Monique Courtade-Saïdi

Human fetal chromaffin cells: a potential tool for cell pain therapy.

Transplantation of adrenal medulla cells has been proposed in the treatment of various conditions. Indeed, these cells possess a bipotentiality: neural and neuroendocrine, which could be exploited for brain repair or pain therapy. In a previous study, we characterized these human cells in vitro over 7-10 gestational weeks (GW) [Zhou, H., Aziza, J., Sol, J.C., Courtade-Saidi, M., Chatelin, S., Evra, C., Parant, O., Lazorthes, Y., and Jozan, S., 2006. Cell therapy of pain: Characterization of human fetal chromaffin cells at early adrenal medulla development. Exp. Neurol. 198, 370-381]. We report here our results on the extension to 23 GW. This developmental period can be split into three stages. During the first stage (7-10 GW), we observed in situ that extra-adrenal surrounding cells display the same morphology and phenotype as the intra-adrenal chromaffin cells. We also found that the intra-adrenal chromaffin cells could be committed in vitro towards an adrenergic phenotype using differentiating agents. During the second stage (11 to 15-16 GW), two types of cells (Type 1 and Type 2 cells) were identified morphologically both inside and outside the gland. Interestingly, we noted that the Type 2 cells stem from the Type 1 cells. However, during this developmental period only the intra-adrenal Type 2 cells will evolve towards an adrenergic phenotype. In the third stage (17-23 GW), we observed the ultimate location of the medulla gland. Both the in situ results and the in vitro experiments indicate that particular procedures need to be implemented prior transplantation of chromaffin cells. First, in order to obtain a large number of immature chromaffin cells, they must be isolated from the intra and extra-adrenal gland and should then be committed towards an adrenergic phenotype in vitro for subsequent use in pain therapy. This strategy is under investigation in our laboratory.

Cell therapy of pain: Characterization of human fetal chromaffin cells at early adrenal medulla development.

Adult adrenal chromaffin cells are being utilized for therapeutic transplantation. With the prospect of using fetal chromaffin cells in pain therapy, we studied their phenotype, proliferative power, function, and growth in vitro and in situ in order to determine the optimal time for implantation. Between 7 and 10 gestational weeks (GW), we isolated, in vitro, two types of chromaffin cells with a noradrenergic phenotype akin to that observed, in situ. Among the adherent chromaffin cells first observed in vitro, only a few samples expressed met-enkephalin, whereas almost all the neurosphere-like colonies, which appeared later, expressed it. However, neither of the two types of populations expressed an adrenergic phenotype in line with that observed in situ. At the upper limits of the voluntary abortion period authorized in France, this phenotype (12 GW) and met-enkephalin expression (13 GW) were evidenced in situ. For the first time in man, we demonstrate the secretion of noradrenaline in vitro by the two populations of cells. Consistent with this result, we also noted dopamine beta hydroxylase (DbetaH) mRNA expression in vitro and in situ within this period. These observations on the expression of these biological factors indicate that 9-10 GW would be the best stage for sampling these cells for preclinical transplantation experiments.

INFLUENCE OF A CONTINUOUS VERY LOW DOSE OF GAMMA-RAYS ON CELL PROLIFERATION, APOPTOSIS AND OXIDATIVE STRESS

We have previously shown a delay of death by lymphoma in SJL/J mice irradiated with continuous very low doses of ionizing radiation. In order to understand the mechanisms involved in this phenomenon, we have irradiated in vitro the Raw264.7 monocytic and the YAC-1 lymphoma cell lines at very low-dose rate of 4cGy.month-1. We have observed a transient increase in production of both free radicals and nitric oxide with a transient adaptive response during at least two weeks after the beginning of the irradiation. The slight decrease of Ki67 proliferation index observed during the second and third weeks of YAC-1 cells culture under irradiation was not significant but consistent with the shift of the proliferation assay curves of YAC-1cells at these same durations of culture. These in vitro results were in good agreement with the slightly decrease under irradiation of Ki67 proliferative index evaluated on lymphomatous lymph nodes of SJL/J mice. A significant decrease of YAC-1 cells apoptotic rate under radiation appeared after 4 weeks of culture. Therefore very small doses of gamma-irradiation are able to modify the cellular response. The main observations did not last with increasing time under irradiation, suggesting a transient adaptation of cells or organisms to this level of irradiation.

Improving the cytological diagnosis of intraocular lymphoma from vitreous fluid

To improve the cytological diagnosis of retinal lymphoma on vitreous fluid using improved cell collection and systematic analyses.

Immunocytochemistry as an adjunct to diagnostic cytology

Immunocytochemistry as a routine ancillary test remains a distant reality for most diagnostic laboratories. Notable barriers to the mass deployment of ICC include: the large variety of specimen preparations, the small specimen size, lack of validation and lack of control specimens. As clinicians constantly strive to answer questions relating to diagnosis, therapy and prognosis with minimally invasive sampling techniques, the cytopathology community must endeavour to adopt ancillary specimen testing by ICC as a core element of diagnostic cytology.

A chronic very low dose of gamma-rays alters cell adhesion

The biological effects of very low doses of ionising radiation are difficult to assess. We previously observed a delay of death by lymphoma in two different mouse strains continuously irradiated ( γ -rays) with a dose rate of 10 cGy year −1 . Cellular mechanisms likely to lead to slowing tumour growth were explored. Human lymphoid and epithelial cell lines (HL60 and MCF7) were irradiated in vitro at very low dose rate of 4 cGy month −1 . Proliferation was not modified in HL60 and MCF7 cells. However, irradiated MCF7 adherent cells showed a lower cell attachment to support partly related to a slight decrease in expression levels of α6 and β4 integrins. We also observed a transient adaptive response during at least two weeks after the beginning of the irradiation in both cell lines. These results demonstrate the ability of tiny amounts of gamma-irradiation to alter cell attachment to support and to induce an adaptive response.

One year of experience using the Paris System for Reporting Urinary Cytology: One-Year Experience Using TPS

The Paris System for Reporting Urinary Cytology (TPS) was published in November 2015. It focuses on the diagnosis of high‐grade urothelial carcinoma (HGUC) and provides criteria with which to define the category of atypical urothelial cells (AUC). The objective of the current study was to compare two 1‐year consecutive periods before and after use of TPS.

Nonlinearity in MCF7 Cell Survival Following Exposure to Modulated 6 MV Radiation Fields: Focus on the Dose Gradient Zone

The study of cell survival following exposure to nonuniform radiation fields is taking on particular interest because of the increasing evidence of a nonlinear relationship at low doses. We conducted in vitro experiments using the MCF7 breast cancer cell line. A 2.4 × 2.4 cm2 square area of a T25 flask was irradiated by a Varian Novalis accelerator delivering 6 MV photons. Cell survival inside the irradiation field, in the dose gradient zone and in the peripheral zone, was determined using a clonogenic assay for different radiation doses at the isocenter. Increased cell survival was observed inside the irradiation area for doses of 2, 10, and 20 Gy when nonirradiated cells were present at the periphery, while the cells at the periphery showed decreased survival compared to controls. Increased survival was also observed at the edge of the dose gradient zone for cells receiving 0.02 to 0.01 Gy when compared with cells at the periphery of the same flask, whatever the isocenter dose. These data are the first to report cell survival in the dose gradient zone. Radiotherapists must be aware of this nonlinearity in dose response.

L’hybridation in situ en fluorescence (FISH) dans les liquides de ponction

L’analyse cytologique des liquides de ponction (liquide cephalorachidien, liquide pleural ou liquide d’ascite) associee ou non a des techniques d’immunocytochimie, permet la detection de nombreuses pathologies tumorales. Toutefois, il est parfois necessaire de completer ces analyses. Au laboratoire, nous avons realise des hybridations in situ en fluorescence (FISH) sur des cytospins de liquides de ponction afin de confirmer un diagnostic ou de mettre en evidence des translocations pouvant faire beneficier le patient de traitements cibles. Le rearrangement du gene c-MYC a ainsi ete mis en evidence dans un liquide cephalo-rachidien d’une patiente atteinte d’un lymphome B diffus a grandes cellules ainsi que dans un liquide d’ascite d’un patient atteint d’un lymphome de Burkitt. Le rearrangement du gene ALK a ete mis en evidence dans le liquide d’ascite d’un patient presentant un adenocarcinome pulmonaire avec des metastases peritoneales. Cette recherche a ete effectuee car la tumeur primitive n’etait pas accessible, et a ainsi permis au patient de profiter d’une therapie ciblee. En conclusion, la detection de rearrangement de genes par FISH est possible sur un petit nombre de cellules tumorales contenues dans des liquides de ponction. Cette technique, realisee sur du materiel cytologique plus facile d’acces, offre la possibilite de poser le diagnostic sur le seul materiel cytologique et de gagner un temps precieux pour permettre aux patients d’acceder a des therapies ciblees.

Specific aspects of immunocytochemistry

L’immunocytochimie (ICC) représente une aide non négligeable pour le diagnostic cytologique. Cependant, son utilisation est parfois très limitée au sein des laboratoires d’anatomie et cytologie pathologiques en raison des difficultés de réalisation et de reproductibilité [1]. Une des contraintes majeures qui limite la standardisation et la généralisation de la pratique de l’ICC est représentée par l’importante diversité des échantillons et des préparations cytologiques que l’on rencontre au sein des laboratoires. Le matériel cytologique peut parvenir sous forme de lames de frottis étalés et séchés à l’air par le clinicien ou de liquides frais fixés ou non fixés qui seront traités selon différents protocoles techniques au laboratoire. Les étalements traditionnels qui parviennent directement au laboratoire peuvent poser des difficultés liées à la présence d’amas tridimensionnels. L’interprétation peut être gênée par un bruit de fond important, notamment lors de prélèvements hémorragiques avec une technique de révélation utilisant la peroxydase [2]. Les liquides fixés ou non fixés se prêtent relativement bien à la réalisation de l’ICC sur cytospins, à condition de respecter au préalable certaines règles de réalisation et de conservation des lames. Les prélèvements issus de milieu liquide (technique en couche mince) sont mieux standardisés mais nécessitent également le respect de certaines règles de confection des lames [3]. Enfin, l’inclusion de culots cellulaires en paraffine (cytoblocs) présente l’avantage de mieux standardiser les étapes de la technique qui sera comparable à celle réalisée sur des prélèvements tissulaires mais augmente le délai de réponse.