Fabrication and characterization of microlens arrays using a cantilever-based spotter.

We present a quantitative study on the fabrication of microlenses using a low-cost polymer dispending technique. Our method is based on the use of a silicon micro-cantilever robotized spotter system. We first give a detailed description of the technique. In a second part, the fabricated microlenses are fully characterized by means of SEM (Scanning Electron Microscope), AFM (Atomic Force Microscopy) non contact optical profilometry and Mach-Zehnder interferometry. Diameters in the range [25-130mum] are obtained with an average surface roughness of 2.02nm. Curvature radii, focal lengths as well as aberrations are also measured for the first time: the fabricated microlenses present focal lengths in the range [55-181mum] and exhibit high optical quality only limited by diffraction behaviour with RMS aberration lower than lambda/14.

Nano-polymer-dispersed liquid crystal as phase modulator for a tunable vertical-cavity surface-emitting laser at 1.55 mum.

We demonstrate what we believe is the first nonmechanical tunable vertical-cavity surface-emitting laser operating in the C band. This was achieved as a result of the combination of an InGaAs quantum well structure with a 6lambda thickness tunable index nano-polymer-dispersed liquid-crystal material. Experimental results exhibited a potential tunable range close to 10 nm, in the preliminary version, and excellent single mode locking due to the side-mode suppression ratio (more than 20 dB) over the whole spectral range. Another decisive advantage, compared to mechanical solutions, was the tuning response time of a few tens of microseconds (>30 micros) to scan the full spectral range (10 nm), making this device appropriate for some access network functions, as well as being robust and low cost. The voltage values are the main limitation to wavelength range extension. We present a first version of the device optically pumped. The next version will be electrically pumped as required for the access network applications targeted here.

Measurement by digital autoradiography with a beta-imager of [35S]methionine incorporated by rat central nervous system cells in primary cultures: a marker of in vitro development.

Mixed glial neuronal cultures prepared from rat embryonic cortical cells were either treated with aracytosine or infected with an adenovirus encoding the Lac-Z gene according to two protocols of infection. In each experiment, 24 h before the end of the incubation period, [35S]methionine was added to one set of cultures which were performed in plastic chamber slides. At 10-13 days in vitro, control and treated cultures were processed either for immunocytochemical detection of neuron-specific enolase (NSE)-stained cells or for measurement of [35S]methionine incorporation. For the latter, cultures grown in the chamber slides were fixed with 4% paraformaldehyde, dehydrated, and air-dried. After removal of the upper structures of the chambers, the slides were directly transferred to a 1200 beta-imager, a gaseous detector which displays a digital image of the cultured cells and permits the quantitative measurement of incorporated [35S]methionine within a few hours. In aracytosine-treated cultures, we observed that the numbers of NSE(+) cells as well as [35S]methionine incorporation were decreased compared with control cultures. After viral infection, the number of NSE(+) neurons and the amount of radioactivity incorporated were either the same in control and infected cultures or decreased for the cultures treated according to the different protocols. In all cases, the amount of [35S]methionine incorporated varied in the same direction as the number of NSE(+) neurons in cultures. The digital imaging of the cultures permitted observation of the layer of cultured cells. It appears that such a rapid and direct measurement of incorporation of a radiolabeled indicator of protein synthesis may be considered as a quick and reliable marker of cell survival and/or proliferation.

Adenoviral-mediated transfection of the Lac-Z gene into rat dissociated embryonic central nervous system cells before and after seeding.

The adenovirus carrying a reporter gene--the Lac Z gene--is known to infect central nervous system (CNS) cells in primary cell cultures. The percentage of infected neurons with respect to the total number of neurons was studied in primary dissociated cultures as a function of the day of inoculation and the age of three rat CNS cultures: spinal cord, mesencephalon and cortex. Two methods of viral inoculation were compared: the first inoculation was performed on the cultured cell at 2, 3 or 6 days in vitro (DIV) whereas the second inoculation was performed on the cell suspensions before seeding. All the infected CNS cells has the same aspect as the control cultures. In the spinal cord and the mesencephalic cultures, the glial cells were preferentially infected, especially when the cells were inoculated at 6 DIV. In the cortical cultures, there were more infected neurons than infected glial cells. The number of CNS cells was lower when inoculation was performed at 6 DIV as compared with 3 DIV. Very few infected GABA cells were found in the cultures. A high percentage of infected neuronal cells relative to the total number of neuronal cells was found when infection of the three types of cultures was performed on the dissociated embryonic cell suspension before seeding.

Functional AMPA/kainate receptors in human embryonic and foetal central nervous system.

Here, functional AMPA/kainate receptors in human embryonic (5.5-7.5 gestational weeks) and foetal (8-10 gestational weeks) central nervous system tissue, shown by the cobalt labeling method, are reported. Specific agonist-induced cobalt incorporation was detected in brainstem and spinal cord cells, even in the youngest embryo studied. T-AMPA or kainate, but also vegetal toxins such as L-BOAA or acromelate, induced accumulation of cobalt. In contrast, no labeling was observed after exposure to KCl or NMDA. Cobalt labeled cells were particularly prominent in motor regions of brainstem and spinal cord. Co-application of the diuretic agent cyclothiazide, a desensitization blocker at AMPA receptors, dramatically increased the number of stained cells, which was particularly obvious in sensory regions, suggesting different receptor properties in motor versus sensory regions. This is the first study providing evidence for functional AMPA/kainate receptors, permeable to divalent cations, in brainstem and spinal cord at an early stage of human central nervous system development. Since many developmental processes are influenced by the modulation of cytosolic calcium, exposure at critical stages of embryogenesis to food or drug substances modifying the activity of AMPA/kainate receptors may alter brain development.

Morphological and morphometric analysis of serotonin-containing neurons in primary dissociated cultures of human rhombencephalon: a study of development.

Primary dissociated cultures of rhombencephalon were prepared from 5-9-week-old human fetuses. Half of some cultures were treated by two non-competitive N-methyl-D-aspartate antagonists, namely 1-(2-thienyl)cyclohexylpiperidine (TCP) and cis-Pip/Mel-[1-(2-thienyl)-2-methyl-cyclohexyl]piperidine (GK11) in negative enantiomeric form, which enhance the survival of human fetal central nervous system cells in culture. At different days in vitro, the treated and the control cultures were processed for immunocytochemical detection of serotonin-containing neurons which were studied by morphological and morphometric analysis. Statistical analysis showed that the surface of the stained neurons increased as a function of two parameters of time, the gestational age of the cells and the duration of the cultures. The complexity of the shape of the serotonin neurons characterized by the shape factor, the number of bifurcations and the morphological feature (bipolar or multipolar) was found to increase with the gestational age. It appears that the in vitro development of the embryonic cells which represents stages of maturation and differentiation can be specifically evaluated. Such an analysis of fetal central nervous system cells improves the knowledge of factors important in grafting experiments. We verified that the two drugs do not appreciably alter the in vitro development of the treated cells; thus they may be considered as promising drugs for human neuroprotection.

An adenovirus vector encoding tyrosine hydroxylase activity may enter human CNS cells in primary dissociated cultures.

An adenovirus encoding tyrosine hydroxylase (TH) activity was inserted in neuronal and glial cultured cells obtained from human fetal central nervous system (CNS) tissue. Using a double fluorescence immunostaining, we characterized inoculated CNS cells, with a TH antiserum and one of the following antibodies: microtubule-associated protein (MAP2) and GABA for neuronal cells, vimentin (Vim) for glial cells and glial fibrillary acidic protein (GFAP) for astrocytes. The characterization of inoculated neuronal cells was established by the detection of TH-MAP2-stained neurons in cultures obtained from the thoracic and lumbar parts of the spinal cord where no intrinsic TH cells are described. Inoculated glial cells were characterized by the detection of TH-Vim and TH-GFAP-stained CNS cultured cells. We also observed GABA neurons expressing TH immunoreactivity which could be considered as inoculated neurons expressing the GABA phenotype. Whatever the time of inoculation, transfection was observed in both neuronal and glial cells, after up to 4 months of culture. Although no precise quantitation was performed, the percentage of inoculation was found on microscopic inspection to be greater in glia than in neurons, as previously reported. We concluded that a gene coding for a key neuronal enzyme can be incorporated in embryonic human glial and neuronal cells through the use of a recombinant adenovirus.

Primary dissociated cultures of human brainstem cells: a useful tool for their characterization and neuroprotection study.

Dissociated cell cultures were prepared from brainstems of 5- to 10-week-old human fetuses. Catecholamine- as well as indolamine-containing cells were visualized using respectively dopamine (DA), noradrenaline (NA) and serotonin (5HT) as immunocytochemical markers. NA-, DA-, and 5HT-stained cells were characterized in the rhombencephalic cultures, representing respectively the fetal localization of the locus coeruleus and raphe nuclei. DA-stained cells were characterized in the mesencephalic cultures; these DA-cells originating from the substantia nigra presented morphological aspects different from the DA-rhombencephalic cells. Two types of GABA neurons and glial cells presenting glial fibrillary acidic protein (GFA-P) reactivity were also found in all the cultures. Two non-competitive N-methyl-D-aspartate antagonists, 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP) and cis-Pip/Me 1-[1-(2-thienyl)-2-methylcyclohexyl]piperidine (GK11) in enantiomeric form (-), have been investigated for survival on rhombencephalic cultured cells. The number of 5HT-cells was found to be greater in the treated cultures than in the control ones. This in vitro system appears to be a useful tool for the investigation of the development of central nervous system (CNS) cells as well as the study of neuroprotection.

Neuroprotective effect of N-methyl-D-aspartate non-competitive antagonists (arylcyclohexylamine derivatives) on human cultured spinal cord cells.

The non-competitive N-methyl-D-aspartate antagonist cis-Pip/Me 1-[1-(2-thienyl)-2-methyl-cyclohexyl] piperidine (GK11) either in racemic(+/-) or (-) enantiomeric form has been investigated on survival of human fetal spinal cord cells in culture. The treated cultured cells were processed for immunocytochemical detection of GABA at different time intervals ranging from 3 to 27 weeks. The number of GABA-stained cells was found to be greater in the treated cultures than in the control ones. The qualitative and quantitative morphological features of both control and treated cells were analysed by computer-assisted methods which allowed us to individualize three different populations: (1) all control and treated small neuritic field neurons, (2) young control and old treated large neuritic field neurons and (3) old control neurons with large neuritic fields.

Adenovirus insertion encoding the Lac Z gene in human nervous cells in primary dissociated cultures.

Primary dissociated cultures of human fetal central nervous system cells were prepared and inoculated at different days in vitro with adenovirus that contained a reporter gene encoding beta-galactosidase. At various time intervals, the cultures were processed for characterization with X-gal histochemistry and additional immunostaining with neurofilament (NF), GABA and glial fibrillary acidic protein (GFA-P). We observed that NF (+) and GABA (+) neuronal as well as GFA-P (+) glial cells could express beta-galactosidase activity after inoculation. The labeling was detected up to 3 months after virus treatment. In addition, neurons cultivated for three months were found to be still permissive for virus infection. We can conclude that adenovirus may be considered as a potential vector to transfer genes to nerve cells.

CD4 expression in neurons of the central nervous system.

CD4 is a member of the Ig gene super family expressed on the surface of many thymocytes and of a subset of T lymphocytes. Human CD4 is the receptor for HIV envelope glycoprotein gp120. Human and mouse CD4 transcripts are expressed in human and mouse central nervous system (CNS), but no corresponding proteins have been reported yet. We have analyzed mRNA expression and carried out immunological experiments on adult mouse brain with probes specific for the long and short CD4 transcripts and with antibodies monospecific for mouse CD4. The main result of these experiments is that the full length CD4 transcript and the CD4 protein are expressed coordinately in neurons throughout the adult mouse brain. CD4 immunoreactivity is also present in brain small vessel walls, ependymal cells, and choroid plexus. The brain mouse CD4 protein is indistinguishable from the thymus protein. In addition, we show that neuronal cells in primary cultures from human fetal CNS are immunoreactive to human CD4 mAbs.

Immunocytochemical demonstration of monoamine-containing cells in human fetal brain stem dissociated cultures.

Primary dissociated cultures were prepared from the brain stems of 7- to 10-week-old human fetuses. Immunocytochemical detection of serotonin (5HT)-, dopamine (DA)-, noradrenaline (NA)- and tyrosine hydroxylase (TH)-containing cells was performed within 4 to 12 weeks in culture. We observed 5HT- and NA-stained neurons in the rhombencephalon. DA-stained cells were observed in the mesencephalon. TH-stained neurons were evidenced in both parts of the brain stem. Our in vitro system appears particularly appropriate for further investigation of grafts transplanted into the lesioned central nervous system (CNS).

GABAergic neurons in human spinal cord cultures: a computer-aided analysis of normal and thienyl-phencyclidine-treated cells.

gamma-Aminobutyric acid (GABA)-containing neurons were studied in dissociated cell cultures of human spinal cords from 6-10-week-old fetuses using immunohistochemistry with anti-GABA antibodies. Light microscopy showed two types of immunoreactive (IR) neurons: (1) IR neurons with short neuritic processes remaining near the cell body (small neuritic tree neurons); and (2) IR neurons with long neuritic processes extending far from the cell body (large neuritic tree neurons). Both types were studied at different ages in vitro, in control and in thienyl phencyclidine (TCP)-treated cultures by means of computer reconstructions and morphometric parameters. A discriminant analysis permitted the recognition of three populations: whatever the age, the control and TCP-treated neurons with small neuritic trees were not discriminated from each other and were considered to be one population whereas the 98 DIV control and both 21 DIV and 98 DIV TCP-treated cells with large neuritic trees were clearly separated from each other and from the small cell population. In all models, an astrocytic labeling, weaker than that of the neurons, was observed. The nature of these neurons (probably interneurons) intrinsic to the spinal cord is discussed in view of previous findings concerning the anatomical distribution and organization of the GABAergic system in the spinal cord.

TCP enhances the survival of human fetal spinal cord cells in culture.

Dissociated cell cultures were prepared from human spinal cords of 7-10-week-old fetuses. After 10 weeks progressive neuronal necrosis was observed in controls whereas N/1-(2-thienyl)cyclohexyl/piperidine (TCP) enhanced the survival time of the cells. After 21 weeks the number of gamma-aminobutyric acid (GABA)ergic and neuron specific enolase (NSE)-stained neurons was higher in the TCP-treated cultures than in controls. TCP appears to be a promising drug for long term survival of neurons.

Effects of ethanol in vitro on some parameters of the immune response.

The action of ethanol (ETH) on murine lymphocyte subpopulations and on human peripheral blood mononuclear cells (PBMC) stimulated in vitro by mitogens was studied. ETH caused a concentration-dependent decrease in DNA synthesis in the different murine cell types. ETH was more immunosuppressive for T lymphocytes than for B lymphocytes. An enhancement of the blastogenic response was observed for B cells at 0.5% ETH. Interleukin 2 synthesis by murine splenocytes was inhibited by ETH in a concentration-related manner; the lowest concentrations of ETH caused an increase in interleukin 2 synthesis. The highest concentrations of ETH tested decreased the size of the cell clusters formed in cultures of mitogen-stimulated lymphocytes, whereas an increase in PBMC cluster size was observed in the presence of 0.5 and 1% ETH.

Cyclooxygenase and lipoxygenase inhibitors act differently on oxidative product formation by immune mononuclear cells: a flow cytometric investigation.

The action of indomethacin, a cyclooxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on oxidative products formed by immune mononuclear cells was studied by flow cytometry. Jurkat T-cells and peripheral blood mononuclear cells were incubated with 2',7'-dichlorofluorescin diacetate; this substance is hydrolyzed in the cells leading to non-fluorescent 2',7'-dichlorofluorescin which is oxidized by oxygen reactive species into highly fluorescent 2,7'-dichlorofluorescein. Using this fluorescent probe, the formation of oxygen reactive species in phytohemagglutinin-stimulated mononuclear cells treated by NDGA or indomethacin was followed by flow cytometry. We observed that NDGA caused a marked decrease, both in the level of fluorescence intensity and in the number of fluorescent cells, whereas indomethacin caused a small increase in fluorescence intensity. On the other hand, NDGA inhibited and indomethacin increased the incorporation of tritiated thymidine by phytohemagglutinin-stimulated lymphocytes. These results suggest that oxygen reactive species are involved in the stimulation of immune mononuclear cells.

Flow cytometric analysis of oxidative product formation in phytohemagglutinin-stimulated ethanol-treated immune mononuclear cells.

Oxidative products formed by immune mononuclear cells were studied by flow cytometry. JURKAT T cells and peripheral blood mononuclear cells were incubated with 2,7-dichlorofluorescin diacetate. This substance was hydrolysed in the cells, leading to a non-fluorescent product which was oxidized into highly fluorescent 2,7-dichlorofluorescein by oxygen reactive species. These latter products were analysed by flow cytometry in phytohemagglutinin (PHA)-stimulated ethanol (ETH)-treated mononuclear cells. The level of fluorescence intensity (FI) was found higher in stimulated cells than in non-stimulated cells. ETH displayed two different effects on the cells: either a decrease of FI associated with a decrease of the number of fluorescent cells (FC) or an increase in FI. Both effects were dose-dependent. ETH is an effective scavenger of .OH radicals, but it is also oxidized by the microsomal ETH oxidizing system with production of oxygen reactive species, which probably explains the opposite effects of ETH. In the presence of desferal, an iron-chelating agent, and nordihydroguaiaretic acid, an inhibitor of the lipooxygenase pathway, the cells showed a decrease of FI and FC. These results suggest that .OH and other oxygen reactive species are involved in stimulation by PHA of ETH-treated immune mononuclear cells.

Sensitivity of human lymphocytes to acetaldehyde: comparison between alcoholic and control subjects.

The action of acetaldehyde (ACH), the first metabolite of ethanol, was studied on human lymphocytes stimulated in vitro by phytohemagglutinin (PHA) or Concanavalin A (Con A). ACH caused a dose-dependent decrease of [3H]thymidine uptake in lymphocytes from both alcoholic and control subjects. The area under the curve of [3H]thymidine incorporation as a function of ACH concentration was determined for each subject and referred to as the lymphocyte sensitivity index. Indexes for alcoholic subjects were found to be higher than those for controls, indicating a lower sensitivity to ACH of lymphocytes from alcoholics. We also found a wide range of sensitivity indexes within the same group. These results are consistent with the current hypothesis that not everybody is at equal risk to develop alcohol related disorders.

[Purine metabolism and blastogenesis in lymphocytes of alcoholic subjects]. / Métabolisme des purines et blastogénèse des lymphocytes chez les sujets éthyliques.

Immune deficiency is characteristic of alcoholic subjects. These subjects usually show altered lymphocyte function. We determined the activities of adenosine deaminase (ADA) and ecto-5'-nucleotidase (ecto-5'N) in lymphocytes from 54 subjects: 15 healthy controls, 28 non-cirrhotic alcoholics, 8 alcoholic cirrhotics and 3 non-alcoholic cirrhotics. Whereas ADA activity was the same for all 54 subjects, ecto-5'N activity was in general lower in alcoholic subjects after cessation of alcohol intake. Following alcohol intoxication, however, ecto-5'N activity increased. The decrease of ecto-5'N activity in alcoholic subjects might be explained by shedding of the ecto-enzyme and alteration of lymphocyte subpopulations. We observed decreased mitogenic-induced lymphoblastic transformation in 3 patients with cirrhosis. All other subjects (including healthy controls) had normal mitogenic-induced blastogenesis. Interestingly, following alcohol intake, non-stimulated lymphoblastic transformation increased, leading to an apparently decreased stimulation index.

Theophylline-like properties of xanthine analogs.

Theophylline and other methylxanthines display a large number of biological effects, some of which are clinically important. The effects of these compounds are commonly ascribed to an inhibition of cyclic AMP breakdown. However, it becomes actually evident that another mechanism, namely adenosine receptor antagonism, could be responsible for certain methylxanthine effects. It could be of interest to find new compounds displaying only one of these mechanisms, either phosphodiesterase inhibition or adenosine receptor antagonism. We have studied several synthetic imidazol[1,2a]pyrazines, some of which display theophylline-like pharmacological properties at lower doses than theophylline. We showed that some of these compounds inhibited mitogen-induced [3H]-thymidine uptake by human lymphocytes, which is consistent with increases in cyclic AMP levels: the most efficient compounds were those which were better phosphodiesterase inhibitors than theophylline and poorer adenosine receptor antagonists.