Suppression of kindled seizures by paracrine adenosine release from stem cell-derived brain implants.

Purpose: Stem cells and their derivatives have emerged as a promising tool for cell-based drug delivery because of (a) their unique ability to differentiate into various somatic cell types, (b) the virtually unlimited donor source for transplantation, and (c) the advantage of being amenable to a wide spectrum of genetic manipulations. Previously, adenosine-releasing embryonic stem (ES) cells have been generated by disruption of both alleles of adenosine kinase (Adk-/-). Lack of ADK did not compromise the cells' differentiation potential into embryoid bodies or glial precursor cells.

Seizure suppression and lack of adenosine A1 receptor desensitization after focal long-term delivery of adenosine by encapsulated myoblasts.

Adenosine is an important inhibitory modulator of brain activity. In a previous ex vivo gene therapy approach, local release of adenosine by encapsulated fibroblasts implanted into the vicinity of an epileptic focus, was sufficient to provide transient protection from seizures (Huber, A., Padrun, V.

Lentiviral nigral delivery of GDNF does not prevent neurodegeneration in a genetic rat model of Parkinson's disease.

Viral delivery of glial cell line-derived neurotrophic factor (GDNF) currently represents one of the most promising neuroprotective strategies for Parkinson's Disease (PD). However, the effect of this neurotrophic factor has never been tested in the newly available genetic models of PD based on the viral expression of mutated alpha-synuclein. In this study, we evaluated the ability of lentiviral vectors coding for GDNF (lenti-GDNF) to prevent nigral dopaminergic degeneration associated with the lentiviral mediated expression of the A30P mutant human alpha-synuclein (lenti-A30P).

Survival of encapsulated human primary fibroblasts and erythropoietin expression under xenogeneic conditions.

Allogeneic cells are the most attractive source for cell transplantation, as the use of xenogeneic cells is hampered by safety concerns and the use of autologous cells involves practical difficulties. The immune rejection of allogeneic cells can be overcome by physical immunoprotection provided by polymer encapsulation. To study the variability of cell and donor sources, we compared different primary human cells as candidates for gene therapy-mediated delivery of human erythropoietin (hEpo).

Encapsulated GDNF-producing C2C12 cells for Parkinson's disease: a pre-clinical study in chronic MPTP-treated baboons.

Glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophic factor with restorative effects in a variety of rodent and primate models of Parkinson's disease (PD), could be of therapeutic value to PD. In this study, we show that intraventricular chronic infusion of low doses of GDNF using encapsulated genetically engineered C2C12 cells can exert: (1) transient recovery of motor deficits (hypokinesia); (2) significant protection of intrinsic striatal dopaminergic function in the immediate vicinity of the site of implantation of the capsule in the caudate nucleus, and (3) significant-long-lasting-neurotrophic properties at the nigral level with an increase volume of the cell bodies. These observations confirm the potent neurorestorative potential of GDNF in PD and the safety/efficacy of the encapsulation technology as a means to deliver in situ this neurotrophic cytokine even using an intraventricular approach.

In vivo calcium deposition on polyvinyl alcohol matrix used in hollow fiber cell macroencapsulation devices.

The encapsulation of genetically modified cells represents a promising approach for the delivery of therapeutic proteins. The functionality of the device is dependent on the characteristics of the biomaterials, the procedures used in its confection and the adaptability of the encapsulated cells in the host. We report conditions leading to the development of calcifications on the polyvinyl alcohol (PVA) matrix introduced in hollow fiber devices for the encapsulation of primary human fibroblasts implanted in mice.

Cell therapy using encapsulated cells producing endostatin.

Despite aggressive surgery and post-operative radiation and chemotherapy, the prognosis is poor for glioblastoma patients. Anti-angiogenic therapy with compounds such as endostatin could delay the onset of relapse. However, the short systemic half-life of this proteins as well as the blood-brain barrier makes the use of this therapy difficult for brain cancer patients.

Prevention of the initial host immuno-inflammatory response determines the long-term survival of encapsulated myoblasts genetically engineered for erythropoietin delivery.

The present study investigates the respective roles of both the host immune response and the metabolic requirements in determining the long-term survival of erythropoietin-secreting myoblasts within encapsulating polymer membranes. Hollow-fiber capsules loaded with a high density of erythropoietin-secreting C(2)C(12) myoblasts survived poorly in the subcutaneous tissue of syngeneic mice, inducing variable hematocrit responses. To determine the role and the nature of the host response, recipients were treated with anti-inflammatory (diclofenac) and immunosuppressive (dexamethasone, FK506) agents.

Lentiviral-mediated RNA interference.

RNA interference (RNAi) is a form of posttranscriptional gene silencing mediated by short double-stranded RNA, known as small interfering RNA (siRNA). These siRNAs are capable of binding to a specific mRNA sequence and causing its degradation. The recent demonstration of a plasmid vector that directs siRNA synthesis in mammalian cells prompted us to examine the ability of lentiviral vectors to encode siRNA as a means of providing long-term gene silencing in mammalian cells.

Sialylated form of the neural cell adhesion molecule (NCAM): a new tool for the identification and sorting of beta-cell subpopulations with different functional activity.

To clarify the relationship between variations in beta-cell mass and pancreatic function, we investigated the possibility to analyze, quantify, and sort beta-cell subpopulations with different functional maturity. To this aim, we tested the reliability of the sialylated form of neural cell adhesion molecule (NCAM) (PSA-NCAM) as a marker of beta-cell functional activity. Islet cells isolated from adult rats were analyzed for their PSA-NCAM abundance using an anti-PSA-NCAM antibody.

Glucose-6-phosphatase in the insulin secreting cell line INS-1.

The glucose-6-phosphatase system of the glucose sensitive insulin secreting rat insulinoma cells (INS-1) was investigated. INS-1 cells contain easily detectable levels of glucose-6-phosphatase enzyme protein (assessed by Western blotting) and have a very significant enzymatic activity. The features of the enzyme (Km and Vmax values, sensitivity to acidic pH, partial latency, and double immunoreactive band) are similar to those of the hepatic form.

Human gingival crevicular fluid contains MRP8 (S100A8) and MRP14 (S100A9), two calcium-binding proteins of the S100 family.

Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects.

Lentivirus-mediated Bcl-2 expression in betaTC-tet cells improves resistance to hypoxia and cytokine-induced apoptosis while preserving in vitro and in vivo control of insulin secretion.

betaTC-tet cells are conditionally immortalized pancreatic beta cells which can confer long-term correction of hyperglycemia when transplanted in syngeneic streptozocin diabetic mice. The use of these cells for control of type I diabetes in humans will require their encapsulation and transplantation in non-native sites where relative hypoxia and cytokines may threaten their survival. In this study we genetically engineered betaTC-tet cells with the anti-apoptotic gene Bcl-2 using new lentiviral vectors and showed that it protected this cell line against apoptosis induced by hypoxia, staurosporine and a mixture of cytokines (IL-1beta, IFN-gamma and TNF-alpha).

Caffeine releases a glucose-primed endoplasmic reticulum Ca2+ pool in the insulin secreting cell line INS-1.

Caffeine mobilized an intracellular Ca2+ pool in intact fura-2-loaded INS-1 cells in suspension exposed to high (16 mM) [glucose], while a minor effect was observed with low (2 mM) [glucose]. Cells were kept in a medium containing diaxozide or no Ca2+ to prevent the influx of extracellular Ca2+. The caffeine-sensitive intracellular Ca2+ pool was within the endoplasmic reticulum since it was depleted by the inhibitor of the reticular Ca2+ pumps thapsigargin and the InsP3-dependent agonist carbachol.

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation.

Short-term insulin-induced glycogen formation in primary hepatocytes as a screening bioassay for insulin action.

We describe a novel bioassay to measure specific insulin-like activity in primary cultures of rat hepatocytes by determination of [3H]glycogen from d-[6-3H]glucose. The dose-response curve of insulin in this assay exhibited an EC50 of 0.42 (+/-0.

Functional analysis of a conditionally transformed pancreatic beta-cell line.

Development of beta-cell lines for cell therapy of diabetes is hindered by functional deviations of the replicating cells from the normal beta-cell phenotype. In a recently developed cell line, denoted betaTC-tet, derived from transgenic mice expressing the SV40 T antigen (Tag) under control of the tetracycline (Tc) gene regulatory system, growth arrest can be induced by shutting off Tag expression in the presence of Tc. Here, we compared differentiated cell functions in dividing and growth-arrested betaTC-tet cells, both in culture and in vivo.

Pancreatic beta-cell regeneration after 48-h glucose infusion in mildly diabetic rats is not correlated with functional improvement.

We investigated the effect of glucose infusion on beta-cell regeneration in rats made mildly diabetic by a single injection of low dosage (35 mg/kg) streptozotocin (STZ). Nondiabetic (ND) and STZ rats were submitted to a 48-h glucose infusion (hyperglycemia approximately 22 mmol/l in both groups: ND and STZ hyperglycemic-hyperinsulinemic [ND HG-HI and STZ HG-HI rats]). Before infusion, beta-cell mass was 65% lower in STZ rats than in ND rats (2.

Histones and basic polypeptides activate Ca2+/cation influx in various cell types.

Histone H2A (1-10 microg/ml) added to Ehrlich ascite cell suspensions promoted: (i) Ca2+ influx, but no apparent intracellular Ca2+ mobilization; (ii) plasma-membrane depolarization and Na+ influx in Ca2+-free medium, which were recovered by Ca2+ readmission; (iii) influx of other cations such as Ba2+, Mn2+, choline+ and N-methyl-d-glucamine+, but not of propidium+, ethidium bromide and Trypan Blue. H2A-induced Ca2+ influx and cell depolarization were: (i) blocked by La3+ and Gd3+, but not by various inhibitors of receptor-activated Ca2+-influx pathways/channels; (ii) mimicked by various basic polypeptides, with Mr>4000; (iii) prevented or reversed by polyanions such as polyglutamate or heparin; (iv) present in other cell types, such as Jurkat, PC12 and Friend erythroleukaemia cells, but virtually absent from rat hepatocytes and thymocytes. We conclude that cationic proteins/polypeptides, by interacting in a cell-specific manner with the cell surface, can activate in those cells putative non-selective Ca2+ channels and membrane depolarization.

Glucose-induced insulin secretion in INS-1 cells depends on factors present in fetal calf serum and rat islet-conditioned medium.

To study the regulation of growth and differentiated function of insulin-secreting cells, the rat insulinoma cell line INS-1 was cultured in a defined serum-free medium containing prolactin, IGF-I, and triiodothyronine, which was originally reported to maintain insulin secretion of islet cells. Growth and viability, as well as cellular insulin content of INS-1 cells in the defined medium, were comparable to the control cells cultured in the complete medium containing 10% fetal calf serum. However, after a 3-day culture in this medium, insulin secretion in response to glucose, pyruvate, and leucine was markedly blunted compared with the control cells (-78, -68, and -56%, respectively), whereas the response to 30 mmol/l K+ was only slightly decreased.

Anti-apoptotic effect of Bcl-2 overexpression in RIN cells infected with Semliki Forest virus.

RIN cells were infected with recombinant Semliki Forest virus (SFV) particles containing the LacZ gene. X-gal staining showed 100% infectivity of the cell cultures and high-level expression of bacterial beta-galactosidase in these cells. The cytopathogenic effects of the SFV infection were studied by measuring the viability of the RIN cells.

Functional N-methyl-D-aspartate receptors in O-2A glial precursor cells: a critical role in regulating polysialic acid-neural cell adhesion molecule expression and cell migration.

The capacity for long-distance migration of the oligodendrocyte precursor cell, oligodendrocyte-type 2 astrocyte (O-2A), is essential for myelin formation. To study the molecular mechanisms that control this process, we used an in vitro migration assay that uses neurohypophysial explants. We provide evidence that O-2A cells in these preparations express functional N-methyl-D-aspartate (NMDA) receptors, most likely as homomeric complexes of the NR1 subunit.

Glucose-stimulated insulin secretion correlates with changes in mitochondrial and cytosolic Ca2+ in aequorin-expressing INS-1 cells.

Nutrient-stimulated insulin secretion is dependent upon the generation of metabolic coupling factors in the mitochondria of the pancreatic B cell. To investigate the role of Ca2+ in mitochondrial function, insulin secretion from INS-1 cells stably expressing the Ca2+-sensitive photoprotein aequorin in the appropriate compartments was correlated with changes in cytosolic calcium ([Ca2+]c) and mitochondrial calcium ([Ca2+]m). Glucose and KCl, which depolarize the cell membrane, as well as the Ca2+-mobilizing agonist, carbachol (CCh), cause substantial increases in [Ca2+]m which are associated with smaller rises in [Ca2+]c.

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells.

The mechanism underlying the generation of cytosolic free Ca2+ ([Ca2+]i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 microM, 61% of cells displayed [Ca2+]i oscillations with variable patterns. The bombesin-induced [Ca2+]i oscillations could last more than 1 h and glucose was required for maintaining these [Ca2+]i fluctuations.

Postreceptor signalling of growth hormone and prolactin and their effects in the differentiated insulin-secreting cell line, INS-1.

Signal transduction of two mitogens for pancreatic beta-cells, GH and PRL, was investigated using the differentiated insulin-secreting cell line, INS-1. Addition of human GH (hGH) or ovine PRL in a serum-substitute medium increased growth, insulin content, and nutrient metabolism evaluated by tetrazolium salt reduction. hGH, bovine GH (bGH), and PRL also stimulated [3H]thymidine incorporation in a dose-dependent manner (1 pM - 1 nM).