Biomedical subjects

H Dreyfus

Publications and source records attributed to H Dreyfus.

At least 37 records · Page 2Linked to original sources

PubMed1998

Adult human retinal neurons in culture: Physiology of horizontal cells.

PURPOSE: Adult postmortem human retinal neurons in long-term monolayer cultures were recorded to characterize the voltage- and transmitter-gated currents in putative human horizontal cells (HCs). METHODS: Enzymatically and mechanically dissociated human retinal cells were seeded on polylysine and laminin- coated coverslips. Cells were identified by immunocytochemistry with cell type-specific antibodies and recorded with the patch-clamp technique. RESULTS: Immunostaining and responses to voltage steps confirmed the survival of various retinal cell types. Horizontal cells were identified by their specific glutamate-modulated anomalous rectifier K+ current conductance. This identification was further confirmed by subsequent immunolabeling of dye-labeled recorded cells with an anti-parvalbumin antibody that selectively stained HCs in frozen human retinal sections. Horizontal cells generated voltage-gated currents classically observed in HCs from fish to mammals: a transient outward K+ current, a sustained outward K+ current, and an L-type (Ca2+ current. Na+ currents were observed in only a few HCs. As in other species, glutamate, gamma-aminobutyric acid (GABA), and glycine generated responses mediated by the activation of kainate/(RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), GABA(A), and glycine receptors, respectively. CONCLUSIONS: Various human retinal cell populations survive in vitro as indicated by immunolabeling with specific cell markers and by the diversity of responses to voltage steps. Human HCs exhibited extensive physiological similarities to HCs from other vertebrate species and a maintained expression of parvalbumin. These results constitute a comprehensive analysis of voltage- and transmitter-gated currents in a primate retinal neuron and validate the use of long-term monolayer culture of adult human neurons as a novel in vitro model for the study of human vision.

Aged↗

PubMed1997

Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro.

Glutamate is the major excitatory neurotransmitter in the retina, but excessive stimulation of its receptors leads to widespread neuronal stress and death. Both growth factors and gangliosides display important influences on responses to neuronal injury and degeneration. In this study, we have investigated the potential protective effects of two well characterized growth factors, epidermal and basic fibroblast growth factor (EGF and bFGF respectively), and the monosialoganglioside GM1, on cultured rat retinal neurons submitted to toxic levels of excitatory amino acids. Application of 1 mM glutamic acid reduced global neuronal viability by 80% when compared to control untreated cultures, whereas treatment with the glutamic acid agonist kainic acid (1 mM) led to specific, large decreases (75% reduction) in amacrine cell numbers. 24 h pretreatment with either EGF or bFGF (500 pM each) prevented the majority of excitatory amino acid-induced neuronal death, whereas similar treatment with 10(-5) M GM1 did not block neuronal degeneration. These findings demonstrate that EGF and bFGF act as neuroprotective agents against retinal excitotoxicity in vitro, whereas ganglioside GM1 is not effective in this particular paradigm.

Animals↗

PubMed1997

Dystrophins in developing retina: Dp260 expression correlates with synaptic maturation.

Dystrophin, the protein altered in Duchenne muscular dystrophy (DMD), is necessary for normal retinal function and exists in several isoforms. We examined the expression of dystrophin and utrophin proteins and transcripts in the rat retina at different developmental stages using Western blots and semi-quantitative RT-PCR. Our results revealed the presence of utrophin (DRP1), G-utrophin and/or DRP2 and four dystrophin isoforms (Dp427, Dp260, Dp140, Dp71) in the normal adult rat retina. Only Dp260 showed a marked progressive increase with age at both protein and mRNA levels. This variation is consistent with the establishment of synaptic functions in the developing retina and suggests a key role for this apo-dystrophin in synaptogenesis.

Animals↗

PubMed1997

Successive isolation and separation of the major lipid fractions including gangliosides from single biological samples.

Currently available techniques concerning extraction and characterization of the different lipids from biological specimens are designed for particular families and do not address consecutive isolation of lipid constituents in their globality. We describe here a simple, nondestructive chromatographic procedure that allows efficient elution and further analysis of the major lipid classes (neutral lipids, phospholipids, nonsialylated sphingolipids, and gangliosides) in their natural states from the same starting material. The procedure describes the use of solvent mixtures adapted to silicic acid column chromatography and permits 90-97% recovery of each of the above lipid groups. We have particularly concentrated on optimizing the efficient recovery of the diverse minor forms of gangliosides, free of other contaminants, from relatively small amounts of neural tissue. As model systems we have used in vivo and in vitro preparations of mammalian retina for which only fragmentary data are available on lipid composition. We show that relative to brain, retina contains, for example, twofold more sphingomyelin and sixfold more GD3 ganglioside. In turn, cultured retinal glial cells contain twofold higher levels of globoside and eightfold higher amounts of GM3 ganglioside with respect to intact retina. Compared to previously published techniques, we obtain improved total ganglioside recovery, with enrichment of poly-sialogangliosides. The technique presented here should be widely applicable to analyze global lipid composition of diverse biological samples.

Animals↗

PubMed1997

Activities of enzymes involved in the metabolism of platelet-activating factor in neural cell cultures during proliferation and differentiation.

Platelet-Activating Factor (PAF) is a potent lipid mediator involved in physiological and pathological events in the nervous tissue where it can be synthesized by two distinct pathways. The last reaction of the de novo pathway utilizes CDPcholine and alkylacetylglycerol and is catalyzed by a specific phosphocholinetransferase (PAF-PCT) whereas the remodelling pathway ends with the reaction catalyzed by lyso-PAF acetyltransferase (lyso-PAF AcT) utilizing lyso-PAF, a product of phospholipase A2 activity, and acetyl-CoA. The levels of PAF in the nervous tissue are also regulated by PAF acetylhydrolase that inactivates this mediator. We have studied the activities of these enzymes during cell proliferation and differentiation in two experimental models: 1) neuronal and glial primary cell cultures from chick embryo and 2) LA-N-1 neuroblastoma cells induced to differentiate by retinoic acid (RA). In undifferentiated neuronal cells from 8-days chick embryos the activity of PAF-PCT was much higher than that of lyso-PAF AcT but it decreased during the period of cellular proliferation up to the arrest of mitosis (day 1-3). During this period no significant changes of lyso-PAF AcT activity was observed. Both enzyme activities increased during the period of neuronal maturation and the formation of cellular contacts and synaptic-like junctions. The activity of PAF acetylhydrolase was unchanged during the development of the neuronal cultures. PAF-PCT activity did not change during the development of chick embryo glial cultures but lyso-PAF AcT activity increased up to the 12th day. RA treatment of LA-N-1 cell culture in proliferation decreased PAF-PCT activity and had no significant effect on lyso-PAF AcT and PAF acetylhydrolase indicating that the synthesis of PAF by the enzyme catalyzing the last step of the de novo pathway is inhibited when the LA-N-1 cells are induced to differentiate. These data suggest that: 1) in chick embryo primary cultures, both pathways are potentially able to contribute to PAF synthesis during development of neuronal cells particularly when they form synaptic-like junctions whereas, during development of glial cells, only the remodelling pathway might be particularly active on synthesizing PAF; 2) in LA-N-1 neuroblastoma cells PAF-synthesizing enzymes coexist and, when cells start to differentiate the contribution of the de novo pathway to PAF biosynthesis might be reduced.

Acetyltransferases↗

PubMed1997

Photoreceptor transplants increase host cone survival in the retinal degeneration (rd) mouse.

Retinal transplants offer a potentially interesting approach to treating human retinal degenerations, but so far little quantitative data are available on possible beneficial effects. We isolated photoreceptor layers from normal-sighted mice and grafted them into the subretinal space of retinal degeneration (rd) mice lacking rod photoreceptors. At 2 weeks after surgery, the numbers of residual host cone photoreceptors outside the graft zone were quantified following specific labelling. Examination of operated retinas revealed highly significantly greater numbers of surviving cones (mean of 38% more at 2 weeks) within the central field compared to sham-operated paired control retinas (p < 0.01). These are the first quantified data indicating a trophic effect of transplanted photoreceptors upon host cone cells. As cone cells are responsible for high acuity and colour vision, such data could have important implications not only for eventual therapeutic approaches to human retinal degenerations but also to understanding underlying interactions between retinal photoreceptors.

Animals↗

PubMed1997

Intravitreal injection of ganglioside GM1 after ischemia reduces retinal damage in rats.

BACKGROUND AND PURPOSE: Gangliosides are normal components of cell membranes and contribute to structural rigidity and membrane function. They have been shown to protect against various insults in the brain. We have shown previously that GM1 administered intraperitoneally before the induction of retinal ischemia provides a protective effect. This study evaluates the protective effect of GM1 administered intravitreally after ischemia on retinal lesions. METHODS: We induced retinal ischemia unilaterally in Long-Evans rats by increasing intraocular pressure to 160 mm Hg for 60 minutes. GM1 (20 microL x 10(-5) mol/L) or saline (20 microL) was injected into the vitreous 15 minutes after ischemia, and the postischemic survival time was either 8 or 15 days. The degree of retinal damage was assessed by histopathological study. RESULTS: Retinal ischemia led to reductions in thickness and cell number, principally in the inner retinal layers (39% to 80%) and to a lesser extent in the outer retinal layers (26% to 45%). Postischemic treatment with intravitreally injected GM1 conferred significant protection against retinal ischemic damage after both 8 and 15 days of survival time. After 8 days of reperfusion, the ischemia-induced loss in overall retinal thickness was reduced by 15% and those of the inner nuclear and plexiform layers by 44% and 17%, respectively. Ischemic-induced ganglion cell and inner nuclear cell density losses were reduced by 37% and 27%, respectively. After 15 days of reperfusion, approximately the same statistically significant differences could be observed in comparison with the 15-day saline-injected group. CONCLUSIONS: GM1 protects the rat retina from pressure-induced ischemic injury when given intravitreally after the insult. The protection provided by GM1 after initiation of retinal damage could be of therapeutic interest.

Animals↗

PubMed1996

Stimulation of endogenous ganglioside metabolism by neurotrophic growth factors in cultured retinal Müller glia.

Neurotrophic factors such as basic fibroblast and epidermal factor (bFGF and EGF respectively) are known to influence many differentiative processes, but their effects on an important group of glycosylated signalling molecules involved in neural differentiation, the gangliosides, are unknown. To study this possibility, we analyzed the effects of exogenously added bFGF and EGF upon the amount and type of endogenous gangliosides extracted from purified cultures of retinal Müller glial cells. A single addition of 500 pM bFGF or EGF for 48 h to such cultures led to significant increases in total ganglioside levels of 30-40%. Analysis of the distribution of specific ganglioside species within control and growth factor treated cells revealed that the precursor form GM3 formed 50-60% of the total ganglioside pool in all cases, the remainder being composed principally of GD1a (20%) with no detectable tri-sialogangliosides. Growth factor treatment for 48 h led to increases mainly in GM3, whereas longer exposure (96 h) of confluent glial cultures to growth factors additionally stimulated synthesis of GT1b. Furthermore, growth factor-induced ganglioside increases were dose-dependent, reaching maximal stimulation at 500 pM for bFGF. Incorporation of radiolabelled [3H]-glucosamine into glial cultures showed that ganglioside synthesis was stimulated 2-fold by the growth factors. To our knowledge these data constitute the first demonstration of neurotrophic factor stimulation of ganglioside levels in cells of central nervous system origin. Such complex interactions between peptide growth factors and gangliosides, if occurring in vivo, could have important consequences for retinal cell behaviour.

Animals↗

PubMed1996

Differential modulation of basic fibroblast and epidermal growth factor receptor activation by ganglioside GM3 in cultured retinal Müller glia.

Polypeptide growth factors and membrane-bound gangliosides are involved in cell signaling, including that observed in cells of neural origin. To analyze possible interactions between these two systems, we investigated the modulation of short- and long-term responses to basic fibroblast and epidermal growth factor (bFGF and EGF, respectively) in cultured retinal Müller glial cells following experimental modification of their ganglioside composition. These glial cells readily incorporated exogenously administered GM3 ganglioside, which was not substantially metabolized within 24 h. Such treatments significantly inhibited bFGF-induced DNA replication and cell migration, while having much less effect on analogous EGF-mediated behaviors. To explore GM3/growth factor interactions further, different aspects of glial metabolism in response to bFGF or EGF stimulation were examined: membrane fluidity, growth factor binding, global and individual changes in growth factor-induced phosphotyrosine levels, and growth factor-induced activation of mitogen-activated protein kinase. GM3 reduced the intensity of immunocytochemical labeling of phosphotyrosine-containing proteins within bFGF-stimulated cells and down-regulated FGF receptor activation and tyrosine phosphorylation of its cellular substrates, whereas similar parameters in EGF-stimulated cells were much less affected. Hence the data reveal a complex relationship in normal neural cells between polypeptide growth factors and membrane-bound gangliosides, which may participate in retinal cellular physiology in vivo.

Animals↗

PubMed1996

Ganglioside effects on basic fibroblast and epidermal growth factor receptors in retinal glial cells.

Gangliosides have long been implicated in cell growth regulation and play an important role as modulators in protein phosphorylation. In order to better understand how glycosphingolipids and growth factors interact, we examined the modulation of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) effects on retinal Müller glial cells (RMG), following modification of their GG composition. Treatment of MG cells with GG (GM1, GT1b) and asialoGM1 resulted in modifications of several aspects of cellular responses to EGF- and FGF-receptor (R) activation: mitogenesis, cell migration, tyrosine phosphorylation of the EGF-R and FGF-R and even their cellular substrates were particularly influenced by GG. Indeed GG caused modifications of EGF-R and FGF-R autophosphorylation kinetics. GG long term effects (mitogenesis and migration) correlate with short term effects (tyrosine phosphorylation) and differences in receptor tyrosine kinase signalling could explain the specificity in growth factor responses.

Animals↗

PubMed1996

Simplified ganglioside composition of photoreceptors compared to other retinal neurons.

PURPOSE: The quantitative and qualitative ganglioside composition of retinal photoreceptor cells is unknown. The aim of this study was to analyze the lipid, especially ganglioside, make-up of photoreceptors compared to other retinal cells. METHODS: Retinas from adult normal rats were mechanically separated into outer (photoreceptors) and inner (other retinal neurons and glia) halves be planar vibratome sectioning. Total lipids were extracted, and each fraction (neural, phospholipids, and glycosphingolipids) was eluted sequentially by column chromatography and quantitated through high-performance thin layer chromatogram analysis. Similar analyses were performed on entire retinas from adult normal rats, adult dystrophic rats lacking photoreceptors (RCS-rdy-p+ strain), and isolated photoreceptor outer segments. RESULTS: Whereas phospholipids were distributed equally between the two halves, inner retina contained significantly more cholesterol (68% total) and gangliosides (74% total) than outer retina on a unit protein basis. The distribution on a percent molar basis of specific gangliosides also was significantly different between the two halves: Outer retina was dominated by GD3 (45% total ganglioside) and contained only trace amounts (<4%) of complex species (GT1b and GQ1b); inner retina was more typical of mature brain tissue exhibiting substantial amounts (approximately 25%) of more complex species. These data were supported by lipid compositional analyses of mutant photoreceptor-less retina. However, isolated outer segments resembled whole retina in containing higher levels of complex gangliosides. CONCLUSIONS: These data indicate that, compared to other central nervous system-derived neurons, photoreceptor cell body membranes exhibit a highly unusual simplified ganglioside composition. Such an unusual neuronal lipid composition may reflect structural adaptations to their specialized function.

Animals↗

PubMed1996

Monosialoganglioside GM1 reduces ischemia--reperfusion-induced injury in the rat retina.

PURPOSE: Gangliosides are normal components of cell membranes, contribute to structural rigidity and membrane function, and have been shown to protect against various insults to the brain. This study evaluates the effect of exogenously administered monosialoganglioside GM1 on retinal damage induced by transient retinal ischemia and reperfusion. METHODS: Retinal ischemia was induced unilaterally in Long Evans rats by increasing intraocular pressure to 160 mm Hg for 60 minutes. GM1 (30 mg/kg, intraperitoneally) or buffer controls were administered at 48 hours, and 15 minutes before ischemia, and survival time after ischemia was either 8 or 15 days. The degree of retinal damage was assessed by histopathologic study according to Hughes' quantification of ischemic damage. RESULTS: Retinal ischemia led to significant reductions in thickness and cell number, principally in the inner retinal layers (30% to 80%), and to a lesser extent in the outer retinal layers (18% to 42%). Pretreatment with intraperitoneally injected monosialoganglioside GM1 conferred significant protection against retinal ischemic damage either 8 or 15 days after ischemic survival time. After 8 days reperfusion, the ischemic-induced loss in overall retinal thickness was reduced by 70%, and those of the inner nuclear and plexiform layers were reduced by 77% and 44%, respectively. Ischemic-induced ganglion cell, inner nuclear, and outer nuclear layer cell density losses were reduced by 45%, 40%, and 57%, respectively. After 15 days of reperfusion, approximately the same statistically significant differences could be observed in comparison with the 15-day ischemic--reperfusion group. CONCLUSIONS: Monosialoganglioside GM1 protects the rat retina from pressure-induced ischemic injury when administered intraperitoneally 2 days before insult. This protection afforded by GM1 can be observed even after 8 days or 15 days of reperfusion.

Animals↗

PubMed1996

[Retinal grafts: biological problems and clinical stakes].

Retinal transplantation, formerly perceived as unrealistic, has become over the past decade a major clinical and biological undertaking in several laboratories and eye clinics. We describe the insights gained through the pioneering experimental works of Del Cerro et al, Turner et al, Gouras et al, Aramant et al, Lund et al e.g. the survival of transplants, the lack of immune response to photoreceptors, their integration and expression of neuronal markers, but also the dysplastic arrangement into rosettes and the lack of a definitive proof for functionality. Our laboratory has undertaken to establish the trophic and synaptic functions of sheets of photoreceptors transplanted, as described by Silverman et al, in the subretinal space of mutant rd mice carrying a retinal degeneration similar to human retinitis pigmentosa. Clinical applications to this condition as well as in cases of end-stage age related macular degeneration are discussed.

Animals↗

PubMed1996

Survival and regeneration of adult human and other mammalian photoreceptors in culture.

PURPOSE: Fully mature neurons of central nervous system origin generally are considered unable to survive for extended periods of time in simple culture conditions. The authors report that adult and aged human, porcine, and rodent retinal neurons, including rod and cone photoreceptors, constitute an exception to this idea. METHODS: Cells were dissociated from human postmortem retinas, adult mammalian retinas, and selected brain regions and were seeded into tissue culture plates and left to develop as monolayer cultures for up to 2 months. A battery of antibody markers was used to identify the nature and morphology of the cells in vitro. RESULTS: Photoreceptor cell survival of rods and cones was observed routinely when the delay between the time of death until culture preparation was 50 hours or less, compatible with current eye bank practice. Two-week-old cultures were formed of rod photoreceptors, representing approximately 50% of neuronal cell types; cone photoreceptors, representing 5% to 30% of neuronal cell types; other retinal neurons (especially amacrine cells approximately 20%); and retinal glial cells, present in variable numbers. Glial cells were essential for long-term photoreceptor survival and neurite outgrowth. Adult mammalian brain neurons isolated under the same conditions did not survive. CONCLUSIONS: Fully adult human and other mammalian retinal neurons, including photoreceptors, exhibit remarkable plasticity in vitro, and such monolayer models may have applications in physiological, pharmacologic, and toxicologic studies of human and other mammalian retina.

Aged↗

PubMed1995

Breast cancer in elderly women: a retrospective analysis of combined treatment with tamoxifen and once-weekly irradiation.

PURPOSE: To evaluate retrospectively the efficacy of combined modality treatment (hormone therapy and hypofractionated radiotherapy) in a population of very elderly women with breast cancer. METHODS AND MATERIALS: Records on 70 patients of median age 81 years, treated between January 1988 and February 1994, whose median follow-up is now 36 months, have been evaluated. Information obtained included clinical stage at diagnosis, histology, tumor grading, hormone receptor levels, details of treatment, type of failure, survival data, and status at last follow-up examination. Treatment consisted of Tamoxifen 20 mg daily and a hypofractionated course of high dose-per-fraction once-weekly radiotherapy. In the majority of cases this consisted of seven exposures of 6.5 Gy (five to the involved breast, and two to the tumor bed) given over 6 weeks, on a 60Co unit. Nodes were treated when clinically involved, to a dose of 27.5-30 Gy in five to six fractions. RESULTS: At median follow-up of 36 months, the overall survival rate is 87% [confidence interval (CI) 78-95%], the disease specific survival rate is 88% (CI 80-96%), and 72% (CI 60-84%) of patients are free of disease. The local control rate at 36 months is 86% (CI 76-95%). When analyzed by T stage, 81% of T1 patients, 96% of T2 patients, 60% of T3 patients and, paradoxically 100% of T4 patients were in local control at 36 months, although at that point there were just four such patients available for consideration in the T4 group. Initial response to hormone therapy does not appear to be a predictive indicator for ultimate loco-regional control. There is a trend towards greater probability of loco-regional failure if total dose delivered to the breast is less than 35 Gy. CONCLUSIONS: Women of elderly age are often denied combined modality therapy, because of coexistant disease or fears held by the responsible physicians that elderly patients are unable to tolerate surgery or protracted courses of radiotherapy. Consequently, many are treated by tamoxifen alone with poor results. This study demonstrates that very high rates of loco-regional control are achievable using hormonal treatment combined with high dose-per-fraction once-weekly radiotherapy.

Aged↗

PubMed1995

Molecular species analysis of 1,2-diglycerides on phorbol ester stimulation of LA-N-1 neuroblastoma cells during proliferation and differentiation.

1,2-Diacyl-sn-glycerol (DAG) is a product of cell activation that has emerged as an important intracellular messenger whose primary function appears to be the activation of protein kinase C. They originate by the activation of phospholipases, which hydrolyze different phospholipids depending on the external stimulus and the nature of the cells, leading to the production of different molecular species. In the present study the quantitative changes in the total mass and the molecular species of DAG formed on phorbol ester (12-O-tetradecanoyl-phorbol 13-acetate) stimulation were investigated in proliferating and retinoic acid (RA)-differentiated human LA-N-1 cells. The TPA treatment of both cell types elicited an increase in the total amount of DAG. The increase was biphasic; i.e., an initial peak at 2-5 min was followed by a sustained increase that persisted for > 30 min. The analysis of the molecular species of DAG and phospholipids showed that in proliferating LA-N-1 cells, the DAG increase corresponds to the production of mainly saturated/monounsaturated (16:0-18:1, 18:0-18:1) and saturated/saturated (16:0-16:0, 16:0-18:0) species, suggesting that they originate essentially from the hydrolysis of phosphatidylcholine. In contrast, RA-differentiated cells responded to TPA treatment by increasing the level of saturated/polyunsaturated (16:1-22:6, 18:0-22:6, 16:0-20:4, 18:0-20:4) and monounsaturated/monounsaturated (18:1-18:1) species, suggesting mainly a phosphatidylethanolamine origin. These findings indicate that the treatment of LA-N-1 cells with TPA generates different molecular species of DAG depending on their physiological state. These observations suggest in turn that different phospholipases are activated by TPA in proliferating and RA-differentiated cells.

Cell Differentiation↗

PubMed1995

Management of breast cancer in the elderly.

This paper examines data which has been published on breast cancer in the elderly and concludes that, wherever possible, combined modality treatment should be offered to elderly breast cancer patients. It appears from an examination of the literature that single modality treatment in the form of hormone treatment often results in very high rates of loco-regional recurrence.

Age Factors↗

PubMed1994

Survival and regeneration of adult human photoreceptors in vitro.

Adult human retina from enucleated ocular tissue was enzymatically dissociated and plated into plastic dishes for cell culture in serum-supplemented medium. Within a few days following seeding, islands of glial-like cells with rounded neurons growing on top of them were visible. Immunocytochemical labelling of these cultures revealed that virtually all the surviving neurons (> 98%) were rod photoreceptors, and that they extended long neurites across the glial cell surface. Hence, adult human photoreceptors retain a remarkable capacity for survival and regrowth, and such preparations may be of value for physiopathological and retinal grafting studies.

Antibodies↗