M-07e human leukemic factor-dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM-CSF and IL-3

We have isolated a subline of the M-07 human megakaryoblastic leukemia cell line, designated M-07e, that requires either interleukin-3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) for growth, even in the presence of fetal calf serum. This cell line will not grow long term in any other cytokine although it responds slightly to IL-2, IL-4, IL-6, IL-9, and interferon-gamma. We have used the M-07e subline to develop a quantitative bioassay for the measurement of levels of either GM-CSF or IL-3. This assay is as sensitive to either factor as the human bone marrow colony assay (CFU-GM) or the chronic myelogeneous leukemic (CML) blast cell proliferation assay for these factors and is much more convenient and reliable than either. With this assay, as little as 25-50 pg/ml of either IL-3 or GM-CSF can be detected, a level that should render the assay useful for analysis of these molecules in samples from patients undergoing colony-stimulating factor therapy and from conditioned media from natural sources of the factors. In these cases, neutralizing antisera to each cytokine are required to demonstrate the specificity of the assay. This assay, in combination with quantitative immunoassays, should greatly facilitate the analysis of the roles of IL-3 and GM-CSF in regulating hematopoiesis both in patients and in natural sources of the cytokines.     

Calcium signals activated by ghrelin and D-Lys-GHRP-6 ghrelin antagonist in developing dorsal root ganglion glial cells

Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys³-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca²⁺]_i followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca²⁺ entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys³-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca²⁺ activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys³-GHRP-6 ghrelin antagonist features ghrelin independent activities.     

Activation of TRPV4 channels reduces migration of immortalized neuroendocrine cells

Calcium is a universal signal, and its capacity to encode intracellular messages via spatial, temporal and amplitude characteristics allows it to participate in most cellular events. In a specific context, calcium plays a pivotal role in migration, although its role has not been elucidated fully. By using immortalized gonadotropin-releasing hormone-secreting neurons (GN11), we have now investigated the role of TRPV4, a member of the vanilloid family of Ca(2+) channels, in neuronal migration. Our results show that TRPV4 channels are present and functional in GN11 cells and their localization is polarized and enriched in lamellipodial structures. TRPV4 activation leads to a retraction of the lamellipodia and to a decrease in migratory behaviour; moreover cells migrate slower and in a more random manner. We therefore provide evidence for a new regulation of gonadotropin-releasing hormone neurons and a new role for calcium at the leading edge of migratory cells.     

Possible relationship between the activity of the adrenal gland and the subcommissural organ in the lizard Lacerta s. sicula raf.

Summary In order to study the possible functional relationship between the adrenal gland and the subcommissural organ (SCO) in the lizard Lacerta s. sicula Raf., ACTH was administered to some specimens of this species in January when both the adrenal gland and the subcommissural organ have a very low activity. In comparison to untreated controls, the adrenals of animals treated with ACTH showed clear signs of stimulation, presenting enlarged blood vessels, very few lipid droplets, numerous polymorphic mitochondria and abundant tubular smooth endoplasmic reticulum. In addition, a distinct increase in secretory material was observed in the subcommissural cells of specimens treated with ACTH. These cells showed large cisternae of the rough endoplasmic reticulum filled with granular material in the basal region, numerous secretory granules of two types in the apical region and a reduced number of microvilli on the free cell surface. These findings, together with the results of preceding studies, lead the authors to the consideration that steroid hormones might play a role in the regulation of the secretory activity of the SCO.     

The subcommissural organ of Lacerta s. sicula Raf.: Functional studies

Summary The secretory activity of the subcommissural organ (SCO) is affected by adrenalectomy, adrenalectomy + castration, and by an increase in ambient temperature in adrenalectomized and adrenalectomized + castrated animals. Adrenalectomy inhibits the activity of the SCO. After adrenalectomy + castration the decrease in the secretory activity of the SCO is more rapid. In contrast, an increase in the ambient temperature in adrenalectomized animals induces a recrudescence of the activity of the SCO. The increase in temperature in castrated + adrenalectomized lizards does not affect the inhibition produced by this type of surgical treatment. The histological changes are discussed on the basis of results obtained in the present study and in previous experiments.This work was presented at the First Colloquium of the European Pineal Study Group, November 20–24, 1978, in Amsterdam     

Towards the Personalized Treatment of Glioblastoma: Integrating Patient-Specific Clinical Data in a Continuous Mechanical Model

Glioblastoma multiforme (GBM) is the most aggressive and malignant among brain tumors. In addition to uncontrolled proliferation and genetic instability, GBM is characterized by a diffuse infiltration, developing long protrusions that penetrate deeply along the fibers of the white matter. These features, combined with the underestimation of the invading GBM area by available imaging techniques, make a definitive treatment of GBM particularly difficult. A multidisciplinary approach combining mathematical, clinical and radiological data has the potential to foster our understanding of GBM evolution in every single patient throughout his/her oncological history, in order to target therapeutic weapons in a patient-specific manner. In this work, we propose a continuous mechanical model and we perform numerical simulations of GBM invasion combining the main mechano-biological characteristics of GBM with the micro-structural information extracted from radiological images, i.e. by elaborating patient-specific Diffusion Tensor Imaging (DTI) data. The numerical simulations highlight the influence of the different biological parameters on tumor progression and they demonstrate the fundamental importance of including anisotropic and heterogeneous patient-specific DTI data in order to obtain a more accurate prediction of GBM evolution. The results of the proposed mathematical model have the potential to provide a relevant benefit for clinicians involved in the treatment of this particularly aggressive disease and, more importantly, they might drive progress towards improving tumor control and patient’s prognosis.     

Human IL-3 (multi-CSF): identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3

A cDNA clone encoding a novel hematopoietic growth factor activity produced by a gibbon T cell line has been identified using a mammalian cell expression cloning system. The sequence of this cDNA proved to have significant homology to the sequence encoding murine interleukin 3 (IL-3). The human gene, which was readily identified because of its high degree of homology to the gibbon sequence, also displayed significant homology with the murine IL-3 sequence. The recombinant gibbon IL-3 protein proved to have multipotent colony stimulating activity when tested with normal human bone marrow cells, proving that this primate hematopoietin is not only structurally but also functionally related to murine IL-3.     

Beneficial effects of estrogen treatment in the HLA-B27 transgenic rat model of inflammatory bowel disease

A well-established model of bowel inflammation is the HLA-B27 transgenic rat that exhibits a spontaneous disease phenotype resulting in chronic diarrhea caused by immune cell activation. Estrogens have previously been shown to modulate the immune system, and both estrogen receptors (ERalpha and ERbeta) are present in the intestine and cells of the immune system. Therefore, the ability of estrogen to ameliorate disease progression in the HLA-B27 transgenic rat was determined. HLA-B27 transgenic rats with chronic diarrhea were treated with 17alpha-ethynyl-17beta-estradiol (EE) for 5 days. EE treatment dramatically improved stool scores after only 3 days. Histological scores of the degree of ulceration, inflammatory cell infiltration, fibrosis, and lesion depth of the colon were also improved by EE treatment. Because neutrophil infiltration into the colon is involved in the development and propagation of disease, myeloperoxidase (MPO) activity was measured. MPO levels were reduced by 80% by EE treatment. Cotreatment with the pure ER antagonist ICI-182780 (ICI) blocked the effects of EE on stool character, MPO activity, and histology scores, strongly suggesting that the activity of EE is mediated through ER. Mast cell proteases can promote neutrophil infiltration, and gene expression analysis demonstrated that mast cell protease 1, 3, and 4 mRNA were all decreased in colons from estrogen-treated rats. In addition, a direct effect of estrogen on bone marrow-derived mast cell activity was demonstrated, suggesting that ER-mediated inactivation of mast cells may contribute to the improvement in the clinical sign and histological scores in this model.