Pleiotropic cell-division defects and apoptosis induced by interference with survivin function.

Here we investigate the role of the control of apoptosis in normal cell division. We show that interference with the expression or function of the apoptosis inhibitor survivin causes caspase-dependent cell death in the G2/M phase of the cell cycle, and a cell-division defect characterized by centrosome dysregulation, multipolar mitotic spindles and multinucleated, polyploid cells. Use of a dominant-negative survivin mutant or antisense survivin complementary DNA disrupts a supramolecular assembly of survivin, caspase-3 and the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 within centrosomes, and results in caspase-dependent cleavage of p21. Polyploidy induced by survivin antagonists is accentuated in p21-deficient cells, and corrected by exogenous expression of p21. These findings show that control of apoptosis and preservation of p21 integrity within centrosomes by survivin are required for normal mitotic progression.

Subretinal Stimulation Chip Set with 3025 Electrodes, Spatial Peaking Filter, Illumination Adaptation and Implant Lifetime Optimization.

A subretinal stimulator chip has been designed and tested, which combines high pixel number with highest simulation voltages, lowest power consumption, spatial peaking and illumination adaptation. A supporting ASIC completes the implantable device electronics. Blind mouse retina has successfully been stimulated in vitro.

Pigmented epithelium induces complete retinal reconstitution from dispersed embryonic chick retinae in reaggregation culture.

Reaggregation of dispersed retinal cells of the chick embryo leads to histotypic retinospheroids in which the laminar organization remains incomplete: photoreceptors form rosettes which are surrounded by constituents of the other retinal layers. Here, for the first time, a complete arrangement of layers is achieved in cellular spheres (stratoids), provided that fully dispersed retinal cells are younger than embryonic day E6, and are reaggregated in the presence of a monolayer of retinal pigmented epithelium (RPE). A remarkable mechanism of stratoid formation from 1 to 15 days in vitro is revealed by the establishment of a radial Müller glia scaffold and of photoreceptors. During the first two days of reaggregation on RPE, rosettes are still observed. At this stage immunostaining with vimentin and F11 antibodies for radial Müller glia reveal a disorganized pattern. Subsequently, radial glia processes organize into long parallel fibre bundles which are arranged like spokes to stabilize the surface and centre of the stratoid. The opsin-specific antibody CERN 901 detects photoreceptors as they gradually build up an outer nuclear layer at the surface. These findings assign to the RPE a decisive role for the genesis and regeneration of a vertebrate retina.

From stem cells towards neural layers: a lesson from re-aggregated embryonic retinal cells.

Cells from dissociated embryonic avian retinae have the capacity to re-aggregate in rotation culture and form cellular spheres reconstituting a complete arrangement of all retinal layers. This exquisite phenomenon is based upon in vitro proliferation of multipotent precursor stem cells and spatial organization of their differentiating descendants. The addition of soluble factors from cultured retinal pigmented epithelial (RPE) or radial glial cells is essential to revert inside-out spheres (rosetted retinal spheres) into correctly laminated outside-out spheres (stratified spheres). Such complete restoration of a laminated brain tissue by cell re-aggregation has been achieved only for the embryonic avian retina, but not the mammalian retina, nor for other brain parts. This review summarises the history of the re-aggregation approach, presents avian retinal re-aggregate models, and analyses roles of the RPE and Müller cells for successful retinal tissue regeneration. It is predicted that these results will become biomedically relevant, as stem cell biology will soon open ways to produce large amounts of human retinal precursors.

A nuclear magnetic resonance spectrometer concept for hermetically sealed magic angle spinning investigations on highly toxic, radiotoxic, or air sensitive materials.

A concept to integrate a commercial high-resolution, magic angle spinning nuclear magnetic resonance (MAS-NMR) probe capable of very rapid rotation rates (70 kHz) in a hermetically sealed enclosure for the study of highly radiotoxic materials has been developed and successfully demonstrated. The concept centres on a conventional wide bore (89 mm) solid-state NMR magnet operating with industry standard 54 mm diameter probes designed for narrow bore magnets. Rotor insertion and probe tuning take place within a hermetically enclosed glovebox, which extends into the bore of the magnet, in the space between the probe and the magnet shim system. Oxygen-17 MAS-NMR measurements demonstrate the possibility of obtaining high quality spectra from small sample masses (~10 mg) of highly radiotoxic material and the need for high spinning speeds to improve the spectral resolution when working with actinides. The large paramagnetic susceptibility arising from actinide paramagnetism in (Th(1-x)U(x))O2 solid solutions gives rise to extensive spinning sidebands and poor resolution at 15 kHz, which is dramatically improved at 55 kHz. The first (17)O MAS-NMR measurements on NpO(2+x) samples spinning at 55 kHz are also reported. The glovebox approach developed here for radiotoxic materials can be easily adapted to work with other hazardous or even air sensitive materials.

Contractile tension and beating rates of self-exciting monolayers and 3D-tissue constructs of neonatal rat cardiomyocytes.

The CellDrum technology (The term 'CellDrum technology' includes a couple of slightly different technological setups for measuring lateral mechanical tension in various types of cell monolayers or 3D-tissue constructs) was designed to quantify the contraction rate and mechanical tension of self-exciting cardiac myocytes. Cells were grown either within flexible, circular collagen gels or as monolayer on top of respective 1-mum thin silicone membranes. Membrane and cells were bulged outwards by air pressure. This biaxial strain distribution is rather similar the beating, blood-filled heart. The setup allowed presetting the mechanical residual stress level externally by adjusting the centre deflection, thus, mimicking hypertension in vitro. Tension was measured as oscillating differential pressure change between chamber and environment. A 0.5-mm thick collagen-cardiac myocyte tissue construct induced after 2 days of culturing (initial cell density 2 x 10(4) cells/ml), a mechanical tension of 1.62 +/- 0.17 microN/mm(2). Mechanical load is an important growth regulator in the developing heart, and the orientation and alignment of cardiomyocytes is stress sensitive. Therefore, it was necessary to develop the CellDrum technology with its biaxial stress-strain distribution and defined mechanical boundary conditions. Cells were exposed to strain in two directions, radially and circumferentially, which is similar to biaxial loading in real heart tissues. Thus, from a biomechanical point of view, the system is preferable to previous setups based on uniaxial stretching.

Natural occurrence of aflatoxins, deoxynivalenol, fumonisins and zearalenone in maize from Entre Ríos Province, Argentina.

In Entre Ríos, Argentina, corn is one of the most important cereal grains produced, being an important income for the regional economy. The aim of this work was to assess aflatoxins, zearalenone, deoxynivalenol (DON) and fumonisins (FB) in corn harvest in 2003 and 2004 in the most contaminated departments found in previous studies in selected sampling places. At the harvest time, when the trucks arrived to store plants, samples of corn were taken from seven different positions of the trucks and from five in the trailer. Composite samples were randomised reduced to 10 kg. The samples were analysed by immunological tests, by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and/or gas liquid chromatography-electron capture detector (GLC-ECD). In 2003 average contamination was 3.19 u.g/kg for aflatoxins, 118.5 µg/kg for deoxynivalenol, 230.8 µg/kg for zearalenone and 10200 µg/kg of total fumonisins (HPLC and ELISA quantification showed a linear correlation (r(2) =0.9618), but RIDASCREEN®FAST values were 1.7 higher than HPLC values); in 2004 deoxynivalenol and zearalenone were not detected and an average of 2.0 µg/kg for aflatoxins and 4700 µg/kg for total fumonisins was found.This province, with the earliest harvested corn in the country each summer, tends to display different contaminations from the rest of the provinces, probably due to climate characteristics, particularly hotter weather.

Photoreceptor plasticity in reaggregates of embryonic chick retina: rods depend on proximal cones and on tissue organization.

Plasticity of photoreceptors and their integration into epithelial structures homologous to an outer nuclear layer (ONL), was investigated in embryonic chick retinal cell reaggregates by immunohistochemistry using an antibody specific for red plus green cones (RG-cones) and an antibody for rods. If reaggregates are raised in the presence of pigmented epithelium (RPE), completely reconstructed, stratified retinal spheres are produced, where all rods and cones are integrated into an outer laminar ONL, similar to a normal retina. In the absence of RPE, 'rosetted' spheres form which contain internal rosettes homologous to an ONL. Only a minor fraction of cones and rods of 'rosetted' spheres are located within rosettes, while a larger fraction is diffusely displaced in nonorganized areas, thus, not contributing to an ONL-like epithelium. In both types of spheres, the total percentage of RG-cones was similar to the in vivo retina, indicating that expression of cones is autonomous. Following cones, after about one day, rods developed only within already existing RG-cone clusters. Thereby, the ratio of rods to RG-cones increases as the tissue organization decreases: for stratified spheres this ratio is, 0.50 (1 rod/2 cones; similar to mature retina); for rosettes, 0.74 (3 rods/4 cones) and for nonorganized areas, 1.09 (1 rod/1 cone) -- a higher ratio under our conditions has never been detected. Thus, rod expression depends strictly on the presence of nearby cones; their relative numbers are distinctively adjusted according to the cytoarchitecture of the tissue environment. The biomedical implications of these findings are briefly discussed.

High affinity cross-reacting mAb generated by minimal mimicry: implications for the pathogenesis of anti-nuclear autoantibodies and immunosuppression.

The antigen recognition of a profoundly immunosuppressive mAb, mAb 2E1, in vivo was investigated. In addition to the 62-kDa effector cell protease receptor 1, mAb 2E1 bound the 32-kDa T cell adhesion receptor E2 (CD99) and the 86-kDa p80 subunit of the nuclear antigen complex Ku. These molecules share no overall sequence similarity. Peptide mapping experiments identified the mAb 2E1 cross-reacting epitopes as the sequences 66GSFSDADLAD75 in E2 and 571GGAHFSVSSLAEG583 in p80 of Ku, sharing a minimal homology motif FSXXXLA, in which X is a nonconserved amino acid. Each of these peptides separately inhibited the binding of mAb 2E1 to E2, effector cell protease receptor 1, and p80 of Ku in a dose-dependent manner. Scatchard plot analysis of 125I-labeled mAb 2E1 binding to peripheral blood mononuclear cells revealed a high-affinity interaction with a dissociation constant of 7 x 10(-10) M. An anti-E2 mAb bound the same epitope 66GSFSDADLAD75 recognized by mAb 2E1 but failed to react with p80 of Ku and was not immunosuppressive. These findings demonstrate that high-affinity cross-reacting mAbs can be generated by mimicry of a minimal surface on unrelated molecules. This model of minimal mimicry may determine the nuclear reactivity of certain autoantibodies to Ku and contribute to aberrant immunosuppression in vivo.

Effect of antigen priming on T-cell suppression. I. Activity of Ly 1+2+ feedback suppressor T-cell precursors after isolation from competing Ly 2-T cells.

Previous experiments have demonstrated that feedback suppression of murine antibody responses occurs in vitro after exposure of unprimed T-cell subsets to suppression-inducing signals from primed cells, resulting in suppression of primary and secondary IgM as well as IgG anti-SRBC responses. However, following priming with antigen when cells appear which are capable of inducing feedback suppression, the ability of unfractionated splenic T-cell populations to mediate detectable feedback suppression in vitro rapidly disappears, suggesting that priming alters the expression of feedback suppression at the same time as providing for its induction. In the present study, we have succeeded in isolating active feedback suppressor T-cell precursors (preTs) in the Ly 1+2+ and L3T4- T-cell populations from SRBC-primed as well as from unprimed mice, demonstrating that preTs are not lost after priming. The preTs isolated from primed mice resemble those isolated from unprimed mice in Ly and L3T4 phenotype, cell dose requirements, kinetics, level of suppression, and requirement for in vitro activation by primed cells. These results imply that antigen priming neither significantly depresses nor enhances the ability of Ly 1+2+ preTs to participate in feedback suppression and that activated suppressor effector cells are not detectable in the Ly 1+2+ splenic T-cell subset. Priming does, however, induce an enhancing activity in Ly 2-, L3T4+ T cells which appears to compete with feedback suppression and thus may account for the absence of detectable feedback suppression when unfractionated T cells from primed mice are the only source of preTs.

Rapid loss of feedback suppressor T-cell activity after priming in vivo.

T cells from antigen-primed mice have a diminished capacity to mediate feedback suppression when compared to T cells from unprimed mice. This was demonstrated using an in vitro model of B-cell induced feedback suppression in which spleen cells from mice primed with sheep erythrocytes (SRBC) activate feedback suppressor T-cell precursors to mediate suppression of the primed spleen cell response. The addition of splenic T cells from unprimed mice to cultures of spleen cells from SRBC-primed mice resulted in suppression of the secondary IgM and IgG anti-SRBC response. In contrast, no suppression was detected when T cells from mice primed with SRBC were added to the primed spleen cell cultures. The loss of suppression by T cells from primed mice was antigen-specific and was detectable by 24 hr after priming, coinciding with the appearance after priming of T-cell enhancing activity. The reduced suppressive activity could be due to changes in the active T-cell subset itself or to the appearance of cells or factors within the T-cell population that block or mask detection of feedback suppression. In either case, the present finding suggests that priming of a host not only activates feedback suppression induction mechanisms, but also rapidly affects the ability of the T-cell population to develop effective feedback suppression.

Müller glia cells reorganize reaggregating chicken retinal cells into correctly laminated in vitro retinae.

Müller cells, that belong to the family of radial glia cells, have central functions during retinogenesis. They form a stabilizing scaffold, they are candidate targets for the mediation of extraneous retinogenetic factors, and they are an important source for retina-borne retinogenetic factors. Reaggregate cultures allow the analysis of retinogenesis from dispersed cells to fully laminated tissues. Reaggregating cells from the embryonic chick retina reassemble to reversed laminated cellular spheres including constituents of all retinal layers, yet the outer nuclear layer is represented by internal rosettes. Using spheroids, we tested whether Müller cells have a decisive function in establishing retinal polarity and in determining the lamination pattern. To this end, we established confluent monolayers of highly enriched Müller cells derived from E6 or E13 chicken retinas, and then let dispersed E5.5 retinal cells reaggregate either in the absence of these monolayers or on top of them. In the presence of Müller cells, the reversed lamina polarity of rosetted spheroids progressively transformed within a week into correctly laminated retinal spheres, whereas all initial rosettes vanished. Moreover, photoreceptors formed a regular outer nuclear layer, as visualized by the rod-specific CERN901 antibody. In correctly laminated spheroids, staining for vimentin and glutamine synthetase was much more pronounced than in rosetted spheroids; in particular, a well-established inner limiting membrane stood out wherever the retinal lamination was complete. Because these effects can be similarly achieved by supernatants derived from Müller cells, direct cell-cell contacts or cellular replenishment from the monolayer do not account for these effects. We conclude that Müller cells are involved in the establishment of a correct retinal lamination and in the arrangement of the cells in the reaggregate cultures. In particular, rosette formation is counteracted and the formation of an inner limiting membrane is induced. Because rosettes are objects of concern in several ophthalmological defects, these results are highly relevant, both biomedically and also for normal retinogenesis.

Modulation in vivo of Lyt 2 antigen expression on T cells by anti-Lyt 2 antibody: effects on Con A-induced unresponsiveness.

After a series of intraperitoneal injections of rat monoclonal anti-Lyt 2 antibody supernatant, BDF1 mice showed a loss of cells bearing Lyt 2 surface antigens but no reduction in the numbers of T cells in the spleen. With overnight culture in vitro, splenic T cells from anti-Lyt 2-treated mice regenerated surface Lyt 2, with the proportion of Lyt 2+ cells returning to control levels. Anti-rat IgG antibody was found in the serum of mice that had received the anti-Lyt 2 treatments. Modulation of the surface Lyt 2 antigens was demonstrable in vitro but only in the presence of mouse anti-rat IgG antibody. Functionally, this series of in vivo anti-Lyt 2 antibody treatments substantially reversed Con A-induced suppression of the anti-sheep red blood cell antibody response.

Inductive effects of the retinal pigmented epithelium (RPE) on histogenesis of the avian retina as revealed by retinospheroid technology.

During eye formation, inductive phenomena occurring between retinal pigmented epithelium (RPE) and retina are not well understood. After briefly summarizing the normal development of retina and RPE, we present three-dimensional in vitro models of the chick embryonic retina which allows elucidation of RPE-retina interactions. In such retinospheroids, a complete arrangement of layers is achieved, provided that dispersed retinal cells are: (1) young enough; and (2) reaggregated on a monolayer of RPE. Thereby, the RPE extends cell proliferation, while differentiation is much delayed. These findings assign to the RPE a decisive role for the genesis and regeneration of a vertebrate retina.

Differential abilities of Th1 and Th2 to induce polyclonal B cell proliferation.

Human gamma globulin-specific T helper cell (Th) clones, activated by HGG in the presence of antigen (Ag)-presenting cells, stimulated polyclonal B cell proliferation. Both Th1 and Th2 clones induced B cell proliferation, but Th1 clones were generally 5- to 10-fold less efficient than Th2 in this capacity. Th1 and Th2 each induced proliferation of both small and large B cells, although Th1 induced less B cell proliferation than Th2, regardless of B cell size. Th1-induced B cell proliferation was increased significantly by stimulating the Th1 clones with immobilized anti-CD3 mAb. The B cell response to Ag-activated Th1 clones was also increased by the addition of rIL-4 or culture supernatants from activated Th2 clones, and this enhancement was abolished by addition of anti-IL-4 mAb. The differential capacity of the Th subsets to stimulate B cells could not be attributed to differences in the degree of Ag-induced activation of the Th clones as reflected by Th proliferation or Th expression of activation markers, RL388 Ag, IL-2R, or TfR. Taken together the results suggest that even though Th1 and Th2 are similarly activated by Ag-presenting cells, Ag-activated Th2 interact more effectively with B cells than Ag-activated Th1. It is possible that inefficient interaction and subsequent intercellular signaling between Th1 and B cells results in inefficient Th1-induced B cell proliferation, and that this deficiency may be circumvented by signals (e.g., lymphokines) provided by Th2, or by the stimulation of Th1 with plate-bound anti-CD3 Ab rather than Ag.

Cerebellar glia cells induce a correct laminar organization in chicken retinal reaggregates.

We investigated the functional role of glia cells during retinogenesis using the rotation culture system. Reaggregating cells from the embryonic chick retina have the unique capacity to reassemble into laminated cellular spheres. These spheres are composed of several compartments holding the constituents of many retinal layers in a topologically correct, yet inverse orientation. However, when these spheres are cultured in the presence of conditioned media derived from monolayers of cerebellar glia cells, the reassembling retinal cells behave totally differently. The anlage of the originally reversed lamina polarity is progressively transformed within a week into a sphere with a compound and correctly laminated orientation. Conditioned media from fibroblasts, other glia cells (except Müller cells) or a set of already characterized retinogenetic factors are not able to produce this dramatic transformation. Additionally, we were able to show that only retinal cells are able to respond with a reorganization process. Reaggregating cells from the chick cerebellum also form spheroids; however, neither in the presence of cerebellar glia cell-derived conditioned medium nor their control counterparts are they able to reassemble histotypically. This indicates that cerebellar glia cells produce diffusible factors to which retinal cells can respond and that these factors can act as important determinants for the correct establishment of the retinal polarity. Since all types of laminar disorganization are of great clinical significance, the knowledge of factors which determine and sustain the normal retinal architecture are biomedically highly relevant.