Involvement of glial cells in the neurotoxicity of parathion and chlorpyrifos.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to investigate the influence of glial cells on the neurotoxicity of two organophosphorus pesticides (OPs), chlorpyrifos and parathion. Mixed-cell aggregate cultures were treated continuously for 10 days between DIV 5 and 15. Parathion induced astrogliosis at concentration at which MAP-2 immunostaining, found here to be more sensitive than neuron-specific enzyme activities, was not affected. In contrast, chlorpyrifos induced a comparatively weak gliotic reaction, and only at concentrations at which neurons were already affected. After similar treatments, increased neurotoxicity of parathion and chlorpyrifos was found in aggregate cultures deprived of glial cells. These results suggest that glial cells provide neuroprotection against OPs toxicity. To address the question of the difference in toxicity between parathion and chlorpyrifos, the toxic effects of their leaving groups, p-nitrophenol and trichloropyridinol, were studied in mixed-cell aggregates. General cytotoxicity was more pronounced for trichloropyridinol and both compounds had similar toxic effects on neuron-specific enzyme activities. In contrast, trichloropyridinol induced a much stronger decrease in glutamine synthetase activity, the enzymatic marker of astrocytes. Trichloropyridinol may exert a toxic effect on astrocytes, compromising their neuroprotective function, thus exacerbating the neurotoxicity of chlorpyrifos. This is in line with the suggestion that glial cells may contribute to OPs neurotoxicity, and with the view that OPs may exert their neurotoxic effects through different mechanisms.

Evaluation of the interactions between multiwalled carbon nanotubes and caco-2 cells

The aim of this study was to determine whether multiwalled carbon nanotubes (MWNCT) are taken up by and are toxic to human intestinal enterocytes using the Caco-2 cell model. Caco-2 cells were exposed to 50 ?g/ml MWCNT (oxidized or pristine) for 24 h, and experiments were repeated in the presence of 2.5 mg/L natural organic matter. Cells displayed many of the properties that characterize enterocytes, such as apical microvilli, basolateral basement membrane, and glycogen. The cell monolayers also displayed tight junctions and electrical resistance. Exposure to pristine and oxidized MWCNT, with or without natural organic matter, did not markedly affect viability, which was assessed by measuring activity of released lactate dehydrogenase (LDH) and staining with propidium iodide. Ultrastructural analysis revealed some damage to microvilli colocalized with the MWCNT; however, neither type of MWCNT was taken up by Caco-2 cells. In contrast, pristine and oxidized MWCNT were taken up by the macrophage RAW 264.7 line. Our study suggests that intestinal enterocytes cells do not take up MWCNT. [Authors]

Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.

Prolonged deprivation of food induces dramatic changes in mammalian metabolism, including the release of large amounts of fatty acids from the adipose tissue, followed by their oxidation in the liver. The nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPARalpha) was found to play a role in regulating mitochondrial and peroxisomal fatty acid oxidation, suggesting that PPARalpha may be involved in the transcriptional response to fasting. To investigate this possibility, PPARalpha-null mice were subjected to a high fat diet or to fasting, and their responses were compared with those of wild-type mice. PPARalpha-null mice chronically fed a high fat diet showed a massive accumulation of lipid in their livers. A similar phenotype was noted in PPARalpha-null mice fasted for 24 hours, who also displayed severe hypoglycemia, hypoketonemia, hypothermia, and elevated plasma free fatty acid levels, indicating a dramatic inhibition of fatty acid uptake and oxidation. It is shown that to accommodate the increased requirement for hepatic fatty acid oxidation, PPARalpha mRNA is induced during fasting in wild-type mice. The data indicate that PPARalpha plays a pivotal role in the management of energy stores during fasting. By modulating gene expression, PPARalpha stimulates hepatic fatty acid oxidation to supply substrates that can be metabolized by other tissues.

Dicarboxylic amino acids and glycine-betaine regulate chaperone-mediated protein-disaggregation under stress.

Active protein-disaggregation by a chaperone network composed of ClpB and DnaK + DnaJ + GrpE is essential for the recovery of stress-induced protein aggregates in vitro and in Escherichia coli cells. K-glutamate and glycine-betaine (betaine) naturally accumulate in salt-stressed cells. In addition to providing thermo-protection to native proteins, we found that these osmolytes can strongly and specifically activate ClpB, resulting in an increased efficiency of chaperone-mediated protein disaggregation. Moreover, factors that inhibited the chaperone network by impairing the stability of the ClpB oligomer, such as natural polyamines, dilution, or high salt, were efficiently counteracted by K-glutamate or betaine. The combined protective, counter-negative and net activatory effects of K-glutamate and betaine, allowed protein disaggregation and refolding under heat-shock temperatures that otherwise cause protein aggregation in vitro and in the cell. Mesophilic organisms may thus benefit from a thermotolerant osmolyte-activated chaperone mechanism that can actively rescue protein aggregates, correctly refold and maintain them in a native state under heat-shock conditions.

Genetic variability of a population of Aedes aegypti from Paraná, Brazil, using the mitochondrial ND4 gene

Genetic variability of a population of Aedes aegypti from Paraná, Brazil, using the mitochondrial ND4 gene. To analyze the genetic variability of populations of Aedes aegypti, 156 samples were collected from 10 municipalities in the state of Paraná, Brazil. A 311 base pairs (bp) region of the NADH dehydrogenase subunit 4 (ND4) mitochondrial gene was examined. An analysis of this fragment identified eight distinct haplotypes. The mean genetic diversity was high (h = 0.702; p = 0.01556). AMOVA analysis indicated that most of the variation (67%) occurred within populations and the F ST value (0.32996) was highly significant. F ST values were significant in most comparisons among cities. The isolation by distance was not significant (r = -0.1216 and p = 0, 7550), indicating that genetic distance is not related to geographic distance. Neighbor-joining analysis showed two genetically distinct groups within Paraná. The DNA polymorphism and AMOVA data indicate a decreased gene flow in populations from Paraná, which can result in increased vectorial competence.

Investigating mineralocorticoid hypertension.

About 3% of our hypertensive patients have high blood pressure induced by corticosteroids. Muscle weakness, tiredness, polyuria and polydipsia may indicate hypokalaemia. Hypokalaemic hypertension in the presence of a low plasma renin activity is the typical finding of corticosteroid hypertension. The most frequent cause of corticosteroid hypertension is primary aldosteronism (Conn's syndrome) due to an adrenal adenoma or bilateral hyperplasia of the adrenal glands. The plasma concentration of aldosterone and the ratio between plasma aldosterone and renin concentrations are high, and the kaliuresis exceeds 30 mmol/24 h in the presence of hypokalaemia. Adrenal carcinomas are rare and very malignant. The localization of an adrenal tumour is made by computer tomography (CT-scan) or nuclear magnetic resonance imaging and by measurement of the aldosterone/cortisol concentrations in the adrenal venous blood. Adenomas are removed under laparoscopy, and adrenal hyperplasias are treated with spironolactone (50-400 mg daily) or amiloride (5-30 mg daily). In rare cases (<1%), excessive stimulation of the mineralocorticoid receptor is due to cortisol (apparent mineralocorticoid excess, Cushing's disease, liquorice, or hereditary deficiency of 11beta-hydroxysteroid dehydrogenase) or to a chimeric gene coding for 11beta-hydroxylase (CYP11B1/CYP11B2). In these rare cases, the synthesis of aldosterone is under the control of the adrenocorticotrophic hormone, so treatment with glucocorticoids (dexamethasone 0.25-1.0 mg daily) is therefore possible (glucocorticoid-remediable aldosteronism). Excessive deoxycorticosterone (DOC) causes the same symptoms and signs as hyperaldosteronism. Excessive DOC is found in patients with adrenal tumours that secrete DOC, in those with hereditary or acquired disorders with dysfunctioning glucocorticoid receptors, or in those with congenital hyperplasia of the adrenal glands (deficiency of 17alpha-hydroxylase or 11beta-hydroxylase). Liddle's syndrome is a constitutive hyperactivity of the transepithelial transport of sodium, which under normal conditions is controlled by the mineralocorticoid receptor. Plasma renin and aldosterone concentrations are suppressed and the plasma potassium concentration may be normal. In contrast, plasma aldosterone and renin concentrations are increased in patients with hypokalaemic hypertension which represents secondary aldosteronism. The increased aldosterone is the consequence of stimulated renin activity due to renal or renovascular or other disorders, antihypertensive drugs or other medications. In conclusion, a work-up for corticosteroid-induced hypertension is indicated in patients with hypokalaemic hypertension and in those with severe hypertension even in the absence of hypokalaemia, and in hypertensive patients with a family history of cardiovascular diseases.

Outcome and patterns of failure in testicular lymphoma: a multicenter Rare Cancer Network study.

PURPOSE: To assess the outcome and patterns of failure in patients with testicular lymphoma treated by chemotherapy (CT) and/or radiation therapy (RT). METHODS AND MATERIALS: Data from a series of 36 adult patients with Ann Arbor Stage I (n = 21), II (n = 9), III (n = 3), or IV (n = 3) primary testicular lymphoma, consecutively treated between 1980 and 1999, were collected in a retrospective multicenter study by the Rare Cancer Network. Median age was 64 years (range: 21-91 years). Full staging workup (chest X-ray, testicular ultrasound, abdominal ultrasound, and/or thoracoabdominal computer tomography, bone marrow assessment, full blood count, lactate dehydrogenase, and cerebrospinal fluid evaluation) was completed in 18 (50%) patients. All but one patient underwent orchidectomy, and spermatic cord infiltration was found in 9 patients. Most patients (n = 29) had CT, consisting in most cases of cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP) with (n = 17) or without intrathecal CT. External RT was delivered to scrotum alone (n = 12) or testicular, iliac, and para-aortic regions (n = 8). The median RT dose was 31 Gy (range: 20-44 Gy) in a median of 17 fractions (10-24), using a median of 1.8 Gy (range: 1.5-2.5 Gy) per fraction. The median follow-up period was 42 months (range: 6-138 months). RESULTS: After a median period of 11 months (range: 1-76 months), 14 patients presented lymphoma progression, mostly in the central nervous system (CNS) (n = 8). Among the 17 patients who received intrathecal CT, 4 had a CNS relapse (p = NS). No testicular, iliac, or para-aortic relapse was observed in patients receiving RT to these regions. The 5-year overall, lymphoma-specific, and disease-free survival was 47%, 66%, and 43%, respectively. In univariate analyses, statistically significant factors favorably influencing the outcome were early-stage and combined modality treatment. Neither RT technique nor total dose influenced the outcome. Multivariate analysis revealed that the most favorable independent factors predicting the outcome were younger age, early-stage disease, and combined modality treatment. CONCLUSIONS: In this multicenter retrospective study, CNS was found to be the principal site of relapse, and no extra-CNS lymphoma progression was observed in the irradiated volumes. More effective CNS prophylaxis, including combined modalities, should be prospectively explored in this uncommon site of extranodal lymphoma.

Licorice-induced hypertension and common variants of genes regulating renal sodium reabsorption.

AIM: To study if gene alterations affecting renal sodium reabsorption associate with susceptibility to licorice-induced hypertension.METHODS: Finnish subjects (n = 30) with a previously documented incident of licorice-induced hypertension were recruited for the study using a newspaper announcement. Their previous clinical and family histories as well as serum electrolyte levels were examined. DNA samples from all individuals were screened for variants of the genes encoding 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) and alpha-, beta-, and gamma-subunits of the epithelial sodium channel (ENaC).RESULTS: Upon licorice predisposition, the patients had a mean blood pressure of 201/118 mmHg. Circulating potassium, renin, and aldosterone levels were low. No significant DNA variations were identified in the 11betaHSD2 gene. Four subjects were heterozygous for beta- and gammaENaC variants previously shown to be associated with hypertension. Furthermore, a novel G insertion (2004-2005insG) in the SCNN1A gene encoding the alphaENaC was identified in two subjects. The frequency of these ENaC variants was significantly higher in subjects with licorice-induced hypertension (6/30 i.e. 20%) than in blood donors (11/301 i.e. 3.7%, P = 0.002).CONCLUSIONS: Defects of the 11betaHSD2 gene do not constitute a likely cause for licorice-induced hypertension. Variants of the ENaC subunits may render some individuals sensitive to licorice-induced metabolic alterations and hypertension.

Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads.

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.

Hemoglobin concentration and cerebral metabolism in patients with aneurysmal subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The optimal hemoglobin (Hgb) target after aneurysmal subarachnoid hemorrhage is not precisely known. We sought to examine the threshold of Hgb concentration associated with an increased risk of cerebral metabolic dysfunction in patients with poor-grade subarachnoid hemorrhage. METHODS: Twenty consecutive patients with poor-grade subarachnoid hemorrhage who underwent multimodality neuromonitoring (intracranial pressure, brain tissue oxygen tension, cerebral microdialysis) were studied prospectively. Brain tissue oxygen tension and extracellular lactate/pyruvate ratio were used as markers of cerebral metabolic dysfunction and the relationship between Hgb concentrations and the incidence of brain hypoxia (defined by a brain tissue oxygen tension <20 mm Hg) and cell energy dysfunction (defined by a lactate/pyruvate ratio >40) was analyzed. RESULTS: Compared with higher Hgb concentrations, a Hgb concentration <9 g/dL was associated with lower brain tissue oxygen tension (27.2 [interquartile range, 21.2 to 33.1] versus 19.9 [interquartile range, 7.1 to 33.1] mm Hg, P=0.02), higher lactate/pyruvate ratio (29 [interquartile range, 25 to 38] versus 36 [interquartile range, 26 to 59], P=0.16), and an increased incidence of brain hypoxia (21% versus 52%, P<0.01) and cell energy dysfunction (23% versus 43%, P=0.03). On multivariable analysis, a Hgb concentration <9 g/dL was associated with a higher risk of brain hypoxia (OR, 7.92; 95% CI, 2.32 to 27.09; P<0.01) and cell energy dysfunction (OR, 4.24; 95% CI, 1.33 to 13.55; P=0.02) after adjusting for cerebral perfusion pressure, central venous pressure, PaO(2)/FIO(2) ratio, and symptomatic vasospasm. CONCLUSIONS: A Hgb concentration <9 g/dL is associated with an increased incidence of brain hypoxia and cell energy dysfunction in patients with poor-grade subarachnoid hemorrhage.

Toxic effects of brake wear particles on epithelial lung cells in vitro

Background: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. Results: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. Conclusion: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress. [Authors]

Growth and metabolic activity of the extramatrical mycelium of endomycorrhizal maize plants

The objective of this experiment was to quantify the extramatrical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus etunicatum (Becker & Gerdemann) grown on maize (Zea mays L. var. Piranão) provided with various levels of phosphate fertilizer and harvested at 30, 60 and 90 days after planting (DAP). Total extramatrical mycelium (TEM) was extracted from soil using a modified membrane filtration method, followed by quantification using a grid intersection technique. Active extramatrical mycelium (AEM) proportion was determined using an enzymatic method which measured dehydrogenase activity by following iodonitrotetrazolium reduction. At low levels of added P, there was relatively less TEM than at high levels of added P, but the AEM proportion at low soil P availability was significantly greater than at high soil P.

Brain lactate metabolism in humans with subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Lactate is central for the regulation of brain metabolism and is an alternative substrate to glucose after injury. Brain lactate metabolism in patients with subarachnoid hemorrhage has not been fully elucidated. METHODS: Thirty-one subarachnoid hemorrhage patients monitored with cerebral microdialysis (CMD) and brain oxygen (PbtO(2)) were studied. Samples with elevated CMD lactate (>4 mmol/L) were matched to PbtO(2) and CMD pyruvate and categorized as hypoxic (PbtO(2) <20 mm Hg) versus nonhypoxic and hyperglycolytic (CMD pyruvate >119 μmol/L) versus nonhyperglycolytic. RESULTS: Median per patient samples with elevated CMD lactate was 54% (interquartile range, 11%-80%). Lactate elevations were more often attributable to cerebral hyperglycolysis (78%; interquartile range, 5%-98%) than brain hypoxia (11%; interquartile range, 4%-75%). Mortality was associated with increased percentage of samples with elevated lactate and brain hypoxia (28% [interquartile range 9%-95%] in nonsurvivors versus 9% [interquartile range 3%-17%] in survivors; P=0.02) and lower percentage of elevated lactate and cerebral hyperglycolysis (13% [interquartile range, 1%-87%] versus 88% [interquartile range, 27%-99%]; P=0.07). Cerebral hyperglycolytic lactate production predicted good 6-month outcome (odds ratio for modified Rankin Scale score, 0-3 1.49; CI, 1.08-2.05; P=0.016), whereas increased lactate with brain hypoxia was associated with a reduced likelihood of good outcome (OR, 0.78; CI, 0.59-1.03; P=0.08). CONCLUSIONS: Brain lactate is frequently elevated in subarachnoid hemorrhage patients, predominantly because of hyperglycolysis rather than hypoxia. A pattern of increased cerebral hyperglycolytic lactate was associated with good long-term recovery. Our data suggest that lactate may be used as an aerobic substrate by the injured human brain.

Futile cycling of intermediates of fatty acid biosynthesis toward peroxisomal beta-oxidation in Saccharomyces cerevisiae.

The flux of fatty acids toward beta-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the beta-oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis of polyhydroxyalkanoate was dependent on the beta-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome. In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid. In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3 Delta 0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3 Delta 0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short- and medium-chain fatty acids by the beta-oxidation cycle. Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production. Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal beta-oxidation pathway.

A cell therapy approach for erythropoietin delivery using encapsulated human primary fibroblasts

Summary Cell therapy has emerged as a strategy for the treatment of various human diseases. Cells can be transplanted considering their morphological and functional properties to restore a tissue damage, as represented by blood transfusion, bone marrow or pancreatic islet cells transplantation. With the advent of the gene therapy, cells also were used as biological supports for the production of therapeutic molecules that can act either locally or at distance. This strategy represents the basis of ex vivo gene therapy characterized by the removal of cells from an organism, their genetic modification and their implantation into the same or another individual in a physiologically suitable location. The tissue or biological function damage dictates the type of cells chosen for implantation and the required function of the implanted cells. The general aim of this work was to develop an ex vivo gene therapy approach for the secretion of erythropoietin (Epo) in patients suffering from Epo-responsive anemia, thus extending to humans, studies previously performed with mouse cells transplanted in mice and rats. Considering the potential clinical application, allogeneic primary human cells were chosen for practical and safety reasons. In contrast to autologous cells, the use of allogeneic cells allows to characterize a cell lineage that can be further transplanted in many individuals. Furthermore allogeneic cells avoid the potential risk of zoonosis encountered with xenogeneic cells. Accordingly, the immune reaction against this allogeneic source was prevented by cell macro- encapsulation that prevents cell-to-cell contact with the host immune system and allows to easy retrieve the implanted device. The first step consisted in testing the survival of various human primary cells that were encapsulated and implanted for one month in the subcutaneous tissue of immunocompetent and naturally or therapeutically immunodepressed mice, assuming that xenogeneic applications constitute a stringent and representative screening before human transplantation. A fibroblast lineage from the foreskin of a young donor, DARC 3.1 cells, showed the highest mean survival score. We have then performed studies to optimize the manufacturing procedures of the encapsulation device for successful engraftment. The development of calcifications on the polyvinyl alcohol (PVA) matrix serving as a scaffold for enclosed cells into the hollow fiber devices was reported after one month in vivo. Various parameters, including matrix rinsing solutions, batches of PVA and cell lineages were assessed for their respective role in the development of the phenomenon. We observed that the calcifications could be totally prevented by using ultra-pure sterile water instead of phosphate buffer saline solution in the rinsing procedure of the PVA matrix. Moreover, a higher lactate dehydrogenase activity of the cells was found to decrease calcium depositions due to more acidic microenvironment, inhibiting the calcium precipitation. After the selection of the appropriate cell lineage and the optimization of encapsulation conditions, a retroviral-based approach was applied to DARC 3.1 fibroblasts for the transduction of the human Epo cDNA. Various modifications of the retroviral vector and the infection conditions were performed to obtain clinically relevant levels of human Epo. The insertion of a post-transcriptional regulatory element from the woodchuck hepatitis virus as well as of a Kozak consensus sequence led to a 7.5-fold increase in transgene expression. Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells allowing to reach 200 IU hEpo/10E6 cells /day. These modified cells were encapsulated and implanted in vivo in the same conditions as previously described. All the mouse strains showed a sustained increase in their hematocrit and a high proportion of viable cells were observed after retrieval of the capsules. Finally, in the perspective of human application, a syngeneic model using encapsulated murine myoblasts transplanted in mice was realized to investigate the roles of both the host immune response and the cells metabolic requirements. Various loading densities and anti-inflammatory as well as immunosuppressive drugs were studied. The results showed that an immune process is responsible of cell death in capsules loaded at high cell density. A supporting matrix of PVA was shown to limit the cell density and to avoid early metabolic cell death, preventing therefore the immune reaction. This study has led to the development of encapsulated cells of human origin producing clinically relevant amounts of human EPO. This work resulted also to the optimization of cell encapsulation technical parameters allowing to begin a clinical application in end-stage renal failure patients. Résumé La thérapie cellulaire s'est imposée comme une stratégie de traitement potentiel pour diverses maladies. Si l'on considère leur morphologie et leur fonction, les cellules peuvent être transplantées dans le but de remplacer une perte tissulaire comme c'est le cas pour les transfusions sanguines ou les greffes de moelle osseuse ou de cellules pancréatiques. Avec le développement de la thérapie génique, les cellules sont également devenues des supports biologiques pour la production de molécules thérapeutiques. Cette stratégie représente le fondement de la thérapie génique ex vivo, caractérisée par le prélèvement de cellules d'un organisme, leur modification génétique et leur implantation dans le même individu ou dans un autre organisme. Le choix du type de cellule et la fonction qu'elle doit remplir pour un traitement spécifique dépend du tissu ou de la fonction biologique atteintes. Le but général de ce travail est de développer .une approche par thérapie génique ex vivo de sécrétion d'érythropoïétine (Epo) chez des patients souffrant d'anémie, prolongeant ainsi des travaux réalisés avec des cellules murines implantées chez des souris et des rats. Dans cette perpective, notre choix s'est porté sur des cellules humaines primaires allogéniques. En effet, contrairement aux cellules autologues, une caractérisation unique de cellules allogéniques peut déboucher sur de nombreuses applications. Par ailleurs, l'emploi de cellules allogéniques permet d'éviter les riques de zoonose que l'on peut rencontrer avec des cellules xénogéniques. Afin de protéger les cellules allogéniques soumises à une réaction immunitaire, leur confinement dans des macro-capsules cylindriques avant leur implantation permet d'éviter leur contact avec les cellules immunitaires de l'hôte, et de les retrouver sans difficulté en cas d'intolérance ou d'effet secondaire. Dans un premier temps, nous avons évalué la survie de différentes lignées cellulaires humaines primaires, une fois encapsulées et implantées dans le tissu sous-cutané de souris, soit immunocompétentes, soit immunodéprimées naturellement ou par l'intermédiaire d'un immunosuppresseur. Ce modèle in vivo correspond à des conditions xénogéniques et représente par conséquent un environnement de loin plus hostile pour les cellules qu'une transplantation allogénique. Une lignée fibroblastique issue du prépuce d'un jeune enfant, nommée DARC 3 .1, a montré une remarquable résistance avec un score de survie moyen le plus élevé parmi les lignées testées. Par la suite, nous nous sommes intéressés aux paramètres intervenant dans la réalisation du système d'implantation afin d'optimaliser les conditions pour une meilleure adaptation des cellules à ce nouvel environnement. En effet, en raison de l'apparition, après un mois in vivo, de calcifications au niveau de la matrice de polyvinyl alcohol (PVA) servant de support aux cellules encapsulées, différents paramètres ont été étudiés, tels que les procédures de fabrication, les lots de PVA ou encore les lignées cellulaires encapsulées, afin de mettre en évidence leur rôle respectif dans la survenue de ce processus. Nous avons montré que l'apparition des calcifications peut être totalement prévenue par l'utilisation d'eau pure au lieu de tampon phosphaté lors du rinçage des matrices de PVA. De plus, nous avons observe qu'un taux de lactate déshydrogénase cellulaire élevé était corrélé avec une diminution des dépôts de calcium au sein de la matrice en raison d'un micro-environnement plus acide inhibant la précipitation du calcium. Après sélection de la lignée cellulaire appropriée et de l'optimisation des conditions d'encapsulation, une modification génétique des fibroblastes DARC 3.1 a été réalisée par une approche rétrovirale, permettant l'insertion de l'ADN du gène de l'Epo dans le génome cellulaire. Diverses modifications, tant au niveau génétique qu'au niveau des conditions d'infection, ont été entreprises afin d'obtenir des taux de sécrétion d'Epo cliniquement appropriés. L'insertion dans la séquence d'ADN d'un élément de régulation post¬transcriptionnelle dérivé du virus de l'hépatite du rongeur (« woodchuck ») ainsi que d'une séquence consensus appelée « Kozak » ont abouti à une augmentation de sécrétion d'Epo 7.5 fois plus importante. De même, l'optimisation de la multiplicité d'infection et la sélection plus drastique des cellules hautement productrices ont permis finalement d'obtenir une sécrétion correspondant à 200 IU d'Epo/10E6 cells/jour. Ces cellules génétiquement modifiées ont été encapsulées et implantées in vivo dans les mêmes conditions que celles décrites plus haut. Toutes les souris transplantées ont montré une augmentation significative de leur hématocrite et une proportion importante de cellules présentait une survie conservée au moment de l'explantation des capsules. Finalement, dans la perspective d'une application humaine, un modèle syngénique a été proposé, basé sur l'implantation de myoblastes murins encapsulés dans des souris, afin d'investiguer les rôles respectifs de la réponse immunitaire du receveur et des besoins métaboliques cellulaires sur leur survie à long terme. Les cellules ont été encapsulées à différentes densités et les animaux transplantés se sont vus administrer des injections de molécules anti-inflammatoires ou immunosuppressives. Les résultats ont démontré qu'une réaction immunologique péri-capsulaire était à la base du rejet cellulaire dans le cas de capsules à haute densité cellulaire. Une matrice de PVA peut limiter cette densité et éviter une mort cellulaire précoce due à une insuffisance métabolique et par conséquent prévenir la réaction immunitaire. Ce travail a permis le développement de cellules encapsulées d'origine humaine sécrétant des taux d'Epo humaine adaptés à des traitements cliniques. De pair avec l'optimalisation des paramètres d'encapsulation, ces résultats ont abouti à l'initiation d'une application clinique destinée à des patients en insuffisance rénale terminale.