Characterization of high osmolarity-induced vacuole fragmentation in "Saccharomyces c."

La majorité des organelles d'une cellule adaptent leur nombre et leur taille pendant les processus de division cellulaire, de trafic vésiculaire ou suite à des changements environnementaux par des processus de fusion et de fragmentation membranaires. Ceci est valable notamment pour le golgi, les mitochondries, les péroxisomes et les lysosomes. La vacuole est le compartiment terminal de la voie endocytaire dans la levure Saccharomyces cerevisiae\ elle correspond aux lysosomes des cellules mammifères. Suite à un choc hyperosmotique, la vacuole se fragmente en plusieurs petites vésicules. Durant ce projet, cette fragmentation a été étudiée en utilisant la technique de microscopie confocale in vivo. J'ai observé que la division de la vacuole se produit d'une façon asymétrique. La première minute après le choc osmotique, les vacuoles rétrécissent et forment des longues invaginations tubulaires. Cette phase est dépendante de la protéine Vps1, un membre de la famille des protéines apparentées à la dynamine, ainsi que d'un gradient transmembranaire de protons. Pendant les 10-15 minutes qui suivent, des vésicules se détachent dans les régions où l'on observe les invaginations pendant la phase initiale. Cette deuxième phase qui mène à la fission des nouveaux compartiments vacuolaires dépend de la production du lipide PI(3,5)P2 par la protéine Fab1. J'ai établi la suite des événements du processus de fragmentation des vacuoles et propose la possibilité d'un rôle régulateur de la protéine kinase cycline-dépendante Pho85.¦En outre, j'ai tenté d'éclaircir plus spécifiquement le rôle de Vps1 pendant la fusion et fission des vacuoles. J'ai trouvé que tous les deux processus sont dépendants de l'activité GTPase de cette protéine. De plus l'association avec la membrane vacuolaire paraît régulée par le cycle d'hydrolyse du GTP. Vps1 peut lier la membrane sans la présence d'un autre facteur protéinique, ce qui permet de conclure à une interaction directe avec des lipides de la membrane. Cette interaction est au moins partiellement effectuée par le domaine GTPase, ce qui est une nouveauté pour un membre de cette famille de protéines. Une deuxième partie de Vps1, nommée insert B, est impliquée dans la liaison à la vacuole, soit par interaction directe avec la membrane, soit par régulation du domaine GTPase. En assumant que Vps1 détienne deux régions capables de liaison aux membranes, je conclus qu'elle pourrait fonctionner comme facteur de « tethering » lors de la fusion des vacuoles.¦-¦La cellule contient plusieurs sous-unités, appelées organelles, possédant chacune une fonction spécifique. Dépendant des processus qui s'y déroulent à l'intérieur, un environnement chimique spécifique est requis. Pour maintenir ces différentes conditions, les organelles sont séparées par des membranes. Lors de la division cellulaire ou en adaptation à des changements de milieu, les organelles doivent être capables de modifier leur morphologie. Cette adaptation a souvent lieu par fusion ou division des organelles. Le même principe est valable pour la vacuole dans la levure. La vacuole est une organelle qui sert principalement au stockage des aliments et à la dégradation des différents composants cellulaires. Alors que la fusion des vacuoles est un processus déjà bien décrit, la fragmentation des vacuoles a jusqu'ici été peu étudiée. Elle peut être induit par un choc osmotique: à cause de la concentration de sel élevé dans le milieu, le cytosol de la levure perd de l'eau. Par un flux d'eau de la vacuole au cytosol, la cellule est capable d'équilibrer celui-ci. Quand la vacuole perd du volume, elle doit réadapter le rapport entre surface membranaire et volume, ce qui se fait efficacement par une fragmentation d'une grande vacuole en plusieurs petites vésicules. Comment ce processus se déroule d'un point de vue morphologique n'a pas été décrit jusqu'à présent. En analysant la fragmentation vacuolaire par microscopie, j'ai trouvé que celle-ci se déroule en deux phases. Pendant la première minute suivant le choc osmotique, les vacuoles rétrécissent et forment des longues invaginations tubulaires. Cette phase dépend de la protéine Vps1, un membre de la famille des protéines apparentées à la dynamine, ainsi que du gradient transmembranaire de protons. Ce gradient s'établit par une pompe membranaire, la V-ATPase, qui transporte des protons dans la vacuole en utilisant l'énergie libérée par hydrolyse d'ATP. Après cette phase initiale, la formation de nouvelles vésicules vacuolaires dépend de la synthèse du lipide PI(3,5)P2.¦Dans la deuxième partie de l'étude, j'ai tenté de décrire comment Vps1 lie la membrane pour effectuer un remodelage de la vacuole. Vps1 est nécessaire pour la fusion et la fragmentation des vacuoles. J'ai découvert que tous les deux processus dépendent de sa capacité d'hydrolyser du GTP. Ainsi l'association avec la membrane est couplée au cycle d'hydrolyse du GTP. Vps1 peut lier la membrane sans la présence d'une autre protéine, et interagit donc très probablement avec les lipides de la membrane. Deux parties différentes de la protéine sont impliquées dans la liaison, dont une, inattendue, le domaine GTPase.¦-¦Numerous organelles undergo membrane fission and fusion events during cell division, vesicular traffic, or in response to changes in environmental conditions. Examples include Golgi (Acharya et al., 1998) mitochondria (Bleazard et al., 1999) peroxisomes (Kuravi et al., 2006) and lysosomes (Ward et al., 1997). In the yeast Saccharomyces cerevisiae the vacuole is the terminal component of the endocytic pathway and corresponds to lysosomes in mammalian cells. Yeast vacuoles fragment into multiple small vesicles in response to a hypertonic shock. This rapid and homogeneous reaction can serve as a model to study the requirements of the fragmentation process. Here, I investigated osmotically induced fragmentation by time-lapse microscopy. I observe that the small fragmentation products originate directly from the large central vacuole by asymmetric scission rather than by consecutive equal divisions and that fragmentation occurs in two distinct phases. During the first minute, vacuoles shrink and generate deep invaginations, leaving behind tubular structures. This phase requires the dynamin-like GTPase Vps1 and the vacuolar proton gradient. In the subsequent 10-15 minutes, vesicles pinch off from the tubular structures in a polarized fashion, directly generating fragmentation products of the final size. This phase depends on the production of phosphatidylinositol- 3,5-bisphosphate by the Fab1 complex. I suggest a possible regulation of vacuole fragmentation by the CDK Pho85. Based on my microscopy study I established a sequential involvement of the different fission factors.¦In addition to the morphological description of vacuole fragmentation I more specifically aimed to shed some light on the role of Vps1 in vacuole fragmentation and fusion. I find that both functions are dependent on the GTPase activity of the protein and that also the membrane association of the dynamin-like protein is coupled to the GTPase cycle. I found that Vps1 has the capacity for direct lipid binding on the vacuole and that this lipid binding is at least partially mediated through residues in the GTPase domain, a complete novelty for a dynamin family member. A second stretch located in the region of insert Β has also membrane-binding activity or regulates the association with the vacuole through the GTPase domain. Under the assumption of two membrane-binding regions I speculate on Vps1 as a possible tethering factor for vacuole fusion.

Ubiquitin-mediated protein degradation and methylation-induced gene silencing cooperate in the inactivation of the INK4/ARF locus in Burkitt lymphoma cell lines.

Burkitt lymphoma is one of the most aggressive tumors affecting humans. Together with the characteristic chromosomal translocation that constitutively activates the c-Myc oncogene, alterations in cellular tumor suppressor pathways are additionally required in order to allow the cells to overcome anti-oncogenic barriers and proliferate in an uncontrolled manner. The INK4a/ARF locus on chromosome 9p21 is considered a safeguard locus since it encodes the two important tumor suppressor proteins, p14 (ARF) and p16 (INK4a) . By regulating the p53 and Rb pathways p14 (ARF) and p16 (INK4a) respectively act as pro-apoptotic and cell cycle inhibitor proteins. The importance of the INK4a/ARF locus has been well documented in several human tumors as well as in Burkitt lymphoma. Although the mechanisms responsible for the transcriptional regulation of the INK4a/ARF locus have been thoroughly characterized, less is known about its posttranscriptional control. In this study we found that p16 (INK4a) and p14 (Arf) are concurrently inactivated in a panel of BL cell lines. We demonstrate that along with the epigenetic silencing of the p16INK4a gene, the complete inactivation of the locus is achieved by the improper turnover of INK4/ARF proteins by the ubiquitin-proteasome system (UPS), as the proteasome inhibitor MG-132 blocks p14 (ARF) degradation and induces a dramatic stabilization of the p16 (INK4a ) protein. We establish that the simultaneous deregulation of both DNA methylation patterns and the ubiquitin-dependent proteolysis system is required to completely inactive the INK4/ARF locus, opening new prospects for the understanding and treatment of Burkitt lymphoma.

CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts.

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.

Central and peripheral metabolic changes induced by gamma-hydroxybutyrate.

STUDY OBJECTIVES: Gamma-hydroxybutyrate (GHB) was originally introduced as an anesthetic but was first abused by bodybuilders and then became a recreational or club drug.1 Sodium salt of GHB is currently used for the treatment of cataplexy in patients with narcolepsy. The mode of action and metabolism of GHB is not well understood. GHB stimulates growth hormone release in humans and induces weight loss in treated patients, suggesting an unexplored metabolic effect. In different experiments the effect of GHB administration on central (cerebral cortex) and peripheral (liver) biochemical processes involved in the metabolism of the drug, as well as the effects of the drug on metabolism, were evaluated in mice. DESIGN: C57BL/6J, gamma-aminobutyric acid B (GABAB) knockout and obese (ob/ob) mice were acutely or chronically treated with GHB at 300 mg/kg. MEASUREMENTS AND RESULTS: Respiratory ratio decreased under GHB treatment, independent of food intake, suggesting a shift in energy substrate from carbohydrates to lipids. GHB-treated C57BL/6J and GABAB null mice but not ob/ob mice gained less weight than matched controls. GHB dramatically increased the corticosterone level but did not affect growth hormone or prolactin. Metabolome profiling showed that an acute high dose of GHB did not increase the brain GABA level. In the brain and the liver, GHB was metabolized into succinic semialdehyde by hydroxyacid-oxoacid transhydrogenase. Chronic administration decreased glutamate, s-adenosylhomocysteine, and oxidized gluthathione, and increased omega-3 fatty acids. CONCLUSIONS: Our findings indicate large central and peripheral metabolic changes induced by GHB with important relevance to its therapeutic use.

Risperidone-induced symptomatic hyperprolactinaemia in adolescents.

Studies performed in adult patients unambiguously demonstrate a marked effect of risperidone on prolactin blood levels, with possible clinical effects related to hyperprolactinemia, such as gynecomastia and galactorrhea. However, the largest study performed in children and adolescents showed a weak effect of risperidone on prolactin concentrations during short-term treatment and a negligible effect during long-term treatment, which was probably because of the relatively low dosages of risperidone used [approximately 0.04 mg/(kg x d)]. Among the 10 psychotic adolescents treated with risperidone in our unit, we had 3 cases of gynecomastia in 3 male patients and 2 cases of galactorrhea in 2 female patients. The prolactin blood levels in these cases and in 3 other patients without apparent prolactin-related side effects were all above the normal range (median, 59 ng/mL; range, 30-123 ng/mL). Thus, risperidone administered to adolescents at doses commonly used for the treatment of psychotic symptoms can strongly increase prolactin levels, with clinical consequences such as gynecomastia and/or galactorrhea. Given that the long-term effects of antipsychotic drug-induced hyperprolactinemia are not well documented, especially regarding osteopenia, infertility, growth, and pubertal delay, risperidone should be administered with caution to children and adolescents.

Local moderate magnetically induced hyperthermia using an implant formed in situ in a mouse tumor model.

Purpose: We investigate a new heat delivery technique for the local treatment of solid tumors. The technique involves injecting a formulation that solidifies to form an implant in situ. This implant entraps superparamagnetic iron oxide nanoparticles (SPIONs) embedded in silica microbeads for magnetically induced moderate hyperthermia. Particle entrapment prevents phagocytosis and distant migration of SPIONs. The implant can be repeatedly heated by magnetic induction. Methods: We evaluated heating and treatment efficacies by means of thermometry and survival studies in nude mice carrying subcutaneous human colocarcinomas. At day 1, we injected the formulation into the tumor. At day 2, a single 20-min hyperthermia treatment was delivered by 141-kHz magnetic induction using field strengths of 9 to 12 mT under thermometry. Results: SPIONs embedded in silica microbeads were effectively confined within the implant at the injection site. Heat-induced necro-apoptosis was assessed by histology on day 3. On average, 12 mT resulted in tumor temperature of 47.8 degrees C, and over 70% tumor necrosis that correlated to the heat dose (AUC = 282 degrees C.min). In contrast, a 9-mT field strength induced tumoral temperature of 40 degrees C (AUC = 131 degrees C.min) without morphologically identifiable necrosis. Survival after treatment with 10.5 or 12 mT fields was significantly improved compared to non-implanted and implanted controls. Median survival times were 27 and 37 days versus 12 and 21 days respectively. Conclusion: Five of eleven mice (45%) of the 12 mT group survived one year without any tumor recurrence, holding promise for tumor therapy using magnetically induced moderate hyperthermia through injectable implants.

Dissociation of AGAT, GAMT and SLC6A8 in CNS: relevance to creatine deficiency syndromes.

AGAT and GAMT, the two enzymes of the creatine synthesis pathway, are well expressed within CNS, suggesting autonomous brain creatine synthesis. This contradicts SLC6A8 deficiency, which causes creatine deficiency despite CNS expression of AGAT and GAMT. We hypothesized that AGAT and GAMT were not co-expressed by brain cells, and that guanidinoacetate must be transported between cells to allow creatine synthesis. We finely analyzed the cell-to-cell co-expression of AGAT, GAMT and SLC6A8 in various regions of rat CNS, and showed that in most structures, cells co-expressing AGAT+GAMT (equipped for autonomous creatine synthesis) were in low proportions (<20%). Using reaggregating brain cell cultures, we also showed that brain cells take up guanidinoacetate and convert it to creatine. Guanidinoacetate uptake was competed by creatine. This suggests that in most brain regions, guanidinoacetate is transported from AGAT- to GAMT-expressing cells through SLC6A8 to allow creatine synthesis, thereby explaining creatine deficiency in SLC6A8-deficient CNS.

The mechanisms of inflammation in gout and pseudogout (CPP-induced arthritis).

Recent advances have stimulated new interest in the area of crystal arthritis, as microcrystals can be considered to be endogenous "danger signals" and are potent stimulators of immune as well as non-immune cells. The best known microcrystals include urate (MSU), and calcium pyrophosphate (CPP) crystals, associated with gout and pseudogout, respectively. Acute inflammation is the hallmark of the acute tissue reaction to crystals in both gout and pseudogout. The mechanisms leading to joint inflammation in these diseases involve first crystal formation and subsequent coating with serum proteins. Crystals can then interact with plasma cell membrane, either directly or via membrane receptors, leading to NLRP3 activation, proteolytic cleavage and maturation of pro-interleukin-1β (pro-IL1β) and secretion of mature IL1β. Once released, this cytokine orchestrates a series of events leading to endothelial cell activation and neutrophil recruitment. Ultimately, gout resolution involves several mechanisms including monocyte differentiation into macrophage, clearance of apoptotic neutrophils by macrophages, production of Transforming Growth Factor (TGF-β) and modification of protein coating on the crystal surface. This review will examine these different steps.

Interleukin-8 secretion by fibroblasts induced by low density lipoproteins is p38 MAPK-dependent and leads to cell spreading and wound closure.

We have previously reported (Dobreva, I., Waeber, G., Mooser, V., James, R. W., and Widmann, C. (2003) J. Lipid Res. 44, 2382-2390) that low density lipoproteins (LDLs) induce activation of the p38 MAPK pathway, resulting in fibroblast spreading and lamellipodia formation. Here, we show that LDL-stimulated fibroblast spreading and wound sealing are due to secretion of a soluble factor. Using an antibody-based human protein array, interleukin-8 (IL-8) was identified as the main cytokine whose concentration was increased in supernatants from LDL-stimulated cells. Incubation of supernatants from LDL-treated cells with an anti-IL-8 blocking antibody completely abolished their ability to induce cell spreading and mediate wound closure. In addition, fibroblasts treated with recombinant IL-8 spread to the same extent as cells incubated with LDL or supernatants from LDL-treated cells. The ability of LDL and IL-8 to induce fibroblast spreading was mediated by the IL-8 receptor type II (CXCR-2). Furthermore, LDL-induced IL-8 production and subsequent wound closure required the activation of the p38 MAPK pathway, because both processes were abrogated by a specific p38 inhibitor. Therefore, the capacity of LDLs to induce fibroblast spreading and accelerate wound closure relies on their ability to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL cholesterol levels, IL-8 production, and extensive remodeling of the vessel wall.

Fas ligand-induced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium tuberculosis.

Mycobacterium tuberculosis-specific cytolytic activity is mediated mostly by CD4+CTL in humans. CD4+CTL kill infected target cells by inducing Fas (APO-1/CD95)-mediated apoptosis. We have examined the effect of Fas ligand (FasL)-induced apoptosis of human macrophages infected in vitro with M. tuberculosis on the viability of the intracellular bacilli. Human macrophages expressed Fas and underwent apoptosis after incubation with soluble recombinant FasL. In macrophages infected either with an attenuated (H37Ra) or with a virulent (H37Rv) strain of M. tuberculosis, the apoptotic death of macrophages was associated with a substantial reduction in bacillary viability. TNF-induced apoptosis of infected macrophages was coupled with a similar reduction in mycobacterial viability, while the induction of nonapoptotic complement-induced cell death had no effect on bacterial viable counts. Infected macrophages also showed a reduced susceptibility to FasL-induced apoptosis correlating with a reduced level of Fas expression. These data suggest that apoptosis of infected macrophages induced through receptors of the TNF family could be an immune effector mechanism not only depriving mycobacteria from their growth environment but also reducing viable bacterial counts by an unknown mechanism. On the other hand, interference by M. tuberculosis with the FasL system might represent an escape mechanism of the bacteria attempting to evade the effect of apoptosis.

Mechanisms of B cell survival induced by BAFF, APRIL and their receptors

RESUME : BAFF est un membre de 1a famille du TNF qui contrôle l'homéostasie des lymphocytes B. BAFF lie les récepteurs TACI, BCMA et BAFF-R sur les cellules B, tandis qu'APRIL, son proche homologue, lie seulement TACI et BCMA. BAFF et APRIL sont des protéines transmembranaires pouvant -être relâchées sous forme de cytokines trimériques solubles suite à un clivage protéolytique. Le BAFF soluble peut s'assembler en 60-mère. Les rôles physiologiques des BAFF membranaires et solubles sont inconnnus. Nous avons étudié la capacité de diverses formes de BAFF et APRIL à activer différents récepteurs. BAFF-R répond à toutes les formes dé BAFF, tandis que TACI nécessite du BAFF ou de l'APRIL membranaire ou oligomérisé pour être activé et pour transmettre des signaux de survie dans les lymphocytes B primaires. TACI ne répond pas aux ligands trimériques bien qu'il puisse les lier. TACI est essentiel pour la réponse humorale aux antigènes présentant des épitoges répétitifs, une réponse qui est indépendante des lymphocytes T (réponse TI-2). Des souris exprimant moins de BAFF ont un pourcentage modérément réduit de lymphocytes B et leur réponse TI-2 est atténuée. Par contre, des souris qui n'expriment que du BAFF membranaire ont encore moins de cellules B mais répondent efficacement aux antigènes TI-2. Ces résultats suggèrent que le BAFF soluble est impliqué dans le maintien de la population des lymphocytes B, alors que le BAFF membranaire peut activer TACI lors d'are réponse TI-2. Le BAFF 60-mère est un autre activateur potentiel de TACI in vivo. Le BAFF 60-mère existe dans des surnageants de cellules productrices de BAFF mais n'est pas détecté dans le plasma de souris saines, même lorsqu'elles présentent des niveaux élevés de BAFF. BAFF 60-mère est néanmoins présent dans le plasma de souris transgéniques pour BAFF et de souris déficientes en TACI. Comme ces deux lignées présentent des signes d'autoimmunité, ces résultats suggèrent que la présence de BAFF 60-mère pourrait être liée à des conditions pathologiques. Summary : The TNF family ligand BAFF is essential for B cell homeostasis. BAFF binds to the receptors TACI, BCMA and BAFF-R on B cells, whereas its close homolog APRIL binds to TACI and BCMA only. BAFF and APRIL are transmembrane proteins, which can be proteolytically processed to release trimeric soluble cytokines. Soluble BAFF 3-mer can further assemble in a 60-mer. The physiological roles of membrane-bound and soluble BAFF are unknown. We studied the ability of various forms of BAFF and APRIL to signal through different receptors. BAFF-R responded to all forms of BAFF, but TACI required membrane-bound, cross-licked or oligomeric BAFF or APRIL in order to transmit productive signals in primary B cells. TACI was unresponsive to trimeric ligands, although it could bind them. TACI is essential for T-cell independent antibody responses to antigens with repetitive epitopes (TI-2 responses). Mice expressing lower than normal levels of BAFF displayed a moderate B cell reduction and impaired TI-2 responses, whereas mice expressing membrane-bound BAFF displayed severe B cell reduction, but unimpaired TI-2 responses. These results suggest that processed BAFF is involved in the maintenance of the B cell pool and that membrane-bound BAFF can activate TACI during T-cell independent humoral responses. BAFF 60-mer is another potential activator of TACI in vivo. BAFF 60-mer was detected in the supernatant of BAFF-producing cells, but not in the plasma of healthy mice with either norma1 or elevated BAFF levels. It was however present in sera of BAFF transgenic mice and TACI-/- mice, both of which suffer from autoimmunity, suggesting that GAFF 60-mer may be linked to pathogenic conditions.

Aganirsen Antisense Oligonucleotide Eye Drops Inhibit Keratitis-Induced Corneal Neovascularization and Reduce Need for Transplantation: The I-CAN Study.

OBJECTIVE: Eye drops of aganirsen, an antisense oligonucleotide preventing insulin receptor substrate-1 expression, inhibited corneal neovascularization in a previous dose-finding phase II study. We aimed to confirm these results in a phase III study and investigated a potential clinical benefit on visual acuity (VA), quality of life (QoL), and need for transplantation. DESIGN: Multicenter, double-masked, randomized, placebo-controlled phase III study. PARTICIPANTS: Analysis of 69 patients with keratitis-related progressive corneal neovascularization randomized to aganirsen (34 patients) or placebo (35 patients). Patients applied aganirsen eye drops (86 μg/day/eye) or placebo twice daily for 90 days and were followed up to day 180. MAIN OUTCOME MEASURES: The primary end point was VA. Secondary end points included area of pathologic corneal neovascularization, need for transplantation, risk of graft rejection, and QoL. RESULTS: Although no significant differences in VA scores between groups were observed, aganirsen significantly reduced the relative corneal neovascularization area after 90 days by 26.20% (P = 0.014). This improvement persisted after 180 days (26.67%, P = 0.012). Aganirsen tended to lower the transplantation need in the intent-to-treat (ITT) population at day 180 (P = 0.087). In patients with viral keratitis and central neovascularization, a significant reduction in transplantation need was achieved (P = 0.048). No significant differences between groups were observed in the risk of graft rejection. However, aganirsen tended to decrease this risk in patients with traumatic/viral keratitis (P = 0.162) at day 90. The QoL analyses revealed a significant improvement with aganirsen in composite and near activity subscores (P = 0.039 and 0.026, respectively) at day 90 in the per protocol population. Ocular and treatment-related treatment-emergent adverse events (TEAEs) were reported in a lower percentage with aganirsen compared with placebo. Only 3 serious TEAEs (2 with aganirsen and 1 with placebo) were considered treatment-related. CONCLUSIONS: This first phase III study on a topical inhibitor of corneal angiogenesis showed that aganirsen eye drops significantly inhibited corneal neovascularization in patients with keratitis. The need for transplantation was significantly reduced in patients with viral keratitis and central neovascularization. Topical application of aganirsen was safe and well tolerated.

Stimulation-induced increases of astrocytic oxidative metabolism in rats and humans investigated with 1-11C-acetate

The purpose of this study was to investigate astrocytic oxidative metabolism using 1-(11)C-acetate. 1-(11)C-acetate kinetics were evaluated in the rat somatosensory cortex using a beta-scintillator during different manipulations (test-retest, infraorbital nerve stimulation, and administration of acetazolamide or dichloroacetate). In humans a visual activation paradigm was used and kinetics were measured with positron emission tomography. Data were analyzed using a one-tissue compartment model. The following features supported the hypothesis that washout of radiolabel (k(2)) is because of (11)C-CO(2) and therefore related to oxygen consumption (CMRO(2)): (1) the onset of (11)C washout was delayed; (2)k(2) was not affected by acetazolamide-induced blood flow increase; (3)k(2) demonstrated a significant increase during stimulation in rats (from 0.014+/-0.007 to 0.027+/-0.006 per minute) and humans (from 0.016+/-0.010 to 0.026+/-0.006 per minute); and (4) dichloroacetate led to a substantial decrease of k(2). In the test-retest experiments K(1) and k(2) were very stable. In summary, 1-(11)C-acetate seems a promising tracer to investigate astrocytic oxidative metabolism in vivo. If the washout rate indeed represents the production of (11)C-CO(2), then its increase during stimulation would point to a substantially higher astrocytic oxidative metabolism during brain activation. However, the quantitative relationship between k(2) and CMRO(2) needs to be determined in future experiments.

Dose reduction of epoetin-alpha in the prevention of chemotherapy-induced anaemia.

INTRODUCTION: Anaemia during chemotherapy is often left untreated. Erythropoiesis-stimulating agents are frequently used to treat overt anaemia. Their prophylactic use, however, remains controversial and raises concerns about cost-effectiveness. Therefore, we assessed the efficacy of a dose-reduction schedule in anaemia prophylaxis. MATERIALS AND METHODS: The study included patients with untreated solid tumours about to receive platinum-based chemotherapy and had haemoglobin (Hb) levels ≥11 g/dL. Epoetin-α was administered at a dose level of 3 × 10,000 U weekly as soon as Hb descended to < 13 g/dL. Dose reductions to 3 × 4,000 U and 3 × 2,000 U weekly were planned in 4-week intervals if Hb stabilised in the range of 11-13 g/dL. Upon ascending to ≥13 g/dL, epoetin was discontinued. Iron supplements of 100 mg intravenous doses were given weekly. Of 37 patients who enrolled, 33 could be evaluated. RESULTS AND DISCUSSION: Their median Hb level was 13.7 (10.9-16.2) g/dL at baseline and descended to 11.0 (7.4-13.8) g/dL by the end of chemotherapy. Anaemia (Hb < 10 g/dL) was prevented in 24 patients (73%). The mean dose requirement for epoetin-α was 3 × 5,866 U per week per patient, representing a dose reduction of 41%. Treatment failed in nine patients (27%), in part due to epoetin-α resistance in four (12%) and blood transfusion in three (9%) patients. CONCLUSION: Dose reduction was as effective as fixed doses in anaemia prophylaxis but reduced the amount of prescribed epoetin substantially.

An introductory review on gravitational-deformation induced structures, fabrics and modeling

Recent studies have pointed out a similarity between tectonics and slope tectonic-induced structures. Numerous studies have demonstrated that structures and fabrics previously interpreted as of purely geodynamical origin are instead the result of large slope deformation, and this led in the past to erroneous interpretations. Nevertheless, their limit seems not clearly defined, but it is somehow transitional. Some studies point out continuity between failures developing at surface with upper crust movements. In this contribution, the main studies which examine the link between rock structures and slope movements are reviewed. The aspects regarding model and scale of observation are discussed together with the role of pre-existing weaknesses in the rock mass. As slope failures can develop through progressive failure, structures and their changes in time and space can be recognized. Furthermore, recognition of the origin of these structures can help in avoiding misinterpretations of regional geology. This also suggests the importance of integrating different slope movement classifications based on distribution and pattern of deformation and the application of structural geology techniques. A structural geology approach in the landslide community is a tool that can greatly support the hazard quantification and related risks, because most of the physical parameters, which are used for landslide modeling, are derived from geotechnical tests or the emerging geophysical approaches.