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PubMedDate not supplied

Characterization of glial cultures from rapid autopsies of Alzheimer's and control patients.

We have developed isolated and mixed cultures of microglia, astrocytes, and oligodendrocytes from rapid (mean of 2 h 55 min) autopsies of nondemented elderly patients and patients with Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Cultures were derived from both the corpus callosum (CC) and superior frontal gyrus (SFG). Cultured microglia phagocytosed latex beads, were reactive for Dil-acetylated low density lipoprotein, were immunoreactive for CD68 and major histocompatibility complex II markers, and were not immunoreactive for fibroblast, astrocyte, or oligodendrocyte markers. Cultured astrocytes included fibrous and protoplasmic types, were immunoreactive for GFAP, and were not immunoreactive for fibroblast, microglia, or oligodendrocyte markers. Cultured oligodendrocytes were poorly adherent, were slow to develop, were immunoreactive for galactocerebroside, and were not immunoreactive for fibroblast, microglia, or astrocyte markers. Because they are readily manipulated under controlled experimental conditions, and because they permit immediate access to individual cells and sets of cells from patients who have actually suffered the disease, these cultures may provide an important new tool for unravelling the etiology and pathogenesis of human CNS disorders.

Aged↗

PubMed1995

Hydrogen peroxide suppresses low-density lipoprotein (LDL) uptake and LDL-supported steroidogenesis by porcine luteal cells.

The present study tested the hypothesis that hydrogen peroxide (H2O2) inhibits low-density lipoprotein (LDL) uptake and LDL-supported steroidogenesis by luteal cells. LDL uptake: dispersed porcine luteal cells from mid-cycle (days 6-11, estrus = day 0) were incubated for 0-120 min at 37 degrees C in F-10 medium + 0.1% BSA containing various concentrations of H2O2 (0-1000 microM). Cells were washed with catalase (2800 U/ml), and then with fresh medium. Cell viability based on trypan blue exclusion was unaltered by H2O2 exposure through 60 min. H2O2-exposed cells were incubated with fluorescent-tagged-LDL (Dil-LDL; 1 microgram/ml) for 10 min at 37 degrees C. Fluorescence of small (SLC) and large (LLC) luteal cells was analyzed by flow cytometry (n = 6 experiments). H2O2 (> or = 10 microM) caused a progressive reduction (P < 0.01) in mean fluorescence intensity (MFI) of SLC and LLC indicative of up to a 30-35% decline in LDL uptake. Progesterone (P) production: dispersed luteal cells (4 x 10(4)/0.2 ml) were pre-cultured in DMEM/F-12 medium overnight (approximately 18 h) in 96-well culture plates. Wells were rinsed and fresh media (0.2 ml) containing H2O2 (0-500 microM) was added. After 30 min, the following treatments were added: human(h)LDL (0 or 50 micrograms/ml), human chorionic gonadotropin (hCG; 0 or 100 ng/ml), hCG + LDL, or 22R-hydroxycholesterol (22[OH]-C; 0 or 25 micrograms/ml). Cells were incubated for an additional 4 h, and P concentrations in final media samples were measured by RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Cholesterol synthesis and lipoprotein reuptake during synaptic remodelling in hippocampus in adult rats.

Apolipoprotein E is synthesized and secreted by astrocytes in the hippocampus following lesions of the entorhinal cortex. It was proposed that apolipoprotein E, by analogy to its role in cholesterol transport in circulation, could be involved in the salvage and reutilization of non-esterified cholesterol released during terminal breakdown. The salvaged cholesterol could then be transported to neurons by apolipoprotein E-complexes and taken up via the apolipoprotein E/apolipoprotein B (low-density lipoprotein) receptor. To test this hypothesis, we have examined low-density lipoprotein receptor binding in brain sections of rats undergoing hippocampal reinnervation. The number of neuronal cells labelled by fluorescent Dil-low-density lipoprotein as well as the density of [125I]low-density lipoprotein binding sites in the dentate gyrus were found to increase in parallel with the extent of cholinergic reinnervation occurring in the deafferented hippocampus. In contrast, hippocampal cholesterol synthesis fell by more than 60% at eight days post-lesion, but eventually returned to control levels at 30 days post-lesion. The transient loss of cholesterol synthesis coincided with a peak in hippocampal apolipoprotein E expression. A concomitant accumulation of sudanophilic lipids (cholesterol esters and phospholipids) was detected in the outer molecular layer of the dentate gyrus and in the hilar region. The present findings suggest that non-esterified cholesterol released during terminal breakdown is esterified, transported via the apolipoprotein E transport system to neurons undergoing reinnervation, and take-up through the low-density lipoprotein receptor pathway where it is presumably used as a precursor molecule for the synthesis of new synapses and terminals.

Afferent Pathways↗

PubMed1995

A new cutaneous nerve fiber connection with the frontal nerve in the frog Rana esculenta: a morphological study.

The frontal nerve arises from the frontal organ, which represents the extracranial component of the pineal complex in some lower vertebrates, and interconnects the frontal organ with the epiphysis and the brain. The existence of a previously unreported nerve branch of the frontal nerve is described here in the frog Rana esculenta and called the lateral nerve. The course of the lateral nerve and its junction with the frontal nerve have been consistently detected by means of different techniques: toluidine blue staining in semithin sections, the Landau-Ignesti method for myelinated nerve fibers, the methylene blue intravital staining for peripheral nerves, and in vitro tracing with the carbocyanine Dil. A method to preserve intact the delicate lateral nerve during dissection is also described. The lateral nerve was consistently found to be unilateral, and to join the frontal nerve at one end (either on the left or the right side), whereas the other extremity was found to be dispersed in the dermis. Thus, the lateral nerve could represent a new pathway interconnecting the skin and the brain and/or the frontal organ in the frog.

Animals↗

PubMed1991

Macrophage-like cells originate from neuroepithelium in culture: characterization and properties of the macrophage-like cells.

Cultures of astroglia from C3H/HeJ mice, which are resistant to bacterial cell wall polysaccharide (LPS), initiated from embryos of Theiler stage 14 (9 days of gestation) up to Theiler stage 25 (17 days of gestation) as well as newborn animals, when subjected to nutritional deprivation, i.e. non-feeding of cultures, form large numbers of macrophage-like cells. These cells express Mac-1, Mac-3, F4/80 and Fc antigens. The cells are negative for GFAP, positive for vimentin, express Ia antigen and take up DiL-Ac-LDL. They are positive to non-specific esterase, secrete lysozyme and are phagocytic. Their morphology and ultrastructure closely resemble those of macrophages. Cultures initiated from neuroepithelium of Theiler stage 13 (8.5 days of gestation), before vascularization, when subjected to nutritional deprivation, also produce macrophage-like cells. Using spleen colony assay and methyl cellulose cultures, we were unable to detect the presence of hemopoietic (macrophage) precursor cells in astroglia cultures. This supports the hypothesis that the macrophage-like cells are of neuroectodermal origin and probably correspond to resident microglia of the CNS. Using nutritionally deprived astroglia cultures, a procedure was developed for isolation of macrophage-like cells and production of highly enriched macrophage-like (microglia) cultures.

Animals↗

PubMed1990

Retinal axon pathfinding in the optic chiasm: divergence of crossed and uncrossed fibers.

In the developing mammalian visual system, retinal fibers grow through the optic chiasm, where one population crosses to the opposite side of the brain and the other does not. Evidence from labeling growing retinal axons with the carbocyanine dye Dil in mouse embryos indicates that the two subpopulations diverge at a zone along the midline of the optic chiasm. At the border of this zone, crossed fibers grow directly across, whereas uncrossed fibers turn back, developing highly complex terminations with bifurcating and wide-ranging growth cones. When one eye is removed at early stages, uncrossed fibers from the remaining eye stall at the chiasm midline. These results suggest that crossed and uncrossed retinal fibers respond differently to cues along the midline of the chiasm and that the uncrossed fibers from one eye grow along crossed fibers from the other eye, both guidance mechanisms contributing to the establishment of the bilateral pattern of visual projections in mammalian brain.

Animals↗

PubMed1992

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.

Animals↗

PubMed1992

Selective dependence of mammalian dorsal root ganglion neurons on nerve growth factor during embryonic development.

We have investigated the NGF dependence of dorsal root ganglion (DRG) neurons in mammals using a paradigm of multiple in utero injections of a high titer anti-NGF antiserum. We have determined the specificity of our antiserum in relation to other members of the NGF neurotrophin family and found no cross-reactivity with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). To identify various classes of DRG neurons, we have stained their characteristic central projections with Dil. We show here that the NGF dependence of DRG neurons is strikingly selective. Although a majority of DRG neurons are lost after NGF deprivation during embryonic life, these are almost exclusively small diameter neurons that project to laminae I and II of the dorsal horn and presumably subserve nociception and thermoreception. Larger neurons that project to more ventral spinal laminae and subserve other sensory modalities do not require NGF for survival. These NGF-independent DRG neurons likely require one of the more recently identified neurotrophins, BDNF or NT-3.

Afferent Pathways↗

PubMed1993

Rhombomere-specific origin of the contralateral vestibulo-acoustic efferent neurons and their migration across the embryonic midline.

The bilateral efferent supply to the inner ear receptor fields is located in the hindbrain. In ovo injections of Dil into the common facial/vestibulo-acoustic nerve root at 3 days of chick development (stage 16) followed by analysis at 7 days has revealed the origin of the contralateral efferent neurons of the inner ear and their relation to the transient hindbrain rhombomeres. These neurons have a rhombomere 4-specific origin and form their commissure not by axonal outgrowth but, unusually, by transmedian cell migration into the contralateral rhombomere 4 and rhombomere 5. Neurons first project their axons from the ipsilateral basal plate through the VII/VIIIth nerve exit point and then migrate in the opposite direction, crossing the floor plate at stage 19-21. This rhombomere-specific cell behavior provides evidence at the cellular level that segmentation is intimately involved in establishing the pattern of this region of the CNS.

Animals↗

PubMed1994

The gene poxn controls different steps of the formation of chemosensory organs in Drosophila.

The gene poxn codes for a transcriptional regulator that specifies poly-innervated (chemosensory), as opposed to mono-innervated (mechanosensory), organs in Drosophila. The ectopic expression of poxn during metamorphosis results in a transformation of the morphology and central projection of adult mechanosensory organs toward those of chemosensory organs. Here we show, by electron microscopy analysis of normal and transformed bristles and by Dil labeling of the innervating neurons, that poxn also controls the number of neurons. To determine whether poxn can transform not only the sense organ precursor cells but also their daughter cells, we examine the effects of the ectopic expression of poxn at different stages of the lineage, and we conclude that poxn can act at a late stage to affect the fate of the undifferentiated neuron.

Animals↗

PubMed1995

huckebein specifies aspects of CNS precursor identity required for motoneuron axon pathfinding.

huckebein encodes a putative zinc finger protein expressed in a subset of Drosophila CNS precursors, including the NB 4-2/GMC 4-2a/RP2 cell lineage. In huckebein mutant embryos, GMC 4-2a does not express the cell fate marker EVEN-SKIPPED; conversely, huckebein overexpression produces a duplicate EVEN-SKIPPED-positive GMC 4-2a. We use Dil to trace the entire NB 4-2 lineage in wild-type and huckebein mutant embryos. Loss of huckebein does not affect the number, position, or type of neurons in the NB 4-2 lineage; however, all motoneurons show axon pathfinding defects and never terminate at the correct muscle. Thus, huckebein regulates aspects of GMC and neuronal identity required for proper motoneuron axon pathfinding in the NB 4-2 lineage.

Animals↗

PubMed1995

Guidance of cerebellofugal axons in the rat embryo: directed growth toward the floor plate and subsequent elongation along the longitudinal axis.

To elucidate guidance mechanisms of brain commissural axons, we examined the navigation of cerebellofugal axons. Axons were labeled by implantation of the fluorescent tracer Dil into the cerebellar plate (CP) of fixed, flat whole-mount embryonic rat brain. Axons initially grew straight toward the ventral midline floor plate (FP) in the rostral hindbrain and then, after crossing it, made a right-angled turn to grow either caudally or rostrally along the longitudinal axis. In collagen gel culture, CP axons showed directed growth toward both FP explants and heterologous cells expressing netrin-1, a FP-derived chemoattractant for spinal commissural axons. These results suggest that CP axons are guided to the midline by FP-derived chemoattractant(s) and then reoriented, possibly by another guidance cue, for longitudinal extension. Considering that the basic structures of the neural tube, including the FP, extend up to the caudal diencephalon, these results suggest that common guidance mechanisms operate for ventrally decussating commissural axons in both the brain and spinal cord.

Animals↗

PubMed1991

Human monoclonal antibodies demonstrate polyreactivity for histones and the cytoskeleton.

Systemic lupus erythematosus (SLE) and other autoimmune diseases are characterized by immune responses to intracellular, highly conserved antigens such as DNA and histone. In this study, peripheral blood lymphocytes (PBL) from a patient with histone autoantibodies were used to prepare IgM human-human hybridoma cell lines. Indirect immunofluorescence (IIF) was used to identify monoclonal antibodies that bound to cytoskeletal and other cytoplasmic constituents. These supernatants did not bind double-stranded or single-stranded DNA. However, immunoblotting revealed that 7/20 hybridomas selected for their binding to cytoskeletal components produced antibodies that also bound mammalian and avian histones. When peptide fragments of histone were used in immunoblotting experiments, it was found that the monoclonal antibodies bound to the carboxyl terminus of H1, a region previously shown to bind autoantibodies from sera of patients with SLE and drug-induced lupus (DIL). When the amino acid sequences of histones and cytoskeletal components were compared using the Swiss-Prot protein data bank, it was confirmed that there are eight regions of similarity. While the significance of polyreactive human monoclonal antibodies to cytoskeletal components and histones is not understood at present, it is possible that the human histone antibodies represent polyreactive antibodies that arise through the mechanism of molecular mimicry.

Amino Acid Sequence↗

PubMed1989

Attenuation of neurotoxicity following anoxia or glutamate receptor activation in EGF- and hippocampal extract-treated neuronal cultures.

Neurotoxicity following anoxia or glutamate receptor activation was studied in primary neuronal cultures grown in serum-free, chemically defined CDM R12 medium. Exposure to 1 mM KCN, 0.5 mM kainic acid and 0.5 mM N-methyl-D-aspartate led to progressive neuronal degeneration. This damage was quantified by measuring lactate dehydrogenase released in the culture medium. The toxic effects were observed early during the development of the neuronal culture (from 4 days in vitro on) and seemed to be neuron-specific since astrocyte cultures were not affected. Chronic treatment of the neuronal cultures with epidermal growth factor at 10 ng/ml and hippocampal extract at dil. 1/833 (w/v) induced morphological alterations, increased beta-adrenergic receptor coupled adenylate cyclase activity, increased level of total lactate dehydrogenase activity in the case of epidermal growth factor-treated cultures, and attenuation of lactate dehydrogenase release following exposure to KCN or glutamate receptor agonists. The alterations observed are probably due to the proliferation and differentiation of glial cells in these treated cultures. This suggests that glial cells protect neurons in vitro from degeneration induced by anoxia or glutamate receptor activation.

Adenylyl Cyclases↗

PubMed1996

Isolation and primary culture of rat cerebral microvascular endothelial cells for studying drug transport in vitro.

To establish the cerebral microvascular endothelial cell (CMEC) culture system for animals commonly utilized in in vivo studies, we developed a method for isolation and culture of rat CMECs, and the model system was used in preliminary in vitro transport experiments. The isolated rat brains were minced. After an incubation with dispase, a fraction of microvessels was obtained by the dextran gradient. The tissue was filtered and dissociated using collagenase/dispase. After the enzyme treatment, the microvessels were layered onto the top of Percoll gradient and centrifuged for purification. Specific enzyme activities of alkaline phosphatase and gamma-glutamyltransferase in the preparations gradually increased as the isolation process progressed. The isolated cells reached confluence after 5-7 days in culture. The cultured cells had Factor VIII-related antigen and an uptake of acetylated-low density lipoprotein labeled with 1,1'-dioctadecyl-3,3, 3',3'-tetramethyl-indocarbocyamine perchlorate (Dil-Ac-LDL). In the transport studies, thawed cells after several months under -80 degrees C were used. The cultured cells after the freezing preservation also had CMEC characteristics, the same as the cells seeded immediately after isolation. The permeability of [14C]-mannitol, an impermeable marker for blood-brain barrier, was considerably reduced when the cell monolayer was present. The transport of [3H]3-O-methyl-D-glucose was significantly inhibited by the unlabeled compound. Furthermore, 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, significantly diminished the transport of [3H]L-proline. These results indicated that the cell monolayer obtained in the present study could be applicable to drug transport studies through the blood-brain barrier in vitro.

3-O-Methylglucose↗

PubMed2004

Afadin- and alpha-actinin-binding protein ADIP directly binds beta'-COP, a subunit of the coatomer complex.

Afadin DIL domain-interacting protein (ADIP) is a novel protein that binds both afadin and alpha-actinin and localizes at adherens junctions, which are formed by nectins and cadherins, cell-cell adhesion molecules. Afadin is an actin filament (F-actin)-binding protein which connects nectins to the actin cytoskeleton. alpha-Actinin is another F-actin-binding protein that is indirectly associated with cadherins through the catenin complex. ADIP is at least partly involved in the physical association of nectins and cadherins. We show here that ADIP furthermore binds beta'-COP, a subunit of the coatomer complex. ADIP co-localizes with beta'-COP at the Golgi complex in Madin Darby canine kidney and normal rat kidney cells. These results suggest that ADIP is involved in vesicle trafficking from the Golgi to the endoplasmic reticulum and through the Golgi complex by interacting with the coatomer complex.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Progressive increase in conduction velocity across human mesenchymal stem cells is mediated by enhanced electrical coupling.

OBJECTIVE: The purpose of the study was to investigate the development of electrical transmission across human adult bone marrow-derived mesenchymal stem cells (hMSCs) during long-term co-incubation with cardiomyocytes (CMCs). METHODS: Neonatal rat CMCs were cultured in multi-electrode array dishes. A conduction block was induced by creating a central acellular channel, yielding two asynchronously beating CMC fields. Enhanced green fluorescent protein (eGFP)-labeled hMSCs from ischemic heart disease patients (n=8), eGFP-labeled hMSCs having RNA interference-mediated connexin43 (Cx43) knockdown (n=6), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil)-labeled CMCs (n=6), or no cells (n=9) were seeded in the channel. Assessment of conduction velocity (CV), Cx expression and localization, gap junctional coupling, and intracellular electrical recordings were performed for up to 14 days. RESULTS: Resynchronization of the two CMC fields occurred within 24 h after seeding of hMSCs. CV across hMSCs increased from 1.4+/-0.4 cm/s at day 7 to 3.5+/-0.1 cm/s (p<0.05) at day 14. CV across seeded CMCs was 16.8+/-0.2 cm/s throughout this period. No resynchronization occurred in the absence of seeded cells. Knockdown of Cx43 in hMSCs abolished conduction across the channel completely. Time-dependent increase of CV across hMSCs was associated with increased Cx43 mRNA and protein expression resulting in increased gap junctional coupling. Intracellular recordings in coupled hMSCs showed increased conducted action potential (AP) amplitude, lower resting membrane potential, and decreased duration of conducted AP (p<0.05, day 14 versus day 1). CONCLUSIONS: CV across hMSCs increases progressively after 7 days of co-incubation with CMCs, most likely via improved electrotonic interaction. This is associated with increased Cx43 expression, increased functional gap junctional coupling, and enhanced intercellular electrical coupling between hMSCs and CMCs.

Action Potentials↗

PubMedDate not supplied

Drug-induced lupus erythematosus.

Among the numerous idiopathic immune-mediated diseases that can be drug-induced, such as pemphigus, psoriasis, lichen, etc, drug-induced lupus is the most widely commented upon and investigated. The terms drug-induced lupus (DIL) and drug-induced lupus erythematosus (DILE) are preferred, but other ones are also used--drug-related lupus, lupus-like syndrome, and lupus erythematosus medicamentosus. This review discusses the general issues in DILE, such as pathogenic mechanisms, clinical forms, and diagnostic criteria, and provides more detailed information for some of the implicated drugs: minocycline, statins, terbinafine, etc.

Drug Eruptions↗