Interactions between cells and functionalized nanoparticles

In this present thesis Superparamagnetic Iron Oxide Nanoparticles (SPIONs) with 9 nm in diameter were selected as nanocarriers in order to study their potential application as drug delivery systems. Therefore the aim of the study was to demonstrate the proof of concept by establishing an efficient system of drug delivery, which would be a valuable tool in biomedical applications, such as the treatement of cancer, by reducing the side effects due to administration of a high concentration of therapeutic agents. As demonstrated in a previous study, the uptake of SPIONs by tumoral human cells was enhanced by the presence of amino groups on their surface. The stabilization of SPIONs were then performed and optimized by the coating of poly(vinylalcohol) and poly(vinylalcohol/vinylamine). Such nanoparticles were known as aminoPVA-SPIONs. The toxicity and the inflammatory reaction of aminoPVA-SPIONs were evaluated in order to establish their potentiel use in the human body. The results demonstrated that the human cells were able to invaginate aminoPVA-SPIONS without revealing any toxicity and inflammatory reaction. The analysis by transmission electron microscopy (TEM), scanning electron microscopy (SEM), cryo-TEM, confocal microscopy and histological staining (i.e. Prussian Blue) showed that the iron oxide core of SPIONs were located in the cytoplasm of cells and concentrated in vesicles. The evaluation of the mechanism of uptake of aminoPVA-SPIONs revealed that their uptake by monolayer cell culture was performed via an active mechanism, which was achieved by a clathrin-mediated endocytosis. Consequently, it was suggested that aminoPVA-SPIONs were good candidates as nanocarriers in drug delivery systems, which were able to reach the cytoplasm of cells. Their incubation with three-dimensional models mimicing tissues, such as differentiated rat brain cell-derived aggregates and spheroids, revealed that aminoPVA-SPIONs were able to invade into deep cell layers according to the stage of growth of these models. In the view of these promising results, drug-SPIONs were prepared by the functionalization of aminoPVA-SPIONs via a biological labile chemical bond by one of these three antineoplastic agents, which are widely used in clinical practice: 5-fluorourdine (Fur) (an antimetabolite), or camptothecin (CPT) (a topoisomerase inhibitor) or doxorubicin (DOX) (an anthracycline which interfere with DNA). The results shown that drug-SPIONs were internalized by human melanoma cells, as it was expected due the previous results with aminoPVA-SPIONs, and in addition they were active as anticancer agents, suggesting the efficient release of the drug from the drug-SPIONs. The results with CPT-SPIONs were the most promising, whereas DOX- SPIONs did not demonstrate a prononced activity of DOX. In conclusion, the results demonstrated that functionalized iron oxide nanoparticles are a promising tool in order to deliver therapeutic agents. - Dans le cadre de ce travail de thèse, les nanoparticules superparamagnétiques d'oxyde de fer (SPIONs) ayant un diamètre de 9 nm ont été choisies, afin d'étudier leur éventuelle utilisation dans un système de délivrance d'agents thérapeutiques. Ainsi le but de la thèse est de démontrer la faisabilité de fabriquer un système efficace de délivrance d'agents thérapeutiques, qui serait un outil intéressant dans le cadre d'une utilisation biomédicale, par exemple lors du traitement du cancer, qui pourrait réduire les effets secondaires provoqués par le dosage trop élevé de médicaments. Comme il a été démontré dans une précédente étude, l'invagination des SPIONs par des cellules humaines cancéreuses est améliorée par la présence de groupes fonctionnels amino à leur surface. La stabilisation des SPIONs est ainsi effectuée et optimisée par l'enrobage de poly(vinylalcool) et de (poly(vinylalcool/vinylamine), qui sont connues sous le nom de aminoPVA-SPIONs. La toxicité et la réaction inflammatoire des aminoPVA-SPIONs ont été évaluées dans le but de déterminer leur potentielle utilisation dans le corps humain. Les résultats démontrèrent que les cellules humaines sont capables d'invaginer les aminoPVAS-SPIONs sans induire une réaction toxique ou inflammatoire. L'analyse par la microscopie électronique en transmission électronique (TEM), la microscopie électronique à balayage (SEM), le cryo-microscopie électronique (SEM), la microscopie confocale et la coloration histologique (par ex, le bleu de Prusse) a montré que l'oxyde de fer des SPIONs est localisé dans le cytoplasme des cellules et est concentré dans des vesicules. L'évaluation du méchanisme d'invagination des aminoPVA-SPIONs ont révélé que leur invagination par des monocultures de cellules est effectué par un méchanisme actif, contrôlé par une endocytose induite par les clathrins. Par conséquent, les aminoPVA-SPIONs sont de bons candidats en tant que transporteurs (nanocamers) dans un système de délivrance d'agents thérapeuthique, capable d'atteindre le cytoplasme des cellules. Leur incubation avec des modèles tridimenstionnels imitant les tissues, tels que les aggrégats de cellules de cerveau différenciées et les sphéroïdes, a montré que les aminoPVA-SPIONs sont capable de pénétrer dans les couches profondes des modèles, selon l'état d'avancement de leur croissance. En vue de ces résultats prometteurs, les drug-SPIONs ont été préparés en fonctionalisant les aminoPVA-SPIONs par le biai d'une liaison chimique labile par un des trois agents thérapeutiques, déjà utilisé en pratique : 5-fluorourdine (Fur) (un antimétabolite), or camptothecin (CPT) (un inhibiteur de la topoisomerase) or doxorubicin (DOX) (un anthracycline qui interfère avec le DNA). Les résultats ont montré que les drug-SPIONs sont capable d'être internalisés par les mélanomes, comme il a été attendu d'après les résultats obtenus précédemment avec les aminoPVA-SPIONs, et de plus, les drug-SPIONs sont actifs, ce qui suggère un relargage efficace de l'agent thérapeutique du drug-SPIONs. Les résultats obtenus avec les CPT-SPIONs sont les plus prometteurs, tandis que ceux avec les DOX-SPIONs, ce n'est pas le cas, dont l'activité thérapeutique de DOX n'a pas été aussi efficace. En conclusion, les résultats ont pu démontrer que les nanoparticules d'oxyde de fer fonctionnalisées sont un outil prometteur dans la délivrance d'agents thérapeutiques.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

The cyclophilin inhibitor alisporivir prevents hepatitis C virus-mediated mitochondrial dysfunction.

Alisporivir (Debio-025) is an analogue of cyclosporine A and represents the prototype of a new class of non-immunosuppressive cyclophilin inhibitors. In vitro and in vivo studies have shown that alisporivir inhibits hepatitis C virus (HCV) replication, and ongoing clinical trials are exploring its therapeutic potential in patients with chronic hepatitis C. Recent data suggest that the antiviral effect is mediated by inhibition of cyclophilin A, which is an essential host factor in the HCV life cycle. However, alisporivir also inhibits mitochondrial permeability transition by binding to cyclophilin D. Because HCV is known to affect mitochondrial function, we explored the effect of alisporivir on HCV protein-mediated mitochondrial dysfunction. Through the use of inducible cell lines, which allow to investigate the effects of HCV polyprotein expression independent from viral RNA replication and which recapitulate the major alterations of mitochondrial bioenergetics observed in infectious cell systems, we show that alisporivir prevents HCV protein-mediated decrease of cell respiration, collapse of mitochondrial membrane potential, overproduction of reactive oxygen species and mitochondrial calcium overload. Strikingly, some of the HCV-mediated mitochondrial dysfunctions could even be rescued by alisporivir. Conclusion: These observations provide new insights into the pathogenesis of HCV-related liver disease and reveal an additional mechanism of action of alisporivir that is likely beneficial in the treatment of chronic hepatitis C. (HEPATOLOGY 2012).

Evolving team compositions by agent swapping

Optimizing collective behavior in multiagent systems requires algorithms to find not only appropriate individual behaviors but also a suitable composition of agents within a team. Over the last two decades, evolutionary methods have emerged as a promising approach for the design of agents and their compositions into teams. The choice of a crossover operator that facilitates the evolution of optimal team composition is recognized to be crucial, but so far, it has never been thoroughly quantified. Here, we highlight the limitations of two different crossover operators that exchange entire agents between teams: restricted agent swapping (RAS) that exchanges only corresponding agents between teams and free agent swapping (FAS) that allows an arbitrary exchange of agents. Our results show that RAS suffers from premature convergence, whereas FAS entails insufficient convergence. Consequently, in both cases, the exploration and exploitation aspects of the evolutionary algorithm are not well balanced resulting in the evolution of suboptimal team compositions. To overcome this problem, we propose combining the two methods. Our approach first applies FAS to explore the search space and then RAS to exploit it. This mixed approach is a much more efficient strategy for the evolution of team compositions compared to either strategy on its own. Our results suggest that such a mixed agent-swapping algorithm should always be preferred whenever the optimal composition of individuals in a multiagent system is unknown.

In vivo study of an injectable poly(acrylonitrile)-based hydrogel paste as a bulking agent for the treatment of urinary incontinence.

Urinary incontinence can be treated by endoscopic injection of bulking agents, however, no optimal therapeutic effect has been achieved upon this treatment yet. In the present study, the development of a injectable poly(acrylonitrile) hydrogel paste is described, and its efficacy and histological behavior, once injected into the submucosal space of the minipig bladder, are evaluated. A device was developed to mix poly(acrylonitrile) hydrogel powder with glycerin, used as carrier, prior to injection into the submucosal space of the bladder. Several paste deposits, depending on the size of the bladder, were injected per animal. The implants were harvested at days 7, 14, 21, 28, 84 and 168 and analyzed morphologically and by histology. The persistence of the implants was demonstrated. However, at later time points the implants were split up and surrounded by granulomatous tissue, which was gradually replaced by histiocytes and adipocytes. Transitory focal urothelial metaplasia was observed only at day 7 and moderate foreign body reaction was detected predominantly between the second and fifth week. This study demonstrated the feasibility to develop an injectable paste of poly(acrylonitrile) hydrogel thought to provide the expected bulking effect, necessary for the treatment of urinary incontinence.

Intratympanic application of an antiviral agent for the treatment of Ménière's disease.

It has been suggested that Ménière's disease is part of a polyganglionitis in which symptoms result from the reactivation of neurotropic virus within the internal auditory canal, and that intratympanic applications of an antiviral agent might be an efficient therapy. In 2002, we performed a pilot study ending with encouraging results. Control of vertigo was achieved in 80% of the 17 patients included. We present here a prospective, double-blind study, with a 2-year follow-up, in 29 patients referred by ENT practitioners for a surgical treatment after failure of a medical therapy. The participation in the study was offered to patients prior to surgery. A solution of ganciclovir 50 mg/ml or of NaCl 9% was delivered for 10 consecutive days via a microwick inserted into the tympanic membrane in the direction of the round window or through a ventilation tube. One patient was withdrawn from the study immediately after the end of the injections. He could not complete the follow-up period, because of persisting vertigo. As he had received the placebo, he was then treated with the solution of ganciclovir. Symptoms persisted and he underwent a vestibular neurectomy. Among the remaining 28 patients, surgery could be postponed in 22 (81%). Surgery remained necessary to control vertigo in 3 patients from the group that received the antiviral agent, and in 3 from the control group. Using an analogical scale, patients of both groups indicated a similar improvement of their health immediately after the intratympanic injections. The scores obtained with a 36-item short-form health survey quality of life questionnaire and the Dizziness Handicap Inventory were also similar for both groups. In conclusion, most patients were improved after the intratympanic injections, but there was no obvious difference between the treated and control groups. The benefit might be due to the middle ear ventilation or reflect an improvement in the patients' emotional state.

Meth Inhibitor FAQs, 2011

This report produced by the Office of Drug Control Policy is a FAQ of Meth Inhibitors.

Systematic assessment of items influencing Crohn's disease patient's preferences in selecting an anti-TNF agent (Choose TNF trial)

Background and Aims: The three anti-TNF agents infliximab (IFX), adalimumab (ADA) andcertolizumab pegol (CZP) have demonstrated similar efficacy in induction and maintenanceof response and remission in Crohn's disease (CD) treatment. Given the comparability ofthese drugs, patient's preferences may influence the choice of the product. However, dataon patient's preferences for choosing anti-TNF agents are lacking. We therefore aimed toassess the CD patient's appraisal to select the drug of his choice and to identify factorsguiding this decision.Methods: A prospective survey among anti-TNF-naive CD patientswas performed. Patients were provided a description of the three anti-TNF agents focusingon indication, application mode (s.c. vs. i.v.), application time intervals, setting of application(hospital vs. private practice vs. patient's home), average time to apply the medication permonth, typical side effects, and the scientific evidence of efficacy and safety available for everydrug. Patients answered a questionnaire consisting of 17 questions, covering demographic,disease-specific, and medication data.Results: Hundred patients (47f/53m, mean age 45±16years) completed the questionnaire. Disease duration was <1year in 7%, 1-5 years in 31%,and >5 years in 62% of patients. Disease location was ileal in 33%, colonic in 40%, andileocolonic in 27%. Disease phenotype was inflammatory in 68%, stenosing in 29%, andinternally fistulizing in 3% of patients. Additionally, 20% had perianal fistulizing disease.Patients were already treated with the following drugs: mesalamines 61%, budesonide 44%,prednisone 97%, thiopurines 78%, methotrexate 16%. In total, 30% had already heardabout IFX, 20% about ADA, and 11% about CZP. Thirty-six percent voted for treatmentwith ADA, 28% for CZP, and 25% for IFX, whereas 11% were undecided. The followingfactors influenced the patient's decision for choosing a specific anti-TNF drug (severalanswers possible): side effects 76%, physician's recommendation 66%, application mode54%, efficacy experience 52%, time to spend for therapy 27%, patient's recommendations21%, interactions with other medications 12%. The single most important factor for choosinga specific anti-TNF was (1 answer): side effect profile 35%, physician's recommendation22%, efficacy experience 21%, application mode 13%, patient's recommendations 5%, timespent for therapy 3%, interaction with other medications 1%.Conclusions: The majority ofpatients preferred anti-TNF syringes to infusions. The safety profile of the drugs and thephysician's recommendation are major factors influencing the patient's choice for a specificanti-TNF drug. Patient's issues about safety and lifestyle habits should be taken into accountwhen prescribing specific anti-TNF formulations.

The vasodilatory effect of juxta-arteriolar microinjection of endothelinA receptor inhibitor in healthy and acute branch retinal vein occlusion minipig retinas.

Purpose. To investigate the effect of the endothelin(A) receptor inhibitor BQ-123 on the retinal arteriolar vasculature in minipig retinas in normal eyes and eyes with acute branch retinal vein occlusion (BRVO). Methods. Seven healthy eyes of seven minipigs and six eyes of six minipigs with experimental BRVO were evaluated under systemic anesthesia. An intravitreal juxta-arteriolar microinjection of 30 microL BQ-123 0.61 microg/mL (pH 7.4) was performed in all but one eye from each group, into which the physiologic saline vehicle alone was injected. Vessel-diameter changes were measured with a retinal vessel analyzer. Results. In healthy minipig retinas (n = 6), arteriolar diameter (+/-SD) increased 6.19% +/- 3.55% (P < 0.05), 25.98% +/- 2.37% (P < 0.001), 23.65% +/- 1.2% (P < 0.001), and 16.84% +/- 1.95% (P < 0.001), at 1, 5, 10, and 15 minutes, respectively, after BQ-123 microinjection. Two hours after experimental BRVO (n = 5), the retinal arteriolar diameter had decreased (13.07% +/- 5.7%; P < 0.01). One, 5, 10, and 15 minutes after BQ-123 microinjection, retinal arteriolar diameter had increased by 7.14% +/- 3.3% (P < 0.01), 26.74% +/- 7.63% (P < 0.001), 23.67% +/- 6.4% (P < 0.001), and 16.09% +/- 3.41% (P < 0.001), respectively. Vehicle only injection had no vasoactive effect on physiologic or BRVO retinas. Conclusions. A significant increase in retinal arteriolar diameter was demonstrated after juxta-arteriolar BQ-123 microinjection in healthy and in acute BRVO minipig retinas. The results suggest a role for endothelin-1 in maintaining retinal basal arteriolar tone. Reversing the BRVO-related vasoconstriction by juxta-arteriolar BQ-123 microinjection could bring a new perspective to the management of BRVO.

Genome sequencing of the plant pathogen Taphrina deformans, the causal agent of peach leaf curl.

Taphrina deformans is a fungus responsible for peach leaf curl, an important plant disease. It is phylogenetically assigned to the Taphrinomycotina subphylum, which includes the fission yeast and the mammalian pathogens of the genus Pneumocystis. We describe here the genome of T. deformans in the light of its dual plant-saprophytic/plant-parasitic lifestyle. The 13.3-Mb genome contains few identifiable repeated elements (ca. 1.5%) and a relatively high GC content (49.5%). A total of 5,735 protein-coding genes were identified, among which 83% share similarities with other fungi. Adaptation to the plant host seems reflected in the genome, since the genome carries genes involved in plant cell wall degradation (e.g., cellulases and cutinases), secondary metabolism, the hallmark glyoxylate cycle, detoxification, and sterol biosynthesis, as well as genes involved in the biosynthesis of plant hormones. Genes involved in lipid metabolism may play a role in its virulence. Several locus candidates for putative MAT cassettes and sex-related genes akin to those of Schizosaccharomyces pombe were identified. A mating-type-switching mechanism similar to that found in ascomycetous yeasts could be in effect. Taken together, the findings are consistent with the alternate saprophytic and parasitic-pathogenic lifestyles of T. deformans. IMPORTANCE: Peach leaf curl is an important plant disease which causes significant losses of fruit production. We report here the genome sequence of the causative agent of the disease, the fungus Taphrina deformans. The genome carries characteristic genes that are important for the plant infection process. These include (i) proteases that allow degradation of the plant tissues; (ii) secondary metabolites which are products favoring interaction of the fungus with the environment, including the host; (iii) hormones that are responsible for the symptom of severely distorted leaves on the host; and (iv) drug detoxification enzymes that confer resistance to fungicides. The availability of the genome allows the design of new drug targets as well as the elaboration of specific management strategies to fight the disease.

Organisational adaptation of multi-agent systems in a peer-to-peer scenario

Organisations in Multi-Agent Systems (MAS) have proven to be successful in regulating agent societies. Nevertheless, changes in agents' behaviour or in the dynamics of the environment may lead to a poor fulfilment of the system's purposes, and so the entire organisation needs to be adapted. In this paper we focus on endowing the organisation with adaptation capabilities, instead of expecting agents to be capable of adapting the organisation by themselves. We regard this organisational adaptation as an assisting service provided by what we call the Assistance Layer. Our generic Two Level Assisted MAS Architecture (2-LAMA) incorporates such a layer. We empirically evaluate this approach by means of an agent-based simulator we have developed for the P2P sharing network domain. This simulator implements 2-LAMA architecture and supports the comparison between different adaptation methods, as well as, with the standard BitTorrent protocol. In particular, we present two alternatives to perform norm adaptation and one method to adapt agents'relationships. The results show improved performance and demonstrate that the cost of introducing an additional layer in charge of the system's adaptation is lower than its benefits.

The caspase 8 inhibitor c-FLIP(L) modulates T-cell receptor-induced proliferation but not activation-induced cell death of lymphocytes.

The caspase 8 inhibitor c-FLIP(L) can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIP(L) in the T-cell compartment (c-FLIP(L) Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIP(L) Tg mice. In contrast, activation-induced cell death of T cells in c-FLIP(L) Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIP(L) Tg mice differed from Fas-deficient mice by showing no accumulation of B220(+) CD4(-) CD8(-) T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIP(L) Tg mice. Thus, a major role of c-FLIP(L) in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.

Bioavailability of repeated oral administration of MDL 100,240, a dual inhibitor of angiotensin-converting enzyme and neutral endopeptidase in healthy volunteers.

BACKGROUND: MDL 100,240 (pyrido[2,1-a] [2]benzazepine-4-carboxylic acid,7-[[2-(acetylthio)-1-oxo-3-phenylpropyl]amino]-1,2,3,4,6,7,8, 12b-octahydro-6-oxo, [4S-[4alpha,7alpha(R(*)),12bbeta]]-) is a molecule possessing an inhibiting ability on both angiotensin converting enzyme (ACE) and neutral endopeptidase, the enzyme responsible for atrial natriuretic peptide (ANP) degradation. Such a dual mechanism of action presents a potential clinical interest for the treatment of hypertension and congestive heart failure. OBJECTIVES: To evaluate the bioavailability of MDL 100,240 and its accumulation over repeated oral administration, using ACE inhibition as a surrogate for plasma drug level and determining its profile after oral and i.v. administration. METHODS: First, in an open, one-period, single-dose study, the ACE inhibition profile was characterised following a 12.5 mg MDL 100,240 i.v. infusion. Second, in a three-group, parallel, randomised, double-blind study, each group of four subjects received q.d., over 8 days, 2.5, 10 or 20 mg of MDL 100,240 orally. The ACE inhibition profile was determined on day 1 and day 8. Trough plasma ACE was measured on days 2, 3 and 4. The recovery of ACE activity was monitored up to 72 h after the last dose of MDL 100,240. RESULTS: ACE inhibition profile was similar on day 1 and day 8, and trough inhibition remained unchanged after the 8 days of treatment with 10 mg or 20 mg. Following repeated 2.5-mg ingestion, trough inhibition increased from 33% to 44% after the eighth dose. The oral bioavailability of MDL 100,240 was estimated at 85%, not statistically different from 100%. The accumulation ratio at steady state was estimated at 112%. Expressing the accumulation ratio in terms of half-life, a t(1/2) of 0.31 days or 7. 5 h was estimated. CONCLUSION: MDL 100,240 (oral solution) has a good bioavailability, as estimated by ACE inhibition, and no drug accumulation seems to occur over 8 days with the 10-mg and 20-mg doses, but a slight rise in the trough level is observed with the 2. 5-mg dose.