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Die Kanzlei BPS Rechtsanwälte Spielmann/Altmann in Hanau kann Sie in folgenden Rechtsgebieten: Arbeitsrecht, Erbrecht, Familienrecht. Volkmar Spielmann. Fachanwalt für Arbeitsrecht. weitere Tätigkeitsschwerpunkte​: Verkehrsrecht, Handels- und Gesellschaftsrecht, Telekommunikationsrecht. Twitter teilen; Auf Google Plus teilen. Volkmar Spielmann. Rechtsanwalt. B.P.S Rechtsanwälte. Hauptstr. Hanau. Postfach Hanau.

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Ra Spielmann Hanau

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Chapter First Online: 29 January This is a preview of subscription content, log in to check access. Kluwer, New York Google Scholar.

Weller 1 Email author 1. Personalised recommendations. These last years, substantial progress were made to characterize distinct subsets of DCs, based on differential phenotype and function, both in humans and mice.

In mice, all DC subsets described so far express CD11c [ 28 ]. Although phenotypic characterization of these DCs does not exactly fit to the in vitro FL-derived DCs presented in this work, latter cells are closely related to spleen DCs of myeloid origin, referred to as population C by Pulendran et al.

Plasmacytoid DCs, also called interferon producing cells IPC were recently characterized in humans and mice [ 30 — 33 ].

Recently, Gilliet et al. Concerning functional aspects, DC-dependent T-cell polarizing activity Th1-, Th2-, Th3- or Tr-type is not related to lineage origin, but rather depends on the stimuli used to activate these cells, probably due to their equipment and subsequent transduction of pathogen associated molecular pattern receptors, such as Toll receptor family [ 34 ].

Model showing the sequential commitment of FL-expanded precursors to macrophages, osteoclasts, dendritic cells, and microglia GCCM: glial cell-conditioned medium; IPC: interferon producing cells.

It would be interesting to test whether DC differentiation from FL cultivated cells is dependent on the presence of GM-CSF, or whether it could be obtained in the presence of proinflammatory cytokines or bacteria-derived products, as recently shown [ 19 ].

Nevertheless, the picture emerging from our study is that diversity within the mononuclear phagocyte system is generated from a continuum of macrophage precursors whose differentiation potential might change as a function of relative ageing and cytokine environment.

However, in vivo experiments have clearly shown that tissue macrophages may differentiate into lymph-node DCs when exposed to a phagocytic stimuli [ 37 ].

Thus, mature macrophages, DCs, or microglia, may activate latent differentiation programs under appropriate stimuli.

We have shown here that macrophage precursors show more limited differentiation potential, raising the attractive possibility that mature cells cells of the mononuclear phagocyte system are more capable of differentiation plasticity than their precursors.

Indeed, preliminary experiments indicated that human FL was as efficient as murine FL in stimulating bone marrow cell proliferation data not shown.

After different durations of culture, as specified in the text, non-adherent cells were recovered following gentle flushing of the culture flasks, and therefore designated as FL-cells.

For FL-dose response studies, cells were cultivated under the same conditions in 0. Proliferation was assessed after 6 days of culture using the MTT assay, which was performed as previously described [ 38 ].

For cell division studies, non-adherent cells were harvested from FL-stimulated cultures at day 6 or day 8, stained with carboxyfluorescein diacetate, succinimidyl ester CFSE as previously described [ 39 ], and cultured for durations as indicated.

In order to ensure optimal growth, culture medium and flask were re-used for culturing CFSE labelled cells. One unit was defined as being able to stimulate one colony from 50, mouse bone marrow cells in standard agar cultures [ 40 ].

Cells were seeded at 1. TRAP activity in osteoclast cultures was identified by using the Leukocyte acid phosphatase kit Sigma.

Following cell removal, dentin slices were stained with toluidin blue to reveal resorption pits under microscope [ 41 ].

Culture supernatant was referred to as GCCM. Following culture in the presence of GCCM, adherent cells were either trypsinized and collected for FACS analysis, acetone-fixed for fluorescence studies or fixed with glutaraldehyde before electron microscopy analysis.

For electron microscopy, cells were fixed directly in the culture flasks for 60 min. Ultrathin sections were contrasted with uranyl acetate and lead citrate.

To visualize actin filaments, acetone-fixed cells were incubated with phalloidin-fluorescein Sigma for 50 min. Signal acquisition and result analysis were performed using Cell Quest software Becton-Dickinson.

For staining with anti-CD clone 2E, ref. After washing in PBS, cells were resuspended in fluorescein conjugated F ab' 2 fragments of goat anti-rat immunoglobulins Caltag for a 40 min.

Gordon S: The macrophage. Perry VH: A revised view of the central nervous system microenvironment and major histocompatibility complex class II antigen presentation.

J Neuroimmunol. J Neurosci. J Exp Med. Teitelbaum SL: Bone resorption by osteoclasts. Wilms H, Wollmer MA, Sievers J: In vitro-staining specificity of the antibody 5-D-4 for microglia but not for monocytes and macrophages indicates that microglia are a unique subgroup of the myelomonocytic lineage.

Exp Hematol. Trends Neurosci. Nat Med. Annu Rev Immunol. J Immunol. Nat Immunol. Bjorck P: Isolation and characterization of plasmacytoid dendritic cells from Flt3 ligand and granulocyte-macrophage colony-stimulating factor-treated mice.

Curr Opin Hematol. Eur J Immunol. Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity.

J Immunol Methods. EMBO J. Arnett TR, Dempster DW: A comparative study of disaggregated chick and rat osteoclasts in vitro: effects of calcitonin and prostaglandins.

Download references. Dentin slices were generous gifts from Dr T. Suda Tokyo, Japan and DrN. Takahashi Nagano, Japan. The authors wish to thank Larry R.

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This website uses cookies to improve your experience while you navigate through the website. Out of these cookies, the cookies that are categorized as. Rechtsanwalt Volkmar Spielmann aus Hanau ist Fachanwalt für Arbeitsrecht und ist tätig in Medienrecht, Telekommunikationsrecht, Gesellschaftsrecht. Anwaltssuche. Alternativen zu diesem Anwalt: Hier gehts zu anderen Anwälten in Hessen. Hier gehts zu anderen Anwälten für ARBEITSRECHT IN HANAU. Beratung in Familienrecht, Verkehrsrecht, Mietrecht & mehr. Jetzt anrufen! Profil von Rechtsanwalt Volkmar Spielmann in Hanau. pcsracing.nu, für Mandanten auf den Erstkontakt in ✅ Hanau spezialisiert.

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Dom 05 Jul Finalizado. FC Lugano. Mar 30 Jun Finalizado. Sab 27 Jun Finalizado. Mar 23 Jun Finalizado. Cells were stained with CFSE then used for reseeding original culture flasks.

Division number is indicated based on sequential halving of CFSE fluorescence. Data are representative of three independent experiments.

Next, we asked whether FL-stimulated growth between day 6 and day 11 could be accounted for by continuous expansion of a common progenitor cell population.

Modulation of cell-surface marker expression suggested that FL-cells might develop various differentiation potentials along with culture duration.

Characterization of FL cultivated cells. A Flow cytometry analyses of day 6-, day 8-, and day FL cells. Filled histograms: expression levels of the markers indicated, dotted line: background staining with irrelevant antibodies.

Results are representative of 3 to 5 independent experiments. Numbers represent the average percentage of positive cells. After 6 days of this treatment, large multinucleated cells were formed and all cells in the cultures were expressing tartrate-resistant acidic phosphatase TRAP activity, a classical osteoclast marker Fig.

Overall, when grown on dentin slices, cells formed typical resorption pits indicating that they were functional osteoclasts Fig. B When grown on top of dentin slices, cells actively resorbed dentin as shown by arrows pointing to the toluidin blue stained resorption pits.

Mature DC differentiation was assessed by phenotypic, morphological and functional analysis. Consistent with mature DC immunophenotype, cells displayed a typical dendritic morphology Fig.

Numbers are the percentages of total viable cells. Controls are positioned by quad limits and dotted histograms.

Data are representative of 5 experiments. They were also potent allostimulatory cells data not shown. Microglia cells share a number of immunological markers with other classes of mononuclear phagocytes but present a unique morphology characterized by the presence of multiple cellular processes and micro-spikes or spines [ 3 ].

By performing electron microscopy on differentiated day FL cells, we were able to ascertain the typical morphological features which characterize microglia in vitro [ 3 ]: thread-like structures of 0.

Characterization of day FL cells differentiated into microglial cells A CD11b staining of day FL cells cultured for 3 days in presence of fresh media alone.

Note the presence of actin-filled spines and pseupodia white arrow. Differentiated cells exhibited cell processes bearing spines of 0. E After 6 days in the presence of GCCM, cells exhibited longer processes expanding from the cell bodies as shown by phase-contrast microscopy.

Insert shows typical thread-like structures of 0. In experiments reported above, FL cells reproducibly differentiated into macrophages in response to M-CSF, whatever duration of the primary culture.

Time-course studies showed that these conditions were optimal Fig. Indeed, osteoclast differentiation sharply decreased between day 6 and day 11 of primary cultures Fig.

These data clearly confirmed that differentiation ability of macrophage precursors was dependent on the duration of the primary cultures.

Differentiation potential of FL cells is time dependent Day 6-, day 8-, and day FL cells were differentiated to osteoclasts, DCs, and microglia using specific cytokine combinations.

Percentage of differentiated cells was measured as specified below. Similar results have been consistently obtained in several experiments.

Results are representative of 3 independent experiments. C Microglia differentiation was assessed by detection of ramified cells after cultivating the cells for 6 days in the presence of GCCM.

This experiment is representative of 4 independent experiments. In particular, this protocol will facilitate investigations on genetically modified cells obtained from transgenic or knockout mice, or following retrovirus-mediated cell transfer.

Both cell types were shown to be macrophage precursors endowed with osteoclast or prominent DC differentiation potential, respectively.

As both proliferation and cell mortality were limited or absent during the differentiation process not shown , we conclude that osteoclast or DC differentiation of FL cells was not preceded by preferential expansion of distinct precursor sub-populations.

Consistent with this model, Miyamoto et al. It remains to be shown whether FL-expanded osteoclast precursors are identical or not to those previously described by Arai et al.

The protocol described in the present paper also represents a powerful tool for studying microglia differentiation control mechanisms.

It now becomes possible to determine which soluble factors in GCCM induce microglia differentiation. It was recently suggested that native microglia are uncommitted precursors of DC and macrophages [ 23 ].

However, it is not clear whether this results from microglia plasticity or de-differentiation to an uncommitted precursor. In that respect, day FL cells, that have no microglia characteristics, may represent a valuable model to delineate the relationship between DC and microglia pathways.

Facilitating in vitro studies on microglia differentiation may help to shed some light on several physiopathological processes.

Indeed, microglia is the principal immunoeffector cell located in the CNS parenchyma, and its involvement in pathogenesis has been established for numerous neurological conditions including stroke, trauma and Alzheimer's disease [ 24 ].

Furthermore, bone marrow-derived progenitors committed to microglial differentiation should be ideally suited for CNS-targeted gene therapy since they may be able to migrate from blood to brain through an intact blood-brain barrier [ 16 , 17 , 25 ].

The unique DC properties of either eliciting immunity or ensuring self-tolerance [ 7 ], make them candidates for enhancing responses against foreign antigens in vaccination protocols [ 26 ] and against tumor antigens in cell immunotherapy [ 27 ], as well as making them targets for the treatment of autoimmunity and allograft rejection [ 6 ].

These last years, substantial progress were made to characterize distinct subsets of DCs, based on differential phenotype and function, both in humans and mice.

In mice, all DC subsets described so far express CD11c [ 28 ]. Although phenotypic characterization of these DCs does not exactly fit to the in vitro FL-derived DCs presented in this work, latter cells are closely related to spleen DCs of myeloid origin, referred to as population C by Pulendran et al.

Plasmacytoid DCs, also called interferon producing cells IPC were recently characterized in humans and mice [ 30 — 33 ]. Recently, Gilliet et al.

Concerning functional aspects, DC-dependent T-cell polarizing activity Th1-, Th2-, Th3- or Tr-type is not related to lineage origin, but rather depends on the stimuli used to activate these cells, probably due to their equipment and subsequent transduction of pathogen associated molecular pattern receptors, such as Toll receptor family [ 34 ].

Model showing the sequential commitment of FL-expanded precursors to macrophages, osteoclasts, dendritic cells, and microglia GCCM: glial cell-conditioned medium; IPC: interferon producing cells.

It would be interesting to test whether DC differentiation from FL cultivated cells is dependent on the presence of GM-CSF, or whether it could be obtained in the presence of proinflammatory cytokines or bacteria-derived products, as recently shown [ 19 ].

Nevertheless, the picture emerging from our study is that diversity within the mononuclear phagocyte system is generated from a continuum of macrophage precursors whose differentiation potential might change as a function of relative ageing and cytokine environment.

However, in vivo experiments have clearly shown that tissue macrophages may differentiate into lymph-node DCs when exposed to a phagocytic stimuli [ 37 ].

Thus, mature macrophages, DCs, or microglia, may activate latent differentiation programs under appropriate stimuli.

We have shown here that macrophage precursors show more limited differentiation potential, raising the attractive possibility that mature cells cells of the mononuclear phagocyte system are more capable of differentiation plasticity than their precursors.

Indeed, preliminary experiments indicated that human FL was as efficient as murine FL in stimulating bone marrow cell proliferation data not shown.

After different durations of culture, as specified in the text, non-adherent cells were recovered following gentle flushing of the culture flasks, and therefore designated as FL-cells.

For FL-dose response studies, cells were cultivated under the same conditions in 0. Proliferation was assessed after 6 days of culture using the MTT assay, which was performed as previously described [ 38 ].

For cell division studies, non-adherent cells were harvested from FL-stimulated cultures at day 6 or day 8, stained with carboxyfluorescein diacetate, succinimidyl ester CFSE as previously described [ 39 ], and cultured for durations as indicated.

In order to ensure optimal growth, culture medium and flask were re-used for culturing CFSE labelled cells. One unit was defined as being able to stimulate one colony from 50, mouse bone marrow cells in standard agar cultures [ 40 ].

Cells were seeded at 1. TRAP activity in osteoclast cultures was identified by using the Leukocyte acid phosphatase kit Sigma.

Following cell removal, dentin slices were stained with toluidin blue to reveal resorption pits under microscope [ 41 ]. Culture supernatant was referred to as GCCM.

Following culture in the presence of GCCM, adherent cells were either trypsinized and collected for FACS analysis, acetone-fixed for fluorescence studies or fixed with glutaraldehyde before electron microscopy analysis.

For electron microscopy, cells were fixed directly in the culture flasks for 60 min. Ultrathin sections were contrasted with uranyl acetate and lead citrate.

To visualize actin filaments, acetone-fixed cells were incubated with phalloidin-fluorescein Sigma for 50 min. Signal acquisition and result analysis were performed using Cell Quest software Becton-Dickinson.

For staining with anti-CD clone 2E, ref. After washing in PBS, cells were resuspended in fluorescein conjugated F ab' 2 fragments of goat anti-rat immunoglobulins Caltag for a 40 min.

Gordon S: The macrophage. Perry VH: A revised view of the central nervous system microenvironment and major histocompatibility complex class II antigen presentation.

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