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PubMed1998

Morphology of P and M retinal ganglion cells of the bush baby.

P/midget ganglion cells mediate red-green color opponency in anthropoids. It has been proposed that these cells evolved as a specialization to subserve color vision in primates. If that is correct, they must have evolved about the same time as the long-wavelength ('red') and medium-wavelength ('green') pigment genes diverged, thirty million years ago. Strepsirhines are another group of primates that diverged from the ancestor of the anthropoids at least 55 million years ago. If P/midget ganglion cells evolved to subserve color vision, they should be absent in strepsirhines. We tested this hypothesis in a nocturnal strepsirhine, the greater bush baby Otolemur. The retinal ganglion cells were labeled with the lipophilic tracer Dil and the results show that bush babies have P/midget and M/parasol cells similar to those found in the peripheral retinas of anthropoids. A number of studies have shown that the P and M pathways of bush babies share many similarities with those of anthropoids, and our results show that the same is true for their retinal ganglion cells. These results support the hypothesis that the P system evolved prior to the emergence of red-green color opponency.

Animals↗

PubMed1989

A sicca syndrome in HIV infection: association with HLA-DR5 and CD8 lymphocytosis.

A sicca syndrome with parotid enlargement, pulmonary insufficiency, and lymphadenopathy was seen in 12 patients infected with human immunodeficiency virus (HIV), only 1 of whom has had an opportunistic infection during 304 patient months of study. There was a striking increase in numbers of circulating CD8 lymphocytes and the prevalence of HLA-DR5 was greatly increased. In patients with this diffuse infiltrative lymphocytosis syndrome (DILS) the CD8 lymphocytosis, which probably depends on histocompatibility antigen status, may influence disease progression in HIV infection.

AIDS-Related Complex↗

PubMed1984

Optimal conditions for the preparation of ferritin-labeled antibodies defined by binding to their antigen in an ELISA.

An ELISA with covalently fixed antigens was used to measure and define ferritin-labeled antibodies. In the presented model, RIG covalently fixed to glass tubes served as antigen. Twenty ferritin-labeled SWaRIG conjugates were prepared with different molar ratios of antibody: ferritin: glutaraldehyde. At various dilutions, their ability to react with the antigen was compared. The amount of FRT in the reactive conjugates was measured using alkaline phosphatase-labeled antibodies against ferritin. The covalent binding of antigens to glass surfaces resulted in a low unspecific binding as shown previously. Besides, these glass tubes with antigens immobilized on their inner surface could be used more than once. This aspect of it renders this test particularly suitable for systems where rare or expensive antigens are used. The range of O.D. values reflecting the amount of reactive SWaRIG/FRT detected in 20 different conjugates (dil 1:1000) was spread over a good range which allowed a specification of optimal conditions for FRT labeling of antibodies. It is concluded that in addition to an electron microscopic evaluation of the conjugates, this assay may be very helpful in defining optimal conditions for the coupling procedure.

Animals↗

PubMed1998

Peripheral axons of Wlds mice, which regenerate after a delay of several weeks, do so readily when transcription is inhibited in the distal stump.

We have raised the hypothesis that differentiated Schwann cells repress regrowth of axons but become permissive upon dedifferentiation. WId(S) mouse is a strain in which severed peripheral nerves do not degenerate for several weeks, and axonal regeneration does not occur either [5,11]. In this strain, we studied the role of resident cells upon axonal regeneration by inhibiting transcription. Regeneration was assessed with the pinch test, electron microscopy and Dil (a fluorescent lipid soluble dye). After a crush, WId(S) axons did not regenerate but they did so when the crush was made through a nerve segment treated with actinomycin D (ActD), an inhibitor of transcription. In contrast, when the crush was made distal to the treated segment no regeneration ensued. Our results support the notion that normal resident cells of peripheral nerves repress axonal growth.

Animals↗

PubMed1999

Isolated axons of Wld(s) mice regrow centralward.

We have conjectured that axons embody a post transcriptional sprouting programme repressed by mature Schwann cells. Injured nerves of Wld(s) mice neither degenerate nor regenerate for several weeks but axons do regrow if the resident cells of the distal stump are destroyed. To test our hypothesis we made an extended crush in Wld(s) nerves to destroy resident cells, transected the nerve at the proximal end of the lesion, and searched for sprouts in the injured domain. These isolated axons regrew centralward as supported by ultrastructure, labelling with horseradish peroxidase, and staining with Dil. This result indicates that: (i) axons embody a post transcriptional sprouting programme; (ii) resident cells of the nerve, probably Schwann cells, repress this programme, and (iii) navigation of regrowing axons is determined by the environment.

Animals↗

PubMed1997

Development of long-distance efferent projections from fetal hippocampal grafts depends upon pathway specificity and graft location in kainate-lesioned adult hippocampus.

Fetal hippocampal cells grafted into the excitotoxically lesioned hippocampus of adult rats are capable of extending axonal projections into the host brain. We hypothesize that the axonal growth of grafted fetal cells into specific host targets, and the establishment of robust long-distance efferent graft projections, require placement of fetal cells in close proximity to appropriate axon guidance pathways. Intracerebroventricular administration of kainic acid in adult rats leads to a specific loss of hippocampal CA3 pyramidal neurons. We grafted 5'-bromodeoxyuridine-labeled embryonic day 19 hippocampal cells into adult hippocampus at four days post-kainic acid lesion, and quantitatively measured the projection of grafted cells into the contralateral hippocampus and the septum after three to four months survival using Fluoro-Gold and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil) tracing. Grafts located in or near the degenerated CA3 cell layer exhibited numerous neurons which established commissural projections with the contralateral hippocampus. However, such projection did not occur in intrahippocampal grafts located away from the CA3 cell layer. In contrast, neurons in all grafts established robust projections into the septum regardless of location within hippocampus although grafts located near the degenerated CA3 cell layer displayed more neurons with such projections. Location of grafted cells clearly influences the development of efferent graft projections into distant targets in the adult host brain, particularly access to axon guidance pathways to facilitate the formation of projections. The establishment of robust long-distance commissural projections of fetal hippocampal grafts is clearly dependent on their placement in or near the degenerated CA3 cell layer, suggesting that appropriate axon guidance pathways for commissural pathways are tightly focussed near this cell layer. However, the establishment of septal projections of these grafts was not dependent on specific location within the CA3 cell layer, suggesting that axonal guidance mechanisms to the septum are more diffuse and not limited to the CA3 dendritic layers. The results underscore that fetal hippocampal grafts are capable of partly restoring lesioned hippocampal circuitry in adult animals when appropriately placed in the host hippocampus.

Animals↗

PubMed2000

Differential taurine effect on outgrowth from goldfish retinal ganglion cells after optic crush or axotomy. Influence of the optic tectum.

The interaction between innervated tissues, targets and nerves is crucial in the maintenance of physiological conditions, and the disturbance of this harmony causes the production of morphological and biochemical changes. After lesion of the optic nerve, several modifications take place in the retina, the optic tectum and the optic nerve. The influence of the tectum on the outgrowth from the goldfish retina and the possible role of taurine was studied. Ganglion retinal cells were identified by retrolabeling with Dil. Crushing the optic nerve 10 days prior to plating retinal cells, as compared with optic axotomy, did not affect the survival of cultured retinal cells, as well as the length of the neurites. However, the number of neurites per cell and the branching of the longest fiber were higher after axotomy than after crushing. The addition of taurine to the medium did not modify this response at 5 days in culture. At early periods in culture, the stimulatory effect on isolated ganglion cell outgrowth produced by taurine was enhanced after axotomy respecting crushing of the optic nerve, but was not affected in retinal explants. The addition of medium from cultured optic tectum several days post-crush of the optic nerve to retinal explants from intact retinas or coming from post-crush retina modified the outgrowth, being inhibitory or stimulatory in a time-dependent manner. The co-culture of optic tectum and retina also affected the outgrowth from the retina with a byphasic shape. The results support the differential response of the retina facing partial or complete interruption with the target and limit the effect of taurine to early periods in culture. In addition, the production of inhibitory factors from the tectum, plus the stimulatory ones, are strongly supported by this work.

Animals↗

PubMed1998

The combined effects of trkB and trkC mutations on the innervation of the inner ear.

Previous research has demonstrated that only the two neurotrophins and their cognate receptors are necessary for the support of the inner ear innervation. However, detailed analyses of patterns of innervation in various combinations of neurotrophin receptor mutants are lacking. We provide here such an analysis of the distribution of afferent and efferent fibers to the ear in various combinations of neurotrophin receptor mutants using the lipophilic tracer Dil. In the vestibular system, trkC+/- heterozygosity aggravates the trkB-/- mutation effect and causes almost complete loss of vestibular neurons. In the cochlea innervation, various mutations are each characterized by specific topological absence of spiral neurons in Rosenthal's canal of the cochlea. trkC-/- mutation alone or in combination with trkB+/- heterozygosity causes absence of all basal turn spiral neurons and afferent fibers extend from the middle turn to the basal turn along inner hair cells with little or no contribution to outer hair cells. Both types of basal turn spiral neurons appear to develop and project via radial fibers to inner and, more sparingly, outer hair cells. Simple trkB-/- mutations show a reduction of fibers to outer hair cells in the apex and, less obvious, in the basal turn. Basal turn spiral neurons may be the only neurons present at birth in the cochlea of a trkB-/- mutant mouse combined with trkC+/- heterozygosity. In addition, the trkB-/- mutation combined with trkC+/- heterozygosity has a patchy and variable loss of middle turn spiral neurons in mice of different litters. Comparisons of patterns of innervation of afferent and efferent fibers show a striking similarity of absence of fibers to topologically corresponding areas. For example, in trkC-/- mutants afferents reach the basal turn, spiraling along the cochlea, rather than through radial fibers and efferent fibers follow the same pathway rather than emanating from intraganglionic spiral fibers. The data presented suggest that there are regional specific effects with some bias towards a specific spiral ganglion type: trkC is essential for support of basal turn spiral neurons whereas trkB appears to be more important for middle and apical turn spiral neurons.

Animals↗

PubMed1998

Emigration of neuroepithelial cells from the hindbrain neural tube in the chick embryo.

It is generally believed that after the emigration of neural crest, the neuroepithelial cells of the neural tube are committed to differentiate only as neurons and supporting cells of the central nervous system. Neural crest cells arise from the dorsal portion of the developing neural tube and contribute to the formation of the peripheral nervous system and a variety of non-neural structures. In contrast to this view we have recently shown, by focal application of the vital dye Dil in duck embryos, that an additional population of cells emigrates from the neural tube. By using an entirely different technique we confirm and extend these observations in the chick embryo. Replication-deficient retroviral vector LZ12 containing the gene LacZ was utilized to label the neural tube cells. The viral concentrate was microinjected into the lumen of the rostral hind-brain neural tube, considerably after the completion of emigration of neural crest cells. The labeled cells were monitored in whole mounts and histological sections. Initially, the labeled cells were restricted to the neuroepithelium of the hindbrain neural tube. Subsequently, they were seen in the neural tube and in the ganglion of the fifth cranial nerve (trigeminal ganglion). Later, they migrated beyond the trigeminal ganglion, i.e., into the mesenchyme of the first pharyngeal arch. Immunostaining with the neural crest cell marker, HNK-1, indicated that the emigrated neuroepithelial cells were HNK-1 negative. It is concluded that in the chick embryo some neuroepithelial cells emigrate at the site of attachment of the trigeminal nerve, migrate into the ganglion and then into the mesenchyme of the first arch. This cell population differs antigenically from the neural crest cells.

Animals↗

PubMed1996

Microglial cells internalize aggregates of the Alzheimer's disease amyloid beta-protein via a scavenger receptor.

Microglia are immune system cells associated with Alzheimer's disease plaques containing beta-amyloid (A beta). Murine microglia internalize microaggregates of fluorescently labeled or radioiodinated A beta peptide 1-42. Uptake was confirmed using aggregates of unlabeled A beta detected by immunofluorescence. Uptake of A beta was reduced by coincubation with excess acetyl-low density lipoprotein (Ac-LDL) or other scavenger receptor (SR) ligands, and Dil-labeled Ac-LDL uptake by microglia was blocked by excess A beta. CHO cells transfected with class A or B SRs showed significantly enhanced uptake of A beta. These results show that microglia express SRs that may play a significant role in the clearance of A beta plaques. Binding to SRs could activate inflammation responses that contribute to the pathology of Alzheimer's disease.

Alzheimer Disease↗

PubMed1998

Establishment of a new murine-phenotypic angiosarcoma cell line (ISOS-1).

A cell line, designated ISOS-1, was established from a tumor formed by transplantation of a human angiosarcoma into mice with severe combined immunodeficiency (SCID). The cells showed endothelial properties, based on the uptake of Dil-Ac-LDL and binding of UEA-I/GSA-I lectins, but were negative for CD11b and Pan Cytokeratin. However, the cells lost differentiated characteristics such as expression of von Willebrand factor, contact inhibition growth and tube formation activity. These findings indicate that ISOS-1 is a poorly-differentiated endothelial cell line. At the 81st passage, all of the cells were positive for H-2Dd in various intensity, but not HLA-ABC. The metaphase chromosomes consistently showed a characteristic mouse, but not human, telocentric form. Furthermore, this cell line produced fatal tumor growth in SCID mice and also in BALB/c mice. These results suggest that ISOS-1 is a murine-phenotypic angiosarcoma cell line.

Animals↗

PubMed2003

Backside contacted field effect transistor array for extracellular signal recording.

A new approach to the design of field-effect transistor (FET) sensors and the use of these FETs in detecting extracellular electrophysiological recordings is reported. Backside contacts were engineered by deep reactive ion etching and a gas phase boron doping process of the holes using planar diffusion sources. The metal contacts were designed to fit on top of the bonding pads of a standard industrial 22-pin DIL (dual inline) chip carrier. To minimise contact resistance, the metal backside contacts of the chips were electroless plated with gold. The chips were mounted on top of the bonding pads using a standard flip-chip process and a fineplacer unit previously described. Rat embryonic myocytes were cultured on these new devices (effective growth area 6 x 6 mm(2)) in order to confirm their validity in electrophysiological recording.

Action Potentials↗

PubMed1997

The in vitro construction of a tissue engineered bioprosthetic heart valve.

PROBLEM: Heart valve replacement with either a nonliving xenograft or a mechanical prosthesis is an effective therapy for valvular heart disease. Both of these approaches have limitations, including their inability to grow, repair, and remodel. In addition, a mechanical prosthesis requires long-term anticoagulation therapy. METHODS: This study demonstrates the in vitro creation of tissue engineered heart valve tissue using cardiovascular cells on degradable polymer matrices, 40 heart valve leaflets were created using this technique from two sources. Xenograft leaflets were created using human dermal fibroblasts and bovine aortic endothelial cells (n = 20) or allograft valve leaflets were created using sheep myofibroblasts and sheep endothelial cells (n = 20). A mixed sheep cell population was obtained consisting of endothelial cells and myofibroblasts. Endothelial cells were labelled with acethylated low density lipoprotein (Ac-Dil-LDL) and cells were separated into two groups using an activated cell sorter: LDL positive cells comprised of a pure endothelial cell population and LDL negative cells comprised of mixed cell population containing myofibroblasts and smooth muscle cells. The LDL negative cells were seeded on a synthetic polyglycolic acid (PGA) mesh and grown in vitro to form a tissue-like fibroblast-mesh core. Endothelial cells were then seeded onto the surface of the fibroblast-mesh core, forming a single monolayer. RESULTS: Histological evaluation of these constructs revealed an inner core of LDL negative cells and outer endothelial-like cells which were factor VIII positive. There was no evidence of capillary formation from endothelial cells invading the myofibroblasts and smooth muscle matrix and the endothelial lining appeared complete. CONCLUSIONS: It is feasible to construct allogenic heart valve tissue which could be used to make a valve.

Animals↗

PubMed1998

Gene transfer into rat heart-derived endothelial cells.

OBJECTIVE: Progressive graft arteriosclerosis is responsible for the majority of late deaths in cardiac transplant recipients. Despite many investigations, the pathogenesis of this disease remains undetermined and its control inadequate. A somatic gene transfer during the cold ischemic time and thus before transplantation might be a new therapeutic tool. This approach allows a long incubation time of the DNA and a safe transfer with liposomes and transferrin with less adverse effects for the organ recipient. METHODS: The target cells (microvascular endothelial cells (MVECs)) for this gene transfer were isolated from rat hearts by perfusion with collagenase via an aortic cannulae. The cells were purified by changing the medium 30 min after subcultivation in order to remove fibroblasts and smooth muscle cells. The endothelial cells (ECs) were identified by typical morphology and the uptake of Dil-Ac-LDL. The gene transfer was carried out with a beta-galactosidase reporter plasmid (pCMVbeta), cationic liposomes (Lipofectin), and transferrin. Different transfection solutions were prepared with or without serum, and with different plasmid-liposome ratios and transferrin concentrations. The transfer rate was monitored with a semiquantitative orthonitrophenyl-beta/-D-galactoside (ONPG) assay and histologically by X-Gal staining. The cytotoxicity of this procedure was determined with a colorimetric ELISA with Alamar blue. The cardioplegic property of the transfection solution was tested in a Langendorff perfusion system monitoring the coronary blood flow over time after a cold ischemic time of 4 h. RESULTS: The maximal gene expression could be detected after transfection with 4 microl Lipofectin, 2 microg pCMVbeta, and 16 microg transferrin/200 microl transfection solution. Under these conditions 60% of the cells showed a blue staining with X-Gal. Only 20% of the cells died during transfection. The lowest cytotoxicity during cold ischemic time for ECs was assessed with normal cell culture medium and the Buckberg solution. The best coronary flow rates after 4 h cold ischemia of the heart were measured for cardioplegia with St. Thomas and Buckberg solutions. In summary, the best transfection solution with a good cardioplegic property was the Buckberg solution. CONCLUSIONS: Finally, the results of this study show that an effective DNA delivery with a low toxicity into ECs is possible with a combination of liposomes and transferrin. This method might be useful for a safe and effective gene transfer into solid organs during the cold ischemic time and thus a therapeutic tool for chronic rejection.

Animals↗

PubMedDate not supplied

Endothelial precursor cells in the peripheral blood of pregnant women.

OBJECTIVE: To determine whether primitive endothelial precursor cells are present in the peripheral blood of pregnant compared with nonpregnant subjects and whether these precursor cells are of fetal or maternal origin. METHODS: Peripheral blood mononuclear cells from 13 pregnant women in the second trimester and from ten nonpregnant women and men were cultured for 8-10 weeks under conditions that promoted endothelial cell development. Early outgrowth (1 week culture) and late outgrowth (4-6 weeks) colonies were observed, their endothelial nature was investigated, and fluorescence in situ hybridization was performed to determine the origin of the colonies from pregnant women's specimens. RESULTS: Peripheral blood mononuclear cell cultures from all pregnant women and all nonpregnant controls yielded early-outgrowth endothelial cells. Late-outgrowth endothelial cells were observed in 61.5% (eight of 13) of pregnant subjects, but in none of the ten nonpregnant controls (chi(2) test; P <.01). The adherent cells stained positively for von Willebrand factor and incorporated Dil-Ac-LDL, confirming their endothelial origin. Fluorescence in situ hybridization analysis showed only X chromosome-specific signals and no Y chromosome-specific signals in the cells from the late-outgrowth endothelial cells in all pregnant women carrying either a male (n = 5) or a female (n = 8) fetus. CONCLUSION: Primitive endothelial precursor cells are present in most pregnant women during the second trimester. These cells appear to be of maternal origin.

Adult↗

PubMed2001

The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm.

Development of the arterial pole of the heart is a critical step in cardiogenesis, yet its embryological origin remains obscure. We have analyzed a transgenic mouse line in which beta-galactosidase activity is observed in the embryonic right ventricle and outflow tract of the heart and in contiguous splanchnic and pharyngeal mesoderm. The nlacZ transgene has integrated upstream of the fibroblast growth factor 10 (Fgf10) gene and comparison with the expression pattern of Fgf10 in pharyngeal mesoderm indicates transgene control by Fgf10 regulatory sequences. Dil labeling shows a progressive movement of cells from the pharyngeal arch region into the growing heart tube between embryonic days 8.25 and 10.5. These data suggest that arterial pole myocardium originates outside the classical heart field.

Animals↗

PubMedDate not supplied

The optic tract in embryonic hamsters: fasciculation, defasciculation, and other rearrangements of retinal axons.

The early development of the optic tract in hamsters was studied by labeling retinal axons with Dil applied to the eye, and then examining the labeled axons in flatmount preparations of the rostral brain stem. This technique permits a panoramic view of the entire retinal projection, from the chiasm to the caudal end of the superior colliculus. In the E11 embryo, retinal axons have reached the chiasm. They defasciculate as they emerge from the nerve, prior to reaching the ventral midline of the diencephalon, then converge again as they pass over to the opposite side. At the midline, many axonal trajectories crisscross, implying some shuffling of relative positions. Retinal axons are tightly bundled within the optic tract. Upon reaching the ventral border of the lateral geniculate body (LGB), they splay out over the nucleus, revealing a wavefront of pioneer axons individually distributed across the rostro-caudal extent of the LGB. Later-emerging retinal axons course over the surface of the thalamus in waves; subsequent waves of axons interdigitate between the lead fibers without fasciculating along them. Past the LGB, the axons undergo a second change in relative positions as the ribbon of fibers swerves caudally, prior to entering the superior colliculus. Retinal axons are tipped with growth cones of varying morphologies. No strong correlation is evident between the structural complexity of the growth cone and its position within the tract. In the majority of cases, ipsilaterally and contralaterally directed axons follow a similar developmental course along the optic tract, without any indication of a temporal lag in the ipsilateral projection as claimed in earlier reports. Understanding the changes in spatial distribution of embryonic retinal axons as they navigate along the optic tract provides a further step towards elucidating how point-to-point projections form in developing sensory systems.

Animals↗

PubMedDate not supplied

An in vitro study of retinotectal transmission in the chick: role of glutamate and GABA in evoked field potentials.

We have developed two brain slice preparations for studying tectofugal visual pathways in the chick: conventional, 400-microns slices ("thin slices"), and "thick slices" which encompass the rostral pole of the optic tectum and the contralateral optic nerve. Stimulation was delivered with a bipolar electrode positioned in stratum opticum in thin slices and in the contralateral optic nerve in thick slices. While the latter preparation provided a means of exclusively and unambiguously activating retinal afferents, several lines of evidence also indicated that the evoked field potentials in thin slices were chiefly consequent to retinal afferent excitation: (1) the similarity of evoked field potentials in thin slices to those in thick slice preparations; (2) their precise localization in retinorecipient layers as shown by prelabeling from retina with FITC-coupled cholera toxin; (3) transmission delays appropriate for retinal afferents as established with the thick slice preparation; (4) patterns of labeled afferents resulting from applications of Dil crystals to slices fixed after recording; and (5) the similarity in transmitter pharmacology between thin and thick slice preparations. Pharmacological manipulations carried out with bath-applied antagonists indicated that glutamate is the principal retinotectal transmitter. The broadly active glutamate receptor blocker, kynurenic acid, reversibly eliminated the postsynaptic component of the field potential as confirmed with 0 Ca2+ saline. A complete block was also effected by the non-NMDA antagonists CNQX and DNQX. The specific NMDA antagonist, AP5, caused a smaller and variable reduction in response amplitude. The GABA antagonist, bicuculline, caused a prolongation of the monosynaptic field epsp in retinorecipient layers and an enhancement of the long-latency, negative wave in cellular layers below, supporting a late, excitation-limiting role for this inhibitory transmitter.

2-Amino-5-phosphonovalerate↗