Characterization of the effects of IL-17F on CHO cell line generation

CHO is the most commonly used mammalian host for the generation of cell lines allowing for the production of high quality therapeutic proteins. The generation of such cell lines is a lengthy and resource-intensive process requiring extensive screening in order to isolate candidates with optimal characteristics, such as growth, stability and productivity. For this reason, the biotechnology industry invests much effort in attempts to optimize CHO expression systems in order to streamline and shorten the cell line selection process. Based on preliminary observations of a facilitated selection of CHO-GS cell lines expressing members of the IL-17 cytokine family, this study investigates the use of IL-17F as a novel enhancing factor for CHO cell line generation. Using two different CHO expression systems (exploiting GS and DHFR-based selection), we demonstrated that IL-17F expression caused a significant increase in the occurrence of colonies during the selection process. All colonies selected produced substantial amounts of IL-17F, suggesting that benefits were conferred, during selection, to those cells expressing the cytokine. Furthermore, transgene expression levels were significantly increased when the selection pressure was raised to a level that would not normally be permissive for colony selection (i.e. 100 |o.M MSX for the CHO-GS expression system or 1000 nM MTX for the CHO-DHFR system). Finally, IL-17F expression was also found to enhance the rate of appearance of clones during single cell subcloning in the absence of selection pressure. Overall, these benefits have the potential to allow a substantial reduction in the length of cell line generation while significantly increasing cell line productivity. Nevertheless, we found that the high IL-17F expression levels required to convey enhancing effects was a limitation when attempting to co-express IL-17F and a recombinant soluble protein of therapeutic interest from independent CMV promoters within the same expression vector. In order to understand and overcome this limitation, studies were designed to characterize the IL-17F enhancing effect at the molecular and cellular level. Regular supplementation of recombinant biologically-active IL-17F into the culture medium during cell line selection was not able to reproduce the enhancing effects of endogenous IL-17F expression. In addition, increased IL-17F expression correlated with increased CHO-GS selection transgene expression at the single cell level. This data suggested a possible effect of IL-17F on viral promoter activity or transgene mRNA stability. It also provided direct evidence that the cells expressing the highest amounts of IL-17F obtained the most benefit. Overall data obtained from these study implied that IL-17F may act through an intracellular mechanism, possibly exerted during secretion. We therefore initiated experiments designed to determine the specific compartment(s) within which IL-17F triggers its effect. This work has identified IL-17F as a potentially powerful tool to optimize the CHO cell line generation process. The characterization of this enhancing effect at the molecular level has given us several insights into overcoming the current limitations, thus paving the way for the development of a viable technology that can be exploited within the biotechnology industry. - La CHO est la cellule hôte de mammifere la plus couramment utilisée dans la création de lignée cellulaire produisant des protéines thérapeutiques de haute qualité. La génération de ces lignées cellulaires est un processus long et exigeant l'utilisation de techniques de sélection robustes afin d'isoler des candidats possédants les caractéristiques optimales de croissance, de productivité et de stabilité d'expression. Les industries biopharmaceutiques ont investi beaucoup d'efforts afin d'optimiser les systèmes d'expression CHO dans le but raccourcir la longueur du procédé de sélection de lignées cellulaires et aussi d'en augmenter l'efficacité. A partir d'observations préliminaires obtenues lors de la génération de lignées cellulaires CHO- GS exprimant une cytokine appartenant à la famille des IL-17, nous avons réalisé une étude portant sur l'utilisation de l'IL-17F humaine (IL-17F) comme nouveau facteur d'optimisation pour la génération de lignées cellulaires CHO. Nous avons démontré, en utilisant les deux systèmes de sélection et d'expression CHO couramment utilisés (le premier exploitant la GS et l'autre basée sur la DHFR), que l'expression de l'IL-17F permet une augmentation significative de la fréquence d'apparition de colonies durant le processus de sélection de lignées cellulaires. Les différentes colonies sélectionnées expriment des quantités substantielles d'IL-17F, suggérant un effet bénéfique lors de la sélection qui serait exclusivement conféré aux cellules exprimant la cytokine. En outre, le niveau d'expression du transgene se trouve significativement augmenté lorsque la pression de sélection est portée à un niveau habituellement trop élevé pour permettre la sélection de colonies (soit 100 |JM MSX pour le système d'expression CHO-GS ou 1000 nM MTX pour le système CHO- DHFR). Enfin, l'expression d'IL-17F permet également d'améliorer la vitesse d'apparition de clones pendant une étape de sous-clonage en l'absence de pression de sélection. L'ensemble de ces effets bénéfiques permettent une réduction substantielle de la durée de génération de lignées cellulaires tout en augmentant considérablement la productivité des lignées obtenues. Néanmoins, nous avons constaté que la nécessité d'exprimer des niveaux élevés d'IL-17F afin obtenir l'ensemble de ses effets bénéfiques devient une contrainte lors de l'utilisation d'un vecteur d'expression composé de deux promoteurs CMV indépendants pour la co-expression de la cytokine et d'une protéine soluble présentant un intérêt thérapeutique. Afin de mieux comprendre et de surmonter cette limitation, plusieurs études ont été effectuées dans le but de mieux caractériser l'effet de IL-17F au niveau subcellulaire. L'apport régulier en IL-17F recombinante et biologiquement active dans le milieu de culture lors de la sélection de lignées cellulaires ne permet pas de reproduire les effets bénéfiques observés par l'expression endogène d'IL-17F. En outre, nous avons constaté que, lors de l'utilisation du système CHO- GS, l'augmentation d'expression de 1TL-17F est corrélée à un accroissement de l'expression du marqueur de sélection au niveau cellulaire. Ces résultats suggèrent un possible effet d'IL- 17F sur l'activité des promoteurs viraux et ainsi fournissent une preuve directe que les cellules exprimant de haut niveau d'IL-17F sont celles qui en profitent le plus. L'ensemble de ces observations mettrait en avant que l'effet d'IL-17F se ferait selon un mécanisme intracellulaire. Nous avons donc étudié le(s) compartiment(s) spécifique(s) dans lequel IL-17F pourrait exercer son effet. Ce travail a permis de définir IL-17F comme un puissant outil pour l'optimisation des procédés de génération de lignées cellulaires CHO. La caractérisation de cette amélioration de l'effet au niveau moléculaire nous a donné plusieurs indications sur la manière de dépasser les limitations actuelles, ouvrant ainsi la voie au développement d'une technologie viable qui peut être exploitée pars l'industrie biotechnologique.

First-line temozolomide combined with bevacizumab in metastatic melanoma: a multicentre phase II trial (SAKK 50/07).

BACKGROUND: Oral temozolomide has shown similar efficacy to dacarbazine in phase III trials with median progression-free survival (PFS) of 2.1 months. Bevacizumab has an inhibitory effect on the proliferation of melanoma and sprouting endothelial cells. We evaluated the addition of bevacizumab to temozolomide to improve efficacy in stage IV melanoma. PATIENTS AND METHODS: Previously untreated metastatic melanoma patients with Eastern Cooperative Oncology Group performance status of two or more were treated with temozolomide 150 mg/m(2) days 1-7 orally and bevacizumab 10 mg/kg body weight i.v. day 1 every 2 weeks until disease progression or unacceptable toxicity. The primary end point was disease stabilisation rate [complete response (CR), partial response (PR) or stable disease (SD)] at week 12 (DSR12); secondary end points were best overall response, PFS, overall survival (OS) and adverse events. RESULTS: Sixty-two patients (median age 59 years) enrolled at nine Swiss centres. DSR12 was 52% (PR: 10 patients and SD: 22 patients). Confirmed overall response rate was 16.1% (CR: 1 patient and PR: 9 patients). Median PFS and OS were 4.2 and 9.6 months. OS (12.0 versus 9.2 months; P = 0.014) was higher in BRAF V600E wild-type patients. CONCLUSIONS: The primary end point was surpassed showing promising activity of this bevacizumab/temozolomide combination with a favourable toxicity profile. Response and OS were significantly higher in BRAF wild-type patients.

A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

Long-term follow-up of four patients with Langer-Giedion syndrome: clinical course and complications.

Long-term observations of individuals with the so-called Langer-Giedion (LGS) or tricho-rhino-phalangeal type II (TRPS2) are scarce. We report here a on follow-up of four LGS individuals, including one first described by Andres Giedion in 1969, and review the sparse publications on adults with this syndrome which comprises ectodermal dysplasia, multiple cone-shaped epiphyses prior to puberty, multiple cartilaginous exostoses, and mostly mild intellectual impairment. LGS is caused by deletion of the chromosomal segment 8q24.11-q24.13 containing among others the genes EXT1 and TRPS1. Most patients with TRPS2 are only borderline or mildly cognitively delayed, and few are of normal intelligence. Their practical skills are better than their intellectual capability, and, for this reason and because of their low self-esteem, they are often underestimated. Some patients develop seizures at variable age. Osteomas on processes of cervical vertebrae may cause pressure on cervical nerves or dissection of cerebral arteries. Joint stiffness is observed during childhood and changes later to joint laxity causing instability and proneness to trauma. Perthes disease is not rare. Almost all males become bald at or soon after puberty, and some develop (pseudo) gynecomastia. Growth hormone deficiency was found in a few patients, TSH deficiency so far only in one. Puberty and fertility are diminished, and no instance of transmission of the deletion from a non-mosaic parent to a child has been observed so far. Several affected females had vaginal atresia with consequent hydrometrocolpos.

Neuro-developmental follow-up of neonates treated with magnesium sulfate for persistent pulmonary hypertension

Rapport de synthèse : DEVENIR NEURO-DEVELOPPEMENTAL DE NOUVEAU-NES TRAITES PAR DU SULFATE DE MAGNESIUM POUR UNE HYPERTENSION PULMONAIRE PERSISTANTE L'hypertension pulmonaire persistante du nouveau-né (HTPP) est un trouble de l'adaptation post-natale de la circulation pulmonaire caractérisé par une défaillance de la diminution normale des résistances vasculaires pulmonaires, accompagné d'un shunt droite-gauche, résultant en une hypoxémie profonde. C'est une pathologie sévère nécessitant des soins intensifs avec un risque augmenté de handicaps neurologiques chez les survivants. Le traitement de l'HTPP du nouveau-né inclut une ventilation mécanique ainsi que différents agents pharmacologiques pour dilater les vaisseaux pulmonaires, dont le sulfate de magnésium (MgSO4) à hautes doses par voie intraveineuse et le monoxyde d'azote par voie inhalée (iN0). Le MgSO4 est une alternative thérapeutique de l'HTPP du nouveau-né avec peu d'effets secondaires et une mortalité basse. Il a aussi été démontré que le MgSO4 est un traitement de l'HTPP du nouveau-né autant efficace que le iN0 et moins coüteux. Des études sur le suivi neuro-développemental de nouveau-nés avec HTPP traités selon différentes méthodes ont été publiées reportant des taux élevés de handicaps majeurs et mineurs. Plus récemment, des études de suivi après traitement par iN0 ont montré des taux plus bas qu'avec des traitements antérieurs. Le devenir neuro-développemental àlong terme d'enfants traités avec du MgSO4 n'a pas été documenté. Le but de cette étude est de décrire le développement des enfants qui ont présenté une HTPP traitée seulement avec du MgS04, de reporter l'incidence de handicaps majeurs et mineurs, et de les comparer à un groupe contrôle d'enfants sains du même âge ainsi qu'aux données de la littérature. La population consiste en 33 nouveau-nés traités pour une HTPP avec seulement du MgSO4 (groupe étude) et 32 nouveau-nés à terme sains (groupe contrôle). Un suivi neurodéveloppemental standardisé et approfondi a été effectué aux âges clés de 18 mois et 5 ans. Les taux de handicaps majeurs à 18 mois et 5 ans dans le groupe étude étaient de 6% et 11,4% respectivement, et de 0% aux deux âges dans le groupe contrôle. Les taux de handicaps mineurs aux mêmes âges étaient de 3% et 26,9% pour le groupe étude, et de 0% et 26,1% pour le groupe contrôle. Les quotients développementaux moyens à 18 mois étaient de 106,6 (DS 1,6) dans le groupe étude et de 118,3 (DS 1,0) dans le groupe contrôle (P < 0,001). L'index général intellectuel en âge préscolaire était de 112.6 (DS 3.7), respectivement de 119.3 (DS 3.1 ), sans différence significative entre les deux groupes. A 18 mois, les taux de handicaps majeurs et mineurs dans les groupes études et contrôle étaient de 6% et 3%. Dans la littérature, des taux entre 0% et 33% ont été décrits. A cet âge, il y avait une différence significative pour tous les scores du test de Griffiths, mëme en tenant compte du status socio-économique de la famille. Ceci suggère un léger retard du développement global et non une altération spécifique. Ces différences n'étaient plus significatives en âge préscolaire, suggérant un rattrapage développemental. Le taux de handicaps majeurs en âge préscolaire pour le groupe étude était de 11.5%, sans aucune infirmité motrice cérébrale. Ces résultats correspondent à ceux d'études de suivi après d'autres traitements jusqu'à l'âge de 24 mois avec des taux variant de 0% à 15%. Le taux de handicaps mineurs était de 26.9% dans le groupe étude et de 26.1% dans le groupe contrôle, sans différence significative entre les deux groupes. L'incidence de handicaps mineurs dans le groupe étude était plutôt élevée en comparaison aux données de la littérature (6 à 22% à 6 ans). Une explication possible est que nous avons considéré des problèmes de langage et de comportement comme handicaps mineurs. Ceci suggère une différence méthodologique et non une plus mauvaise issue dans nos deux groupes. Les évaluations cognitives des enfants des deux groupes se trouvaient dans la norme, ce qui est aussi le cas dans la littérature. En conclusion, cette étude longitudinale non randomisée d'enfants traités avec du MgSO4 seul pour une HTPP sévère ne montre pas de conséquences sur le devenir neuro-développemental à long terme. Cette étude le démontre pour la première fois. Malgré le fait que iN0 soit le traitement actuellement recommandé pour l'HTPP du nopuveau-né, le MgSO4 reste largement utilisé, en particulier dans des pays en voie de développement. L'absence de complications neuro-développementales majeures à long terme permet de considérer l'administration du MgSO4 pour le traitement de l'HTPP du nouveau-né en cas de non réponse ou d'inaccessibilité au iNO.

Recombinant " Physcomitrella patens " as a sensitive tool to study heat sensing and heat-shock signal transduction in plants

SUMMARY When exposed to heat stress, plants display a particular set of cellular and molecular responses, such as chaperones expression, which are highly conserved in all organisms. In chapter 1, I studied the ability of heat shock genes to become transiently and abundantly induced under various temperature regimes. To this aim, I designed a highly sensitive heat-shock dependent conditional gene expression system in the moss Physcomitrella patens, using the soybean heatinducible promoter (hsp17.3B). Heat-induced expression of various reporter genes was over three orders of magnitude, in tight correlation with the intensity and duration of the heat treatments. By performing repeated heating/cooling cycles, a massive accumulation of recombinant proteins was obtained. Interestingly, the hsp17.3B promoter was also activated by specific organic chemicals. Thus, in chapter 2, I took advantage of the extreme sensitivity of this promoter to small temperature variations to further address the role of various natural and organic chemicals and develop a plant based-bioassay that can serve as an early warning indicator of toxicity by pollutants and heavy metals. A screen of several organic pollutants from textile and paper industry showed that chlorophenols as well as sulfonated anthraquinones elicited a heat shock like response at noninducing temperatures. Their effects were synergistically amplified by mild elevated temperatures. In contrast to standard methods of pollutant detection, this plant-based biosensor allowed to monitor early stress-responses, in correlation with long-term toxic effect, and to attribute effective toxicity thresholds for pollutants, in a context of varying environmental cues. In chapter 3, I deepened the study of the primary mechanism by which plants sense mild temperature variations and trigger a cellular signal leading to the heat shock response. In addition to the above described heat-inducible reporter line, I generated a P. patens transgenic line to measure, in vivo, variations of cytosolic calcium during heat treatment, and another line to monitor the role of protein unfolding in heat-shock sensing and signalling. The heat shock signalling pathway was found to be triggered by the plasma membrane, where temperature up shift specifically induced the transient opening of a putative high afimity calcium channel. The calcium influx triggered a signalling cascade leading to the activation of the heat shock genes, independently on the presence of misfolded proteins in the cytoplasm. These results strongly suggest that changes in the fluidity of the plasma membrane are the primary trigger of the heatshocksignalling pathway in plants. The present thesis contributes to the understanding of the basic mechanism by which plants perceive and respond to heat and chemical stresses. This may contribute to developing appropriate better strategies to enhance plant productivity under the increasingly stressful environment of global warming. RÉSUME Les plantes exposées à des températures élevées déclenchent rapidement des réponses cellulaires qui conduisent à l'induction de gènes codant pour les heat shock proteins (HSPs). En fonction de la durée d'exposition et de la vitesse à laquelle la température augmente, les HSPs sont fortement et transitoirement induites. Dans le premier chapitre, cette caractéristique aété utilisée pour développer un système inductible d'expression de gènes dans la mousse Physcomitrella patens. En utilisant plusieurs gènes rapporteurs, j'ai montré que le promoteur du gène hsp17.3B du Soja est activé d'une manière. homogène dans tous les tissus de la mousse proportionnellement à l'intensité du heat shock physiologique appliqué. Un très fort taux de protéines recombinantes peut ainsi être produit en réalisant plusieurs cycles induction/recovery. De plus, ce promoteur peut également être activé par des composés organiques, tels que les composés anti-inflammatoires, ce qui constitue une bonne alternative à l'induction par la chaleur. Les HSPs sont induites pour remédier aux dommages cellulaires qui surviennent. Étant donné que le promoteur hsp17.3B est très sensible à des petites augmentations de température ainsi qu'à des composés chimiques, j'ai utilisé les lignées développées dans le chapitre 1 pour identifier des polluants qui déclenchent une réaction de défense impliquant les HSPs. Après un criblage de plusieurs composés, les chlorophénols et les antraquinones sulfonés ont été identifiés comme étant activateurs du promoteur de stress. La détection de leurs effets a été réalisée seulement après quelques heures d'exposition et corrèle parfaitement avec les effets toxiques détectés après de longues périodes d'exposition. Les produits identifiés montrent aussi un effet synergique avec la température, ce qui fait du biosensor développé dans ce chapitre un bon outil pour révéler les effets réels des polluants dans un environnement où les stress chimiques sont combinés aux stress abiotiques. Le troisième chapitre est consacré à l'étude des mécanismes précoces qui permettent aux plantes de percevoir la chaleur et ainsi de déclencher une cascade de signalisation spécifique qui aboutit à l'induction des gènes HSPs. J'ai généré deux nouvelles lignées afin de mesurer en temps réel les changements de concentrations du calcium cytosolique ainsi que l'état de dénaturation des protéines au cours du heat shock. Quand la fluidité de la membrane augmente après élévation de la température, elle semble induire l'ouverture d'un canal qui permet de faire entrer le calcium dans les cellules. Ce dernier initie une cascade de signalisation qui finit par activer la transcription des gènes HSPs indépendamment de la dénaturation de protéines cytoplasmiques. Les résultats présentés dans ce chapitre montrent que la perception de la chaleur se fait essentiellement au niveau de la membrane plasmique qui joue un rôle majeur dans la régulation des gènes HSPs. L'élucidation des mécanismes par lesquels les plantes perçoivent les signaux environnementaux est d'une grande utilité pour le développement de nouvelles stratégies afin d'améliorer la productivité des plantes soumises à des conditions extrêmes. La présente thèse contribue à décortiquer la voie de signalisation impliquée dans la réponse à la chaleur.

Solitary fibrous tumor of the lateral ventricle: CT appearances and pathologic correlation with follow-up.

Intracranial solitary fibrous tumors are rare, and intraventricular fibrous tumors are even more unusual. We report a case of solitary fibrous tumor in the region of trigone and body of the left lateral ventricle and discuss the clinical presentation, CT characteristics, and histopathologic features with 1-year follow-up. We speculate that the tumor arose from the perivascular connective tissue of the choroid plexus.

Liquid line of descent of a basanitic liquid at 1.5 GPa: Constraints for metasomatic vein formation

The metasomatism observed in the oceanic and continental lithosphere is generally interpreted to represent a continuous differentiation process forming anhydrous and hydrous veins plus a cryptic enrichment in the surrounding peridotite. In order to constrain the mechanisms of vein formation and potentially clarify the nature and origin of the initial metasomatic agent, we performed a series of high-pressure experiments simulating the liquid line of descent of a basanitic magma differentiating within continental or mature oceanic lithosphere. This series of experiments has been conducted in an end-loaded piston cylinder apparatus starting from an initial hydrous ne-normative basanite at 1.5 GPa and temperature varying between 1,250 and 980°C. Near-pure fractional crystallization process was achieved in a stepwise manner in 30°C temperature steps and starting compositions corresponding to the liquid composition of the previous, higher-temperature glass composition. Liquids evolve progressively from basanite to peralkaline, aluminum-rich compositions without significant SiO2 variation. The resulting cumulates are characterized by an anhydrous clinopyroxene + olivine assemblage at high temperature (1,250-1,160°C), while at lower temperature (1,130-980°C), hydrous cumulates with dominantly amphibole + minor clinopyroxene, spinel, ilmenite, titanomagnetite and apatite (1,130-980°C) are formed. This new data set supports the interpretation that anhydrous and hydrous metasomatic veins could be produced during continuous differentiation processes of primary, hydrous alkaline magmas at high pressure. However, the comparison between the cumulates generated by the fractional crystallization from an initial ne-normative liquid or from hy-normative initial compositions (hawaiite or picrobasalt) indicates that for all hydrous liquids, the different phases formed upon differentiation are mostly similar even though the proportions of hydrous versus anhydrous minerals could vary significantly. This suggests that the formation of amphibole-bearing metasomatic veins observed in the lithospheric mantle could be linked to the differentiation of initial liquids ranging from ne-normative to hy-normative in composition. The present study does not resolve the question whether the metasomatism observed in lithospheric mantle is a precursor or a consequence of alkaline magmatism; however, it confirms that the percolation and differentiation of a liquid produced by a low degree of partial melting of a source similar or slightly more enriched than depleted MORB mantle could generate hydrous metasomatic veins interpreted as a potential source for alkaline magmatism by various authors.

The role of ceramide trihexoside (globotriaosylceramide) in the diagnosis and follow-up of the efficacy of treatment of Fabry disease: a review of the literature.

Fabry disease is caused by a deficiency of a-galactosidase A which leads to the progressive intra-lysosomal accumulation of ceramide trihexoside (CTH), also known as globotriaosylceramide (Gb3), in different cell types and body fluids. The clinical manifestations are multisystemic and predominantly affect the heart, kidney and central nervous system. The role of CTH in the pathophysiological process of Fabry disease is not established, and the link between the degree of accumulation and disease manifestations is not systematic. The use of CTH as a diagnostic tool has been proposed for several decades. The recent introduction of a specific treatment for Fabry disease in the form of enzyme replacement therapy (ERT) has led to the need for a biological marker, in place of a clinical sign, for evaluating the efficacy of treatment and also as a tool for following the long term effects of treatment. The ideal biomarker must adhere to strict criteria, and there should be a correlation between the degree of clinical efficacy of treatment and a change in its concentration. This review of the literature assesses the utility of CTH as a diagnostic tool and as a marker of the efficacy of ERT in patients with Fabry disease. Several techniques have been developed for measuring CTH; the principles and the sensitivity thresholds of these methods and the units used to express the results should be taken into consideration when interpreting data. The use of CTH measurement in Fabry disease should be re-evaluated in light of recent published data.

Congenital Myasthenic Syndromes with COLQ mutations: Long term follow-up

Congenital myasthenic syndromes (CMS) are clinically and genetically heterogeneous inherited disorders characterized by impaired neuromuscular transmission. Mutations in the acetylcholinesterase (AChE) collagenlike tail subunit gene (ColQ) cause recessive forms of synaptic CMS with end plate AChE deficiency. We report the time course of clinical manifestations in 15 COLQ-mutated patients followed from 1987 to 2010. All patients suffered from a muscle weakness with onset at birth or in childhood. Ocular and bulbar signs were found in 60% of the patients and delayed pupillary light response in 20% of our patients. EMG study demonstrated a decrement on repetitive nerve stimulation and repetitive compound muscle action potential in all patients. Clinical symptoms strongly fluctuated daily, weekly, monthly or even yearly. Severe relapses were characterized by a general motor weakness associated with pain which resolved spontaneously after a few months whereas the relapses with these symptoms and bulbar signs could last up to several years. Genetic analyses identified 16 different mutations including 9 novel ones. There was no genotype-phenotype correlation. Our study confirms the predominance of oculobulbar signs and the frequency of respiratory distress in COLQrelated CMS. At the end of the follow up of 23 years, interesting findings were (i) the spontaneous reversibility of severe relapses, some of them lasting for up to 5 years (ii) the good prognosis of COLQ-related CMS, since at the end of the follow-up 80% of patients were ambulant and 87% of patients had no respiratory trouble (iii) the efficacy of Ephedrine and, to a lesser extend, of 3-4 DAP. The triggering factors of relapses were esterase inhibitors, effort, puberty, pregnancy and delivery highlighting the importance of hormonal factors in CMS. In conclusion, patients diagnosed with unknown congenital myopathy should undergo an electrophysiological study of neuromuscular junction to identify ColQ-related CMS.

Sawhorse-type diruthenium tetracarbonyl complexes containing porphyrin-derived ligands as highly selective photosensitizers for female reproductive cancer cells.

Diruthenium tetracarbonyl complexes of the type [Ru2(CO)4(l2-g2-O2CR)2L2] containing a Ru-Ru backbone with four equatorial carbonyl ligands, two carboxylato bridges, and two axial two-electron ligands in a sawhorse-like geometry have been synthesized with porphyrin-derived substituents in the axial ligands [1: R is CH3, L is 5-(4-pyridyl)-10,15,20-triphenyl-21,23H-porphyrin], in the bridging carboxylato ligands [2: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is PPh3; 3: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is 1,3,5-triaza-7-phosphatricyclo [3.3.1.1]decane], or in both positions [4: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is 5-(4-pyridyl)-10,15,20-triphenyl-21,23H-porphyrin]. Compounds 1-3 were assessed on different types of human cancer cells and normal cells. Their uptake by cells was quantified by fluorescence and checked by fluorescence microscopy. These compounds were taken up by human HeLa cervix and A2780 and Ovcar ovarian carcinoma cells but not by normal cells and other cancer cell lines (A549 pulmonary, Me300 melanoma, PC3 and LnCap prostate, KB head and neck, MDAMB231 and MCF7 breast, or HT29 colon cancer cells). The compounds demonstrated no cytotoxicity in the absence of laser irradiation but exhibited good phototoxicities in HeLa and A2780 cells when exposed to laser light at 652 nm, displaying an LD50 between 1.5 and 6.5 J/cm2 in these two cell lines and more than 15 J/cm2 for the others. Thus, these types of porphyric compound present specificity for cancer cell lines of the female reproductive system and not for normal cells; thus being promising new organometallic photosensitizers.

Bone anabolic potential of soy isoflavones and plant extracts and genomic changes during differentiation of hPOB-tert osteoblast-like cells

Summary For the nutritional management of bone health and the prevention of osteoporosis it is important to identify nutrients that positively influence the bone remodeling process at the cellular level. Soy isoflavones show promising osteoprotective effects in animals and humans but their mechanism of action in bone cells is yet poorly understood. Firstly, soy tissue cultures were characterized as a new and optimized source of isoflavones. A large variability in the isoflavone content was observed and high-producing strains (46.3 mg/g dry wt isoflavones) were identified. In the Ishikawa cells bioassay, the estrogenicity of isoflavones was confirmed to be 1000 to 10000 less than 17Mestradiol and that of the malonyl forms was shown for the first time (EC50 of 350 nM and 1880 nM for malonylgenistin and malonyldaidzin, respectively). The estrogenic activity of soya tissue culture extracts correlated to their isoflavone content. Secondly, the effects of phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway, as key mediators of bone formation, were investigated. Dietary achievable concentrations of genistein and daidzein (10vM), and statins (4xM) but not 17M estradiol (10nM), induced BMP-2 gene expression (by up to 3-fold) and inhibited the cholesterol biosynthetic pathway (by up to 50%) in the human osteoblastic cell line hP0B¬tert. In addition, several plant extracts (Cyperus rotundus, Lindera benzoin and Cnidium monnieri) induced BMP-2 gene expression but this induction was not restricted to the inhibition of the cholesterol synthesis pathway neither to the estrogenicity. Finally, the gene expression profiles during hP0B-tert differentiation induced by vitamin D and dexamethasone were analyzed with the Affymetrix human GeneChip. 1665 different genes and 98 ESTs were significantly regulated. The expression profiles of bone-related genes was largely in agreement with previously documented patterns, supporting the physiological relevance of the genomic results and the hP0B-tert cell line as a valid model of human osteoblast differentiation. The expression of alternative differentiation markers during the osteogenic treatment of hP0B-tert cells indicated that the adipocyte and myoblast differentiation pathways were repressed, confirming that these culture conditions allowed only osteoblast differentiation. The gene ontology analysis identified further sub-groups of genes that may be involved in the bone formation process. Aims of the thesis In order to define new strategies for the nutritional management of bone health and for the prevention of osteoporosis the major goal of the present work was to investigate the potential of phytonutrients to positively modulate the bone formation process at the cellular level and, in particular: 1.To select and optimise alternative plant sources containing high levels of isoflavones with estrogenic activity (Chapter 3). 2.To compare the effects of statins and phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway and to select new plant extracts with a bone anabolic potential (Chapter 4). 3.To further characterize the new human periosteal cell line, hP0B-tert, as a bone- formation model, by elucidating its gene expression profile during differentiation induced by vitamin D and dexamethasone (Chapter 5).