Adult human progenitor cells from the temporal lobe: another source of neuronal cells.

OBJECTIVES: In the adult human brain, neurogenesis occurs in the SVZ and the dentate gyrus of the hippocampus, but it is still unclear whether persistent neural progenitor/stem cells are also present in other brain areas. The present work studies the possibility of obtaining neural progenitor/stem cells from the temporal lobe and investigates their potential to differentiate into neuronal cells. METHODS: Human biopsies from the temporal lobe of epileptic patients were used to isolate potential neural progenitors. Differentiation was induced in the presence of different agents (NGF, NT3, RA) and immunocytochemistry was then performed for quantitative analysis. RESULTS: It was shown that a significant number of cells in the temporal lobe are also capable of expansion and multi-potency. These cells can be amplified as neurospheres and have the potential to differentiate naturally in vitro into neurons, astrocytes and oligodendrocytes. Quantitative analyses show that the progenitor cells of the temporal lobe exhibit a better rate of neuronal differentiation in vitro than the cells from the SVZ, particularly in the presence of NGF. CONCLUSION: This study indicates that neural progenitors are also present in the human temporal lobe. Studying them could be of great interest for cell therapy in neurological disorders.

Cut-off values and significance of Oil Red O-positive cells in bronchoalveolar lavage fluid.

OBJECTIVE: To evaluate the percentage and predictive value of Oil Red O-positive macrophages (ORO-PM) to identify lipid-laden macrophages in bronchoalveolar lavage fluids (BALF) from patients with different pathologies. METHODS: The percentage and absolute numbers of ORO-PM were evaluated in 305 BALF. The patients were separated into ten groups: corticosteroid treatment (n = 18), amiodarone treatment (n = 8), interstitial fibrosis (n = 11), human immunodeficiency virus (HIV)-positive (n = 25), infectious pneumonia (n = 43), severe haematological disorder (n = 25), interstitial syndrome (n = 109), suspicion of cancer (n = 17), transplant recipients (n = 50) and controls (n = 43). The total and differential cell counts in BALF were recorded. The presence of specific pathogens was also noted. Parametric and non-parametric tests were used to compare the values between groups. Receiver-operating characteristics (ROC) curves were established in order to determine a cut-off value. RESULTS: The percentages of ORO-PM were (mean +/- standard deviation) 21.67 +/- 29.12 in the corticosteroid group, 10.00 +/- 12.49 in the amiodarone group, 19.45 +/- 20.72 in the interstitial fibrosis group, 47.80 +/- 30.46 in the HIV group, 19.72 +/- 26.26 in the infectious pneumonia group, 27.42 +/- 30.04 in the severe haematological disorder group, 25.18 +/- 30.63 in the interstitial syndrome group, 17.64 +/- 27.76 in the suspicion of cancer group, 22.50 +/- 27.27 in the transplanted recipients group and 2.63 +/- 3.48 in the control group. Significantly higher values were found in all groups when compared with the control group (P < 0.001). Only the HIV group showed higher numbers of ORO-PM when compared with the interstitial syndrome group (P < 0.01). According to ROC curves, > 6% ORO-PM was suggested as the positive cut-off value. CONCLUSION: Significantly increased numbers of ORO-PM were associated with various lung pathologies. However, the higher numbers observed in HIV patients require further investigations.

Estrogen and antiestrogen-dependent regulation of breast cancer cell proliferation in multicellular spheroids: Influence of cell microenvironment.

Multicellular tumor spheroids, an in vitro 3-D model that simulates malignant-cell contacts within a tumor, can be used to evaluate tumor response to therapeutic agents. We found that MELN (derived from MCF-7 cells) cells grown in 3-D as spheroids, remain highly sensitive to estradiol in terms of growth, down-regulation of ERalpha expression and ERalpha-induced transcriptional activity. Estradiol induces cyclin D1 and CDK1 proteins in Ki-67 positive proliferating cells, whereas survivin is up-regulated in both Ki-67 positive proliferative outer layer of cells and around the necrotic zone in non-proliferating cells. OH-Tam inhibits both estradiol-induced transcriptional activity and estradiol-dependent growth of MELN spheroids. Consistent with its antiproliferative effect, we observed that OH-Tam induces an important decrease in the proportion of proliferating cells, positive for Ki-67, cyclin D1 and CDK1. But, in contrast to what was expected, OH-Tam treatment resulted in a decrease in the proportion of p21 positive cells. Furthermore, despite its ability to down-regulate survivin in MELN spheroids, OH-Tam did not trigger apoptosis. Taken together, these results indicate that this model, is more relevant to an in vivo situation than monolayer cultures. It could be useful to identify new markers of the response to endocrine treatment and to investigate the effects of drugs combination.

Effect of continuous irradiation with a very low dose of gamma rays on life span and the immune system in SJL mice prone to B-cell lymphoma.

Ionizing radiation has been shown to have dose- and dose-rate-dependent carcinogenic effects on the hematopoietic and lymphoreticular systems. We report here that continuous exposure to a low dose of gamma rays influences the course of spontaneous B-cell lymphoma in SJL mice. We studied the biological effects of 10 cGy year(-1) gamma rays on the life span of 560 4-week-old SJL/J female mice and on various parameters of the cell-mediated immune response. Life span was slightly prolonged. The mean survival was 397 days for controls and 417 days for irradiated mice that died with lymphoma (P = 0.34). In lymph nodes and spleen, lower percentages of CD4+ and CD8+ T cells were observed in irradiated mice before 32 weeks. Interestingly, the percentages of CD49+ NK cells were increased in the spleens of irradiated mice at 28 weeks (0.61 +/- 0.08% compared to 0.43 +/- 0.12% in controls, P = 0.01) and at 32 weeks (0.62 +/- 0.24% compared to 0.33 +/- 0.09%, P = 0.02), while NK cell activity remained unchanged in exposed mice. These results provide further support for the absence of harmful effects of a continuous very low dose of radiation on life span and incidence of lymphoma in SJL mice.

Influence of 12-O-tetradecanoylphorbol-13-acetate on proliferation and maturation of human breast carcinoma cells (MCF-7): relationship to cell cycle events.

Exposure of MCF-7 human mammary carcinoma cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) results in changes in cell morphology and arrest of cell growth. The inhibition of cell proliferation and the increase in cell volume are concentration dependent; these effects are reversible upon removal of the tumor promoting agent. Electron microscopic studies reveal that TPA increases endoplasmic reticulum and induces the appearance of secretory granules. MCF-7 cells treated by TPA therefore present morphological characteristics of secretory cells. These effects of TPA on MCF-7 cells are accompanied by specific disruption of cell cycle events, a block of cells in G1 at the expense of S base, and a delayed passage through G2. Studies in which a cell cycle lock in G1 is produced by tamoxifen show that exposure of such cells to PA produces cell morphological changes and an inability to progress through the cell cycle when estradiol is added.

Intrathecal grafting of porcine chromaffin cells reduces formalin-evoked c-Fos expression in the rat spinal cord.

Chromaffin cells from the adrenal gland secrete a combination of neuroactive compounds including catecholamines, opioid peptides, and growth factors that have strong analgesic effects, especially when administered intrathecally. Preclinical studies of intrathecal implantation with xenogeneic bovine chromaffin cells in rats have provided conflicting data with regard to analgesic effects, and recent concern over risk of prion transmission has precluded their use in human clinical trials. We previously developed a new, safer source of adult adrenal chromaffin cells of porcine origin and demonstrated an in vivo antinociceptive effect in the formalin test, a rodent model of tonic pain. The goal of the present study was to confirm porcine chromaffin cell analgesic effects at the molecular level by evaluating neural activity as reflected by spinal cord c-Fos protein expression. To this end, the expression of c-Fos in response to intraplantar formalin injection was evaluated in animals following intrathecal grafting of 10(6) porcine or bovine chromaffin cells. For the two species, adrenal chromaffin cells significantly reduced the tonic phases of the formalin response. Similarly, c-Fos-like immunoreactive neurons were markedly reduced in the dorsal horns of animals that had received injections of xenogeneic chromaffin cells. This reduction was observed in both the superficial (I-II) and deep (V-VI) lamina of the dorsal horn. The present study demonstrates that both xenogeneic porcine and bovine chromaffin cells transplanted into the spinal subarachnoid space of the rat can suppress formalin-evoked c-Fos expression equally, in parallel with suppression of nociceptive behaviors in the tonic phase of the test. These findings confirm previous reports that adrenal chromaffin cells may produce antinociception by inhibiting activation of nociceptive neurons in the spinal dorsal horn. Taken together these results support the concept that porcine chromaffin cells may offer an alternative xenogeneic cell source for transplants delivering pain-reducing neuroactive substances.

Modulation of cisplatin cytotoxicity by human recombinant interferon-gamma in human ovarian cancer cell lines.

Cytotoxic interactions between recombinant human interferon-gamma (IFN gamma) and cisplatin have been studied in six ovarian cell lines (IGROV1, NIHOVCAR3, SKOV3, OVCCR1, 2008 and its cisplatin resident variant 2008/C13*). Studies were performed using a cell survival assay. Results were assessed using median effect analysis. Synergy between these two drugs was observed in cell lines sensitive to IFN gamma, whatever their relative sensitivity or resistance to cisplatin, suggesting that IFN gamma enhances the cytotoxic activity of cisplatin. This interaction is not due to an increase in platinum accumulation in cells. This combination of drugs should be evaluated against human ovarian cancer xenografts in nude mice before its use in clinical practice.

Effect of prostaglandin E2 and A2 on steroid synthesis by the rat adrenal gland.

Prostaglandin E2 increased aldosterone output by superfused capsular adrenal glands obtained from sodium-repleted, hypophysectomized rats but corticosterone did not show a statistically significant increase. Prostaglandin A2 increased corticosterone but not aldosterone production by incubated capsular glands obtained from sodium-repleted, hypophysectomized rats. Both aldosterone and corticosterone production rates were increased by PGA2 after previous sodium restriction. Corticosterone production rate of the decapsulated adrenal gland was not significantly modified by prostaglandin A2 in a concentration effective on the capsular adrenal gland. A possible role of prostaglandins in the regulation of aldosterone secretion is discussed.

Dissociation between protein kinase C content and biological responsiveness to phorbol esters in tumor promoter-sensitive (MCF-7) and resistant (RPh-4) cells.

A cell line (RPh-4) insensitive to the effects of phorbol esters has been isolated from MCF-7 human breast cancer cells. The growth pattern of RPh-4 cells in the presence of 50 ng/mL (80 nM) 12-O-tetradecanoylphorbol 13-acetate (TPA) is similar to that of parental MCF-7 cells in the absence of TPA. While phorbol esters inhibit MCF-7 cell proliferation and increase cell volume and protein content, no such effects are observed in RPh-4 cells. TPA affects MCF-7 but not RPh-4 cell cycle in two ways: a G1 block and a delayed passage through G2 phase. Profound alterations in protein kinase C content and activity are observed in RPh-4 versus MCF-7 cells, i.e. (i) a dramatic decline in the cellular enzyme content; (ii) a loss of the capacity to translocate upon acute TPA stimulation for the remainder enzyme; and (iii) a lack of stimulation by phorbol esters of the endogenous Mr 28,000 substrate. However, these striking changes are only transient and rapidly reverse when RPh-4 cells are subcultured in TPA-free medium, with a 60% and an almost total recovery, respectively, after 15 days and 3 months. By contrast, a much lower rate of reversion is observed in terms of cell growth responsiveness to TPA with a total insensitivity to phorbol ester after 80 days and a 50% inhibition of RPh-4 cell proliferation after 3.5 months. Our data clearly demonstrate an apparent dissociation between the cellular protein kinase C content and the biological responsiveness to phorbol ester in the variant RPh-4 cells. Moreover, they suggest that the Mr 28,000 protein phosphorylation event is not directly related to the cell growth arrest induced by phorbol esters in MCF-7 cells.

Influence of continuous, very low-dose gamma-irradiation on the mouse immune system.

PURPOSE: To investigate whether continuous, very low-dose gamma-irradiation (10 cGy/year) modifies immune parameters in mice. MATERIAL AND METHODS: C57BL/6 female mice, 4 weeks old, were irradiated for 24 months and compared with control mice living in the same room. B- and T-cell subsets were evaluated by flow cytometry before and after stimulation with lectins; subclasses of immunoglobulins were determined by ELISA 2, 4, 6, 8, 12, 18 and 24 months after the beginning of the irradiation. RESULTS: No difference was found in the percentage of CD4(+) and CD8(+) cells in the thymus and the spleen, or in the reactivity of T-cells to lectins. While the number of B-cells in the spleen remained unchanged, a significant decrease of IgG1, IgG2b and IgG2a was observed after respectively 12, 18 and 24 months of irradiation. CONCLUSION: The parameters of cellular immunity studied were not affected by this chronic low-dose of irradiation, but this dose rate is probably too low to induce the hormetic effect previously described. Further investigations are necessary to assess whether the decline of immunoglobulin secretion is indicative of a lower rate of infectious diseases or a defect in B-cell function.

GABAergic pathway in a rat model of chronic neuropathic pain: modulation after intrathecal transplantation of a human neuronal cell line.

Current understanding of chronic pain points a decrease in level of the inhibitory neurotransmitter GABA, in the spinal dorsal horn, leading to an imbalance between excitatory and inhibitory pathways. A subcloned derivative of the human NT2 cell line (hNT2.17) which, after neuronal differentiation, secretes different inhibitory neurotransmitters such as GABA and glycine has been recently isolated. In this study, we have investigated the effect of this new cell line on peripheral nerve injury induced by chronic constriction (CCI) and notably the effect on the cellular GABAergic pathway. Our data show that the decrease in GABA expression in the spinal dorsal horn of injured animals is concomitant with a decline of its synthetic enzyme GAD67-Ir and mRNA but not GAD65. Interestingly, in transplanted animals we observed a strong induction of GAD67 mRNA with one week after graft, which is followed by a recovery of GAD67 and GABA Ir. This effect paralleled a reduction of hindpaw hypersensitivity and thermal hyperalgesia induced by CCI. These results suggest that hNT2.17 GABA cells can modulate neuropathic pain after CCI certainly by minimizing the imbalance and restoring the cellular GABAergic pathway.

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.

[Mechanism of action of a triphenylethylene type antiestrogen on growth of the human breast cancer cell line. MCF-7 in culture]. / Etude du mécanisme d'action d'un antioestrogène du groupe triphényléthylène sur la croissance de la lignée cellulaire de cancer du sein humain MCF-7 en culture.

The antiestrogen of the triphenylethylene type, Taxomifen (Tx), exerts its inhibitory action on cell multiplication of the human breast cancer cell line MCF-7 in the complete absence of estrogens in the culture medium. These cells contain saturable, high affinity (KA = 7 x 10(8) LM(-1)) binding sites for Tx and specific for antiestrogens (12,000 sites/cell). The concentration of these sites is significantly decreased in Tx-resistant variants of this cell line (2,700 sites/cell). In contrast, the concentration of estrogen receptors is similar in this population of variants and in the wild population (43,000 and 48,000 sites/cell respectively). These data support the concept of a role of these antiestrogen binding sites in the molecular mechanism controlling the multiplication of these breast cancer cells.

Different effects of oestradiol, oestriol, oestetrol and of oestrone on human breast cancer cells (MCF-7) in long term tissue culture.

The effects of oestradiol (Oe2), oestrone (Oe1), oestriol (Oe3), oestetrol (Oe4) on the induction of the progesterone receptor (PgR) and growth of MCF-7 cells are compared. All the four oestrogens increased cell PgR concentration. Analysis of the dose-response curves shows induction by Oe2 to be 10 times and 50 times greater than Oe3 and Oe4, respectively. Oe1 and Oe2 are equally effective, even with consideration of metabolic conversion of O31 into Oe2. When compared with untreated cells, Oe2, Oe3, and Oe4 do not influence significantly the plating efficiency but all 3 hormones increase thymidine incorporation of the cells in log phase growth. Oe2, Oe3 and Oe4 are able to rescue the growth inhibition induced by antioestrogens. The respective potency compared to Oe2 is again in the range of 10 and 50 times lower for Oe3 and Oe4, respectively. On the other hand Oe1 decreases plating efficiency, thymidine incorporation and does not rescue the growth inhibition induced by antioestrogens when the metabolic conversion of Oe1 into Oe2 is prevented. Thus, Oe3 and Oe4 behave like complete Oe2 agonists whereas Oe1 has dissociated effects, agonist on PgR induction and antagonist on cell growth.

[Results of the transplantation of 2 lines of human endometrial adenocarcinoma into the nude mouse: takes of the graft and true tumors]. / Résultats de la transplantation de deux lignées d'adénocarcinome endométrial humain à la souris nue: prises de greffe et tumeurs vraies.

Two human endometrial adenocarcinoma cell lines, isolated and routinely cultivated in our laboratory were transplanted into nude mice. MOQ cells, isolated in 1979, are devoid of estrogen (E) receptors; GUS cells, isolated in 1981, are bearing E and progesterone receptors. Cell inoculation was realized in 504 nude mice in various conditions (male or female mice, pre or post puberal, normal or castrated, with or without E supplementation). The aim of these experiments was to establish cell tumorigenicity and to compare growth characteristics in vivo and in vitro. Our results show that the age of the mice and of the cells is important for the "take" of the injected cells. Young mice seem to possess thymo-independent graft resistance stronger than old mice; "young cells", near re-cultivation from a nude mice tumour, are more tumorigenic than "old" cells, always cultivated in vitro from the time of first explantation. There is also evidence that E improve the "take" of the cells in the two cell lines, regardless of the presence of E receptors. This is suggesting of an indirect action of E on tumour growth. Finally, the most striking result is the fact that castration is the decisive factor leading to true tumours, suggesting direct growth stimulation by hypophyseal factors.

Caryometry and nuclear ultrastructure of cultured human breast cancer cells (FAM) after oestradiol administration.

In a breast cancer cell line (FAM) isolated and characterized in our laboratory, we previously observed that oestradiol (E2) did not affect cell multiplication in vitro. In this work, we examined whether these cells bearing oestrogen and progesterone receptors, were oestrogen (E)-sensitive in spite of their lack of growth response. We observed that E2 administration resulted in an increase in protein synthesis characterized by enhancement of progesterone receptors, creatine-phosphokinase and plasminogen activator. Thus these FAm cells were true E-responsive target cells and we studied their nuclear size and ultrastructure to determine if E2 stimulation induced the same important changes as described in vivo. We observed no significant differences between control and E2-treated cells. In these breast cancer cells in vitro, E does not act on DNA-auto-reproduction but only on transcriptional activity. Thus, there is only a limited number of activated genes and no gross nuclear morphological changes. In these cultured oestrogen target cells, ultrastructural nuclear changes are not a marker of hormone action, unlike the situation in vivo where E also acts in triggering cell multiplication.

Evidence of specific nuclear binding sites for T3 in the mouse cultured fibroblast.

Mouse L 929 cultured fibroblasts revealed saturable and high affinity nuclear receptors for triiodothyronine. 125I-T3 bound rapidly to intact cells, with a steady state achieved between 1-2 h at 37 C. By Scatchard estimation, the maximal binding capacity averaged 18 fentomoles of T3 bound per 100 microgram of DNA, which approximated to 2,000 sites per cell nucleus; the apparent equilibrium association constant Ka averaged 4.90 x 109 LM-1. The relative affinity of T3 analogs tested correlated with their respective thyromimetic potency. Limited capacity, high affinity and specificity of T3 nuclear receptors in these fibroblasts were found to have properties similar to those previously observed in other cell lines or tissues. Such cultured fibroblasts may provide a useful experimental model to investigate the intracellular biological effects of thyroid hormones.

[Variation in the number of estrogen and progesterone receptors in normal human endometrial cells in culture]. / Variation du taux récepteurs des oestrogènes et de la progestérone dans les cellules endométriales humaines normales en culture.

As estrogens are in vivo potent growth factors of their target cells but are ineffective in vitro on human endometrial cell multiplication, an eventual role of steroïd sex hormone receptors has been suspected. These receptors have been assayed in human endometrium, and cytoplasmic concentrations compared between fresh tissue, short term and long term cultures. Receptors remain present in culture but at a lower level than in vivo. Estrogen receptors keep stable with the duration of the culture while progesterone receptors progressively diminish. More, these receptors are active: estradiol administration induces a new progesterone receptor synthesis in culture. So, steroïd sex hormone receptors do not seem to be involved in the ineffectiveness of estrogens on cell multiplication in culture.