Pi*-pi* bonding interactions generated by halogen oxidation of zirconium(IV) redox-active ligand complexes.

The new complex, [Zr(pda)2]n (1, pda2- = N,N'-bis(neo-pentyl)-ortho-phenylenediamide, n = 1 or 2), prepared by the reaction of 2 equiv of pdaLi2 with ZrCl4, reacts rapidly with halogen oxidants to afford the new product ZrX2(disq)2 (3, X = Cl, Br, I; disq- = N,N'-bis(neo-pentyl)-ortho-diiminosemiquinonate) in which each redox-active ligand has been oxidized by one electron. The oxidation products 3a-c have been structurally characterized and display an unusual parallel stacked arrangement of the disq- ligands in the solid state, with a separation of approximately 3 A. Density functional calculations show a bonding-type interaction between the SOMOs of the disq- ligands to form a unique HOMO while the antibonding linear combination forms a unique LUMO. This orbital configuration leads to a closed-shell-singlet ground-state electron configuration (S = 0). Temperature-dependent magnetism measurements indicate a low-lying triplet excited state at approximately 750 cm-1. In solution, 3a-c show strong disq--based absorption bands that are invariant across the halide series. Taken together these spectroscopic measurements provide experimental values for the one- and two-electron energies that characterize the pi-stacked bonding interaction between the two disq- ligands.

Aqueous dynamic and histological findings after deep sclerectomy with collagen implant in an animal model.

AIM: The use of an animal model to study the aqueous dynamic and the histological findings after deep sclerectomy with (DSCI) and without collagen implant. METHODS: Deep sclerectomy was performed on rabbits' eyes. Eyes were randomly assigned to receive collagen implants. Measurements of intraocular pressure (IOP) and aqueous outflow facility using the constant pressure method through cannulation of the anterior chamber were performed. The system was filled with BSS and cationised ferritin. Histological assessment of the operative site was performed. Sections were stained with haematoxylin and eosin and with Prussian blue. Aqueous drainage vessels were identified by the reaction between ferritin and Prussian blue. All eyes were coded so that the investigator was blind to the type of surgery until the evaluation was completed. RESULTS: A significant decrease in IOP (p<0.05) was observed during the first 6 weeks after DSCI (mean IOP was 13.07 (2.95) mm Hg preoperatively and 9.08 (2.25) mm Hg at 6 weeks); DS without collagen implant revealed a significant decrease in IOP at weeks 4 and 8 after surgery (mean IOP 12.57 (3.52) mm Hg preoperatively, 9.45 (3.38) mm Hg at 4 weeks, and 9.22 (3.39) mm Hg at 8 weeks). Outflow facility was significantly increased throughout the 9 months of follow up in both DSCI and DS groups (p<0.05). The preoperative outflow facility (OF) was 0.15 (0.02) micro l/min/mm Hg. At 9 months, OF was 0.52 (0.28) microl/min/mm Hg and 0.46 (0.07) micro l/min/mm Hg for DSCI and DS respectively. Light microscopy studies showed the appearance of new aqueous drainage vessels in the sclera adjacent to the dissection site in DSCI and DS and the apparition of spindle cells lining the collagen implant in DSCI after 2 months. CONCLUSION: A significant IOP decrease was observed during the first weeks after DSCI and DS. DS with or without collagen implant provided a significant increase in outflow facility throughout the 9 months of follow up. This might be partly explained by new drainage vessels in the sclera surrounding the operated site. Microscopic studies revealed the appearance of spindle cells lining the collagen implant in DSCI after 2 months.

Promoter IV of the class II transactivator gene is essential for positive selection of CD4+ T cells.

Major histocompatibility complex class II (MHCII) expression is regulated by the transcriptional coactivator CIITA. Positive selection of CD4(+) T cells is abrogated in mice lacking one of the promoters (pIV) of the Mhc2ta gene. This is entirely due to the absence of MHCII expression in thymic epithelia, as demonstrated by bone marrow transfer experiments between wild-type and pIV(-/-) mice. Medullary thymic epithelial cells (mTECs) are also MHCII(-) in pIV(-/-) mice. Bone marrow-derived, professional antigen-presenting cells (APCs) retain normal MHCII expression in pIV(-/-) mice, including those believed to mediate negative selection in the thymic medulla. Endogenous retroviruses thus retain their ability to sustain negative selection of the residual CD4(+) thymocytes in pIV(-/-) mice. Interestingly, the passive acquisition of MHCII molecules by thymocytes is abrogated in pIV(-/-) mice. This identifies thymic epithelial cells as the source of this passive transfer. In peripheral lymphoid organs, the CD4(+) T-cell population of pIV(-/-) mice is quantitatively and qualitatively comparable to that of MHCII-deficient mice. It comprises a high proportion of CD1-restricted natural killer T cells, which results in a bias of the V beta repertoire of the residual CD4(+) T-cell population. We have also addressed the identity of the signal that sustains pIV expression in cortical epithelia. We found that the Jak/STAT pathways activated by the common gamma chain (CD132) or common beta chain (CDw131) cytokine receptors are not required for MHCII expression in thymic cortical epithelia.

Biological activity of ectodysplasin A is conditioned by its collagen and heparan sulfate proteoglycan-binding domains.

Mutations in the TNF family ligand EDA1 cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition characterized by defective development of skin appendages. The EDA1 protein displays a proteolytic processing site responsible for its conversion to a soluble form, a collagen domain, and a trimeric TNF homology domain (THD) that binds the receptor EDAR. In-frame deletions in the collagen domain reduced the thermal stability of EDA1. Removal of the collagen domain decreased its activity about 100-fold, as measured with natural and engineered EDA1-responsive cell lines. The collagen domain could be functionally replaced by multimerization domains or by cross-linking antibodies, suggesting that it functions as an oligomerization unit. Surprisingly, mature soluble EDA1 containing the collagen domain was poorly active when administered in newborn, EDA-deficient (Tabby) mice. This was due to a short stretch of basic amino acids located at the N terminus of the collagen domain that confers EDA1 with proteoglycan binding ability. In contrast to wild-type EDA1, EDA1 with mutations in this basic sequence was a potent inducer of tail hair development in vivo. Thus, the collagen domain activates EDA1 by multimerization, whereas the proteoglycan-binding domain may restrict the distribution of endogeneous EDA1 in vivo.

Calmodulin-dependent protein kinase IV during T-cell development.

In a primary cell culture system of fetal rat brain, the calmodulin-dependent protein-kinase IV (CaMKIV) could be induced by the thyroid hormone T3 in a time- and concentration-dependent manner, provided the tissue was excised not later than day 15 of gestation (E15) (Krebs et al., J. Biol. Chem. 271, 11055, 1996). We report here that in the fetal thymus CaMKIV could not be detected earlier than day 16 of gestation and that the expression of this enzyme was fully upregulated at day 18. In mouse fetal thymus organ culture (FTOC) of day 14 embryonic thymus, CaMKIV could not be detected, even after several days of culture if a minimal culture medium lacking fetal calf serum was used. However, after addition of fetal calf serum to the culture medium the expression of CaMKIV could be specifically induced. Furthermore, it could also be shown that during T-cell development in the adult murine thymus the expression of CaMKIV was tightly regulated. Taken together, these results demonstrate that the expression of CaMKIV, an enzyme involved in the regulation of Ca(2+)-dependent gene expression, is itself under stringent regulatory control during tissue development.

Long-lasting antidiabetic effect of a dipeptidyl peptidase IV-resistant analog of glucagon-like peptide-1.

Glucagon-like peptide-1(7-37) (GLP-1) is the most potent insulinotropic hormone characterized thus far. Because its activity is preserved in non-insulin-dependent diabetes mellitus (NIDDM) patients, it is considered a potential new drug for the treatment of this disease. One limitation in its therapeutic use is a short half-life in vivo (5 minutes), due in part to a fast degradation by the endoprotease dipeptidylpeptidase IV (DPPIV). Recently, it was reported that GLP-1 became resistant to DPPIV when the alanine residue at position 8 was replaced by a glycine (GLP-1-Gly8). We report here that this change slightly decreased the affinity of the peptide for its receptor (IC50, 0.41 +/- 0.14 and 1.39 +/- 0.61 nmol/L for GLP-1 and GLP-1-Gly8, respectively) but did not change the efficiency to stimulate accumulation of intracellular cyclic adenosine monophosphate (cAMP) (EC50, 0.25 +/- 0.05 and 0.36 +/- 0.06 nmol/L for GLP-1 and GLP-1-Gly8, respectively). Second, we demonstrate for the first time that this mutant has an improved insulinotropic activity compared with the wild-type peptide when tested in vivo in an animal model of diabetes. A single injection of 0.1 nmol GLP-1-Gly8 in diabetic mice fed a high-fat diet can correct fasting hyperglycemia and glucose intolerance for several hours, whereas the activity of 1 nmol GLP-1 vanishes a few minutes after injection. These actions were correlated with increased insulin and decreased glucagon levels. Interestingly, normoglycemia was maintained over a period that was longer than the predicted peptide half-life, suggesting a yet undescribed long-term effect of GLP-1-Gly8. GLP-1-Gly8 thus has a markedly improved therapeutic potential compared with GLP-1, since it can be used at much lower doses and with a more flexible schedule of administration.

BMP-2 and TGF-beta1 differentially control expression of type II procollagen and alpha 10 and alpha 11 integrins in mouse chondrocytes.

Bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1 are multifunctional cytokines both proposed as stimulants for cartilage repair. Thus it is crucial to closely examine and compare their effects on the expression of key markers of the chondrocyte phenotype, at the gene and protein level. In this study, the expression of alpha 10 and alpha 11 integrin subunits and the IIA/IIB spliced forms of type II procollagen have been monitored for the first time in parallel in the same in vitro model of mouse chondrocyte dedifferentiation/redifferentiation. We demonstrated that TGF-beta1 stimulates the expression of the non-chondrogenic form of type II procollagen, IIA isoform, and of a marker of mesenchymal tissues, i.e. the alpha 11 integrin subunit. On the contrary, BMP-2 stimulates the cartilage-specific form of type II procollagen, IIB isoform, and a specific marker of chondrocytes, i.e. the alpha 10 integrin subunit. Collectively, our results demonstrate that BMP-2 has a better capability than TGF-beta1 to stimulate chondrocyte redifferentiation and reveal that the relative expressions of type IIB to type IIA procollagens and alpha 10 to alpha 11 integrin subunits are good markers to define the differentiation state of chondrocytes. In addition, adenoviral expression of Smad6, an inhibitor of BMP canonical Smad signaling, did not affect expression of total type II procollagen or the ratio of type IIA and type IIB isoforms in mouse chondrocytes exposed to BMP-2. This result strongly suggests that signaling pathways other than Smad proteins are involved in the effect of BMP-2 on type II procollagen expression.

Ten years follow-up after deep sclerectomy with collagen implant

RAPPORT DE SYNTHESE : BUT : Le but de ce sujet de recherche est d'évaluer le taux de succès et les complications à long terme de la sclérectomie profonde non pénétrante avec implant de collagène (SPIC) chez les patients atteints de glaucome à angle ouvert. METODES ET PATIENTS : Il s'agit d'une étude clinique, prospective, monocentrique, non-randomisée, effectuée sur 105 patients atteints d'un glaucome à angle ouvert médicalement non-contrôlé. Ces patients ont tous bénéficiés d'une SPIC, effectuée selon le geste chirurgicale standard (technique décrite dans l'article). Dans le cadre de cette étude, nous avons effectué un bilan ophtalmologique complet avant l'acte chirurgical puis un suivi postopératoire à 1 et 7 jours ; 1,2,3,6,9,12 mois et ensuite tous les 6 mois durant les dix années suivantes. RESULTATS : Le suivit moyen de cette étude s'étend sur 101.5 ± 43.1, [3-144] mois (moyenne ± écart type, [étendue]). La pression intraoculaire (PIO) préopératoire était élevée à 26.8 ± 7.7, (14-52] mmHg, et l'acuité visuelle corrigée à 0.71 ± 0.33, [0.02-1.5]. Au terme des dix années après le traitement chirurgical, le nombre de patients suivit était de 52 avec une pression intraoculaire abaissée à 12.2 ± 4.7, [6-20] mmHg et une acuité visuelle corrigée de 0.63 ± 0.34, [0.01-1.2]. Le nombre de médicaments par patient a diminué de 2.3 ± 0.7, [ 1-4] à 1.3 ± 1.1, [0-3]. Dix ans après la SPIC, une pression intraoculaire <_ 21 mmHg sans médicaments (succès complet) était obtenue chez 47.7 % des patients et 89 % avec ou sans traitement médicamenteux (succès relatif). Les gestes postopératoires additionnels par gonioponcture ont été effectués sur 61 yeux (59.8%) et les injections sous-conjonctival de 5-fluorouracil ont été pratiquées sur 25 yeux dont 5 incluant un needling. CONCLUSIONS : Le suivit à long terme sur une période de dix ans, démontre que la sclérectomie profonde avec implant de collagène (SPIC) est efficace dans le contrôle de la pression intraoculaire et présente peu de complications postopératoires.

L'implication des cellules de type cellules souches cancéreuses dans la résistance aux drogues du neuroblastome

Le neuroblastome (NB), tumeur spécifique de l'enfant, se situe au second rang en terme de¦fréquence des tumeurs solides dans la population pédiatrique (1). Il dérive des cellules¦primitives de la crête neurale, une population de cellules embryonnaires dotées d'une¦capacité de différentiation en une panoplie de tissus très variés, dont le système nerveux¦sympathique (2). Cette origine explique la très grande hétérogénéité du NB, tant du point de¦vue biologique que clinique (3). Malgré un traitement intensif et multimodal (chirurgie,¦chimiothérapie à haute dose, greffe de moelle osseuse et immunothérapie), seuls 30 % des¦patients de haut risque (stade IV) survivent sans rechute. La forte résistance du¦neuroblastome de haut grade aux diverses thérapies est une des causes probable du¦pronostic sombre de cette tumeur. Les thérapies actuelles étant insuffisamment efficaces, il¦est primordial de comprendre les mécanismes impliqués dans le processus de résistance¦afin d'élaborer de nouveaux traitements, mieux ciblés, capables de contrer toute résistance¦(4).¦Il a été démontré que certains cancers, tels que les tumeurs du poumon, du sein, de la¦prostate ou du colon, possédaient des cellules souches cancéreuses (CSCs) (5). Ces¦dernières, définies comme étant une petite sous-population de cellules malignes, jouent un¦rôle prépondérant dans l'initiation et la progression tumorale. Elles partagent certaines¦propriétés avec les cellules souches physiologiques, telles que la capacité d'autorenouvellement,¦un potentiel de prolifération indéfini, une dépendance à un¦microenvironnement spécifique, une faculté de pluripotence et une résistance accrue aux¦drogues (6). Ce modèle de CSCs a également été étudié pour le NB (7), permettant ainsi¦d'avancer l'hypothèse selon laquelle cette population de CSCs serait responsable de la¦résistance aux chimiothérapies des cellules tumorales du NB.¦Afin de tenter d'éclaircir le caractère résistant aux drogues des CSCs du NB, nous avons¦sélectionné des sous-populations cellulaires résistantes, en traitant par divers agents¦cytotoxiques (cisplatine, doxorubicine, rapamycine et vincristine) cinq lignées différentes de¦neuroblastes. Dans le but d'établir un potentiel enrichissement en CSCs au sein de ces¦sous-populations par rapport aux populations contrôles non traitées, nous avons testé leurs¦fonctions d'auto-renouvellement et de clonogénicité. Ces propriétés ont été respectivement¦mises en évidence par la capacité des cellules à former des sphères de plusieurs¦générations dans des conditions de culture inhibant l'adhésion cellulaire et par la mesure de¦la croissance cellulaire en milieu semi-solide (soft agar assay). Une analyse d'expression¦génique effectuée préalablement par microarray (Human Genome U133Plus 2.0 Affymetrix¦GeneChip oligonucleotide) dans le laboratoire avait révélé une liste de gènes surexprimés¦dans les CSCs, dont fait partie mdr1 (8). Ce gène code la protéine de transport Pgp (Pglycoprotein),¦impliquée dans le mécanisme de résistance (9,10). Une étude par cytométrie¦en flux de l'expression de MDR1 dans nos diverses populations a également été réalisée¦afin de mettre en évidence une potentielle surexpression de ce gène au sein des cellules¦résistantes aux chimiothérapies.

Evaluation des résultats du traitement chirurgical des luxations acromio-claviculaires de type III selon un score cinématique

Introduction : Le traitement des entorses acromio-claviculaires (AC) est aujourd'hui encore controversé. Les luxations AC avec lésion du fascia delto-trapézoidale (grade IV, V et VI) sont généralement traitées par une chirurgie de stabilisation. A l'inverse les entorses sans luxation de la clavicule (grade I et II) sont traitées conservativement avec de bons résultats. Il reste une interrogation concernant le traitement des luxations AC sans lésion du fascia delto-trapézoidale (grade III). Le but de notre étude est d'évaluer les résultats du traitement chirurgical des entorses AC de grade III selon un score cinématique. Matériel et Méthode : 30 patients avec une entorse AC de grade III ont été opérés d'une stabilisation de la clavicule entre 2003 et 2011 par le service d'Orthopédie et traumatologie du CHUV. Tous ont été cliniquement évalués selon le score de Constant. L'évaluation cinématique a été effectuée à l'aide d'un iPod touch, fixé sur l'humérus. Cet outil de mesure, décrit et validé par l'EPFL, prend en considération l'accélération et la vitesse angulaire du membre supérieur pour 7 différents mouvements des deux bras. L'évaluation cinématique a été effectuée en comparant le côté opéré par rapport au côté sain selon 2 scores (RAV et P) provenant de ces variables. Les scores RAV et P sont calculés par l'application installée sur l'iPod touch, ils sont donnés en pourcentage par rapport à l'épaule saine. Nous avons défini un score de Constant relatif de plus de 60 et un score cinématique de plus de 75% comme satisfaisant. Résultats : Nous avons revus dix patients avec un recul moyen de 36 mois (6 à 72 mois) d'un âge moyen de 42 ans (27 à 62 ans). Le score de Constant moyen est de 75.9 ± 21.7. Le score P moyen est de 89.3% ± 23.4 et le score RAV moyen est de 91.8% ± 15.8 (tab.1). Quatre sujets obtiennent un excellent score de Constant pour le bras opéré, 2 sujets obtiennent un bon score et un sujet obtient un score moyen, tandis que 3 sujets obtiennent un mauvais score. Huit patients obtiennent un score cinématique satisfaisant alors que nous observons 2 résultats non satisfaisants. Les mauvais résultats tant cliniques que cinématiques ont été observés chez des patients travailleurs de force, nécessitant d'effectuer des mouvements de l'épaule au-dessus du niveau du buste. Discussion et Conclusion : Sur la base d'une évaluation clinique et cinématique, le traitement chirugical des entorses AC de grade III donne des résultats satisfaisants. Notre étude ne comportant pas de groupe contrôle et notre série étant non homogène, avec un nombre limité de sujet, nous ne pouvons conclure que le traitement chirurgical est le traitement le mieux adapté aux patients avec une entorse acromio-claviculaires de type III. Nous recommandons toutefois un traitement chirugical chez les patients actifs, et les patients exerçant un métier avec nécessité de mobilisation de l'épaule au dessus du buste. Un travail manuel lourd représente un facteur de mauvais pronostic.

Proximity of excitatory synapses and astroglial gap junctions in layer IV of the mouse barrel cortex.

Neurons and astrocytes, the two major cell populations in the adult brain, are characterized by their own mode of intercellular communication--the synapses and the gap junctions (GJ), respectively. In addition, there is increasing evidence for dynamic and metabolic neuroglial interactions resulting in the modulation of synaptic transmission at the so-called "tripartite synapse". Based on this, we have investigated at the ultrastructural level how excitatory synapses (ES) and astroglial GJ are spatially distributed in layer IV of the barrel cortex of the adult mouse. We used specific antibodies for connexin (Cx) 30 and 43 to identify astroglial GJ, these two proteins are known to be present in the majority of astroglial GJ in the cerebral cortex. In electron-microscopic images, we measured the distance between two ES, between two GJ and between a GJ and its nearest ES. We found a ratio of two GJ per three ES in the hollow and septal areas. Taking into account the size of an astrocyte domain, the high density of GJ suggests the occurrence of reflexive type, i.e. GJ between processes of the same astrocyte. Interestingly, the distance between an ES and an astroglial GJ was found to be significantly lower than that between either two synapses or between two GJ. These observations indicate that the two modes of cell-to-cell communication are not randomly distributed in layer IV of the barrel cortex. Consequently, this feature may provide the morphological support for the recently reported functional interactions between neuronal circuits and astroglial networks.

Cell type-specific expression and localization of cytochrome P450 isoforms in tridimensional aggregating rat brain cell cultures.

Within the Predict-IV FP7 project a strategy for measurement of in vitro biokinetics was developed, requiring the characterization of the cellular model used, especially regarding biotransformation, which frequently depends on cytochrome P450 (CYP) activity. The extrahepatic in situ CYP-mediated metabolism is especially relevant in target organ toxicity. In this study, the constitutive mRNA levels and protein localization of different CYP isoforms were investigated in 3D aggregating brain cell cultures. CYP1A1, CYP2B1/B2, CYP2D2/4, CYP2E1 and CYP3A were expressed; CYP1A1 and 2B1 represented almost 80% of the total mRNA content. Double-immunolabeling revealed their presence in astrocytes, in neurons, and to a minor extent in oligodendrocytes, confirming the cell-specific localization of CYPs in the brain. These results together with the recently reported formation of an amiodarone metabolite following repeated exposure suggest that this cell culture system possesses some metabolic potential, most likely contributing to its high performance in neurotoxicological studies and support the use of this model in studying brain neurotoxicity involving mechanisms of toxication/detoxication.