Adult human and rodent brains contain neural stem and progenitor cells, and the presence of neural stem cells in the adult rodent spinal cord has also been described. Here, using electron microscopy, expression of neural precursor cell markers, and cell culture, we investigated whether neural precursor cells are also present in adult human spinal cord. In well-preserved nonpathological post-mortem human adult spinal cord, nestin, Sox2, GFAP, CD15, Nkx6.1, and PSA-NCAM were found to be expressed heterogeneously by cells located around the central canal. Ultrastructural analysis revealed the existence of immature cells close to the ependymal cells, which display characteristics of type B and C cells found in the adult rodent brain subventricular region, which are considered to be stem and progenitor cells, respectively. Completely dissociated spinal cord cells reproducibly formed Sox2(+) nestin(+) neurospheres containing proliferative precursor cells. On differentiation, these generate glial cells and gamma-aminobutyric acid (GABA)-ergic neurons. These results provide the first evidence for the existence in the adult human spinal cord of neural precursors with the potential to differentiate into neurons and glia. They represent a major interest for endogenous regeneration of spinal cord after trauma and in degenerative diseases.
Neurospheres (NSs) are clonal cellular aggregates composed of neural stem cells and progenitors. A comprehensive description of their proliferation and differentiation regulation is an essential prerequisite for their use in biotherapies. Cytokines are essential molecules regulating cell precursor fate. Using a gene-array strategy, we conducted a descriptive and functional analysis of endogenous cytokines and receptors expressed by spinal cord-derived NSs during their growth or their differentiation into neuronal and glial cells. NSs were found to express approximately 100 receptor subunits and cytokine/ secreted developmental factors. Several angiogenic factors and receptors that could mediate neural precursor cell-endothelial cell relationships were detected. Among them, receptor B for endothelins was highly expressed, and endothelins were found to increase NS growth. In contrast, NSs express receptors for ciliary neurotrophic factor (CNTF), bone morphogenetic protein (BMP), interferon (IFN)-␥, or tumor necrosis factor (TNF)-␣, which, when added in the growth phase, led to a dramatic growth reduction followed by a reduction or a loss of oligodendrocyte formation on differentiation. In addition, NSs synthesize fibroblast growth factor 2/epidermal growth factor (FGF2/EGF)-regulated endogenous cytokines that participate in their growth and differentiation. Notably, BMP-7 and CNTF were expressed during expansion, but upon differentiation there was a remarkable switch from BMP-7 to BMP-4 and -6 and a sharp increase of CNTF. Reintroduction of growth factors reverses the BMP expression profile, indicating growth factor-BMP cross-regulations. The role of endogenous CNTF was investigated by deriving NSs from CNTF knockout mice. These NSs have an increased growth rate associated with reduction of apoptosis and generate astrocytes with a reduced glial fibulary acidic protein (GFAP) content. These results demonstrate the combined role of endogenous and exogenous cytokines in neural precursor cell growth and differentiation. STEM CELLS 2006;24:748 -762
In vitro antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used in vitro antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.Malaria is a major global health problem, with an estimated 250 to 300 million clinical cases annually and 3.3 billion people at risk, causing nearly a million deaths, mostly among children under 5 years old in sub-Saharan Africa (18, 47). The resistance of Plasmodium falciparum, the most deadly malaria parasite to most antimalarial drugs, is a major obstacle to the eradication of this disease (46). It is also of considerable concern in the light of a recent report on decreased sensitivity to artemesinin drugs in Southeast Asia (14, 28). New chemotherapeutic approaches are thus urgently needed, based on optimization of current drugs and, more importantly, on the discovery of new antimalarial drugs. The latter implies systematic screening of drug libraries, a series of natural compounds, or a structure-based drug design targeting novel targets. In all cases, in vitro evaluation of the thousands of new molecules for their antimalarial activity is an early and necessary step. This early step aims at detecting the antimalarial potential of individual or series of compounds. It is performed in vitro against P. falciparum laboratory strains and, at a later stage, against field isolates, including multidrug-resistant strains. Assays must provide a first indication on the potency of the pharmacological activity, usually expressed as the concentration required to inhibit the parasite viability ...
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