantitatively determined an extensive immunologic phenotype of endothelial cells through a large panel of antibodies directed against i) well-known endothelial molecules CD31, CD34, CD49b, e, f, CD51, CD54, CD55, CD62E and P, CD105, CD106, HLA class I and HLA class II; ii) molecules defined by monoclonal antibodies newly clustered during the 6th workshop of Human Leukocyte Differentiation Antigen (HLDA) CD109, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146 and CD147; iii) molecules defined by unclustered monoclonal antibodies. The expression of these molecules was quantified on human umbilical vein endothelial cells (HUVEC) cultured in resting conditions and after stimulation with IL-1beta (10 U/ml), TNF-alpha (10 ng/ml) and phorbol myristate acetate (60 ng/ml). Some molecules were constitutively expressed, and others were negative, which served to determine the basal phenotype. After cell stimulation, the molecules showed weak or strong expression modulation, leading to the definition of an activated phenotype. Changes in the kinetics and the amplitude of expression served to characterize poorly defined molecules and may be useful to determine their physiologic role. Also, we compared the phenotypes of endothelial cell lines EA.hy 926 and ECV 304 to that of HUVEC to assess their reliability as an endothelial cell model. Each cell line displayed a specific repertoire of molecules expressed at different levels, which could have significant implications for cell line behavior as endothelial cells.

Quantitative analysis of leukocyte membrane antigen expression on human fetal and cord blood: normal values and changes during development.

We studied the antibody binding capacity (ABC) of various cell-surface antigens in normal human fetuses and term neonates on lymphocyte, monocyte, and polymorphonuclear (PMN) cells by quantitative flow cytometry also designated by quantimetry. Analysis of changes of expression level on these leukocytes during the developmental process was also investigated. The results indicated that the ABC values of most studied markers change during the maturational process. The ABC of lymphocyte-associated antigens studied such as CD5 and CD7 showed only a decrease from fetus to adult, whereas according to the type of molecule on monocyte and PMN there was either an increase or a decrease of ABC values dependent on the stage of the developmental process, from fetus to neonate or from neonate to adult. However, the ABC values of leukocyte membrane antigens such as CD16, CD46, and CD55 on all leukocytes and CD11b, CD11c, and CD35 on myeloid cells did not change. Their expression level was already mature in fetuses compared with adult cells. In addition, in this quantimetric approach, the analysis of the results for CD11a and CD8 suggested that the changes of CD11a expression level on lymphocyte subsets can depend on one mechanism, whereas there are probably at least two for CD8. Furthermore, the expression patterns of CD5, CD7, and CD11a change during maturation. We concluded that, even if the neonate response pattern to immunological challenge differs from an adult and this is based primarily on the relative numbers and functional activity of lymphocyte T subsets (especially TH1/TH2) and their cytokine profiles, these quantitative and qualitative phenotypical differences might also contribute to explain the functional peculiarities of leukocyte fetal and cord blood cells. All these findings support the notion of immaturity and maturity of ABC expression.

Rickettsia conorii infection enhances vascular cell adhesion molecule-1- and intercellular adhesion molecule-1-dependent mononuclear cell adherence to endothelial cells.

Leukocyte adherence to the endothelium is an essential component of the inflammatory response during rickettsial infection. In vitro, Rickettsia conorii infection of endothelial cells enhances the expression of adhesive molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in a time- and dose-dependent manner. Rickettsial lipopolysaccharide does not seem to be involved, because polymyxin B does not reduce their expression. The intracellular presence of the organism and de novo host protein synthesis are required for expression of cell adhesive molecules, since rickettsial inactivation by formol and pretreatment of cells with cycloheximide inhibits an increase in expression. The contribution of interleukin-1alpha (IL-1alpha) to this endothelial adhesive phenotype was shown by inhibitory experiments 8 and 24 h after infection with IL-1 receptor antagonist and IL-1alpha blocking antibodies. Enhanced adherence of mononuclear cells to infected endothelial cells involved VCAM-1- and ICAM-1-dependent mechanisms at the late phase of the inflammatory response. This endothelial adhesive phenotype may constitute a key pathophysiologic mechanism in R. conorii-induced vascular injury.

S-Endo 1, a pan-endothelial monoclonal antibody recognizing a novel human endothelial antigen.

A murine monoclonal antibody (mAb) S-Endo 1 has been produced to detect circulating endothelial cells detached from blood vessels in pathological conditions. We have demonstrated that the associated-antigen (S-Endo 1 Ag) was highly expressed on human vascular structure irrespective of tissue origin or vessel caliber. Its expression was not restricted to endothelium, since it was also detected at low level on smooth muscle cells, stroma cells and follicular dendritic cells. But its absence on hematopoietic cells made S-Endo 1 a helpful reagent to specifically discriminate endothelium from hematopoietic tissues. Biochemical characterization showed that S-Endo 1 recognizes a monomeric structure of approximately 118 kDa on cultured endothelial cells. S-Endo 1 was submitted to the 5th International Workshop (Boston, 1993) and did not cluster in any of the old or new endothelial clusters discussed at the conference, indicating its unique reactivity. Together with the data presented in this paper, this suggested that S-Endo 1 defines a previously undescribed endothelial molecule.

Transplantation of fetal liver cells corrects accumulation of lipids in tissues and prevents fatal neuropathy in cholesterol-storage disease BALB/c mice.

The cholesterol storage disease (CSD) BALB/c mouse represents a model of the Niemann-Pick type C (NPC) disease. It is characterized by the accumulation of unesterified cholesterol within various tissues, resulting in fatal neurological lesions. Transplantation of 6x10(6) fetal liver cells from normal allogeneic CBA mice into lethally irradiated CSD mice led to reconstitution of the recipient mice with donor cells. As a result of this stable chimerism, deposition of lipids in tissues was decreased, neuropathy was prevented, and survival was significantly prolonged (over 190 days on average in transplanted mice versus 70 days in untreated mice). Foamy cells containing unesterified cholesterol, observed by filipin staining, were numerous in most tissues from untreated CSD mice; they were significantly fewer in CSD mice treated with fetal liver transplantation at the age of 36-45 days.

Quantitative analysis of leukocyte membrane antigen expression: normal adult values.

In this paper the normal ranges of the expression of various differentiation antigens, referred to by their cluster of differentiation (CD) numbers, are described on the lymphoid, monocytic, and polymorphonuclear (PMN) blood populations in normal healthy individuals. The values expressed as antibody binding capacity per cell (ABC/cell) are related to the density of antigenic molecules expressed by these cells. These values have been quantitated by the quantitative indirect immunofluorescence (QIFI) test which renders the ABC/ cell values for the different antigens directly comparable and defines a "league table," i.e., an enumeration of antigen expression on the three main cell types studied. The values for occasional antigen that are expressed differently in adults and the elderly or men and women (CD5, CD8, and CD18) are also shown. Furthermore, the QIFI test is used in two-color immunofluorescence for defining the subset heterogeneity within the T lineage for the CD2 and CD7 antigen within the separately analyzed CD45RA and CD45RO subsets. These quantitative immune phenotype analyses, also referred to as quantimetry, show variations in ABC values if different monoclonal antibodies (MAbs) are used, although these differences are frequently minor. Therefore, using whole blood and well-characterized MAbs, we established values of antigen density in normal adults which can be regarded as control values for the various pathological conditions where CD antigen expression may be altered.

[Detection of circulating endothelial cells: a new diagnostic test of angina at rest]. / Détection des cellules endothéliales circulantes: un nouveau test diagnostique de l'angor de repos.

The diagnosis of spontaneous angina depends on the recording of per-critical electrocardiographic changes. There is no simple biological test to make its retrospective diagnosis. The attack is usually triggered by instability of an atheromatous plaque which fissures and liberates endothelial cells in the blod stream. The detection of these cells cold therefore be a biological sign of this condition. The technique of detection of circulating endothelial cells by immuno-magnetic method was used in 3 groups of patients admitted to hospital within 24 hours: group I comprised 11 patients with acute myocardial infarction, group II comprised 23 patients who had suffered from spontaneous angina with ST segment depression during the attack and significant coronary arterial stenosis, group III comprised 6 patients with chest pain for which coronary angiography is normal and provocative test of spasm is negative. Circulating endothelial cells were detected in all patients of group I (100%), in 18 of the 23 patients of group II (78%) and only in one of group III (18%). These results confer on this biological test for spontaneous angina a specificity and predictive positive value of 83 and 95% and a sensitivity and negative predictive value of 78 and 50%. Therefore the detection of circulating endothelial cells could be used as a simple and reliable test for retrospective diagnosis of spontaneous angina. The mediocre sensitivity and negative predictive value may be explained by a mechanism other than fissuration of atheromatous plaque in some cases of spontaneous angina.

Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis.

Programmed cell death or apoptosis is characterized by typical morphological alterations. By transmission electron microscopy, apoptotic cells are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragmentation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display an early desintegration of cytoplasmic membrane and swelling of mitochondria. In this study we assessed by flow cytometry the sequential alterations of forward angle light scatter, 90 degrees light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by sodium azide. At the same time intervals, cells were examined by transmission electron microscopy and by UV microscopy after staining with Hoechst 33342. We report that sequential changes in light scatters and fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed decrease of mitochondrial activity as assessed by rhodamine 123 staining. Surprisingly a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragmentation of their nuclei. It is concluded that uptake of propidium iodide is a very early marker of cell death which does not discriminate between necrosis and apoptosis. Along with biochemical criteria, nuclear morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis.