Cutaneous leishmaniasis mimicking a pyogenic granuloma.

The Leishmania genus is formed by parasitic protozoa which are transmitted by the bite of infected female sand flies. Cases of sexual, vertical or transfusional transmission or via infected needles have also been described. In humans, 4 forms of this disease have been described: localised cutaneous (LC), diffuse cutaneous, mucocutaneous and visceral (1). LC counts for 50–75% of all cases (2), it is the mildest form of the disease and can be caused by any species of Leishmania. In Spain, the most frequent form is the oriental sore caused by L. infantum (2). Most cases resolve spontaneously within one year. In United States and Europe, the incidence is increasing due to tourism and co-infection with HIV. The morphological spectrum of LC is very wide; multiple forms of clinical presentation have been described, although the most characteristic one is the nodular ulcerative lesion, characterised by painless crater-like ulcers with a necrotic base and covered by an adhesive crust. The main complication of LC is its progression in some strains towards the other 3 forms of the disease (3). In patients with AIDS and other diseases associated with immunosuppression the risk of dissemination is much higher than in the immunocompetent. We present a case of LC with clinical and histopathological features similar to a pyogenic granuloma.

Pooling of dentin microtensile bond strength data improves clinical correlation.

Purpose: To evaluate whether the correlation between in vitro bond strength data and estimated clinical retention rates of cervical restorations after two years depends on pooled data obtained from multicenter studies or single-test data. Materials and Methods: Pooled mean data for six dentin adhesive systems (Adper Prompt L-Pop, Clearfil SE, OptiBond FL, Prime & Bond NT, Single Bond, and Scotchbond Multipurpose) and four laboratory methods (macroshear, microshear, macrotensile and microtensile bond strength test) (Scherrer et al, 2010) were correlated to estimated pooled two-year retention rates of Class V restorations using the same adhesive systems. For bond strength data from a single test institute, the literature search in SCOPUS revealed one study that tested all six adhesive systems (microtensile) and two that tested five of the six systems (microtensile, macroshear). The correlation was determined with a database designed to perform a meta-analysis on the clinical performance of cervical restorations (Heintze et al, 2010). The clinical data were pooled and adjusted in a linear mixed model, taking the study effect, dentin preparation, type of isolation and bevelling of enamel into account. A regression analysis was carried out to evaluate the correlation between clinical and laboratory findings. Results: The results of the regression analysis for the pooled data revealed that only the macrotensile (adjusted R2 = 0.86) and microtensile tests (adjusted R2 = 0.64), but not the shear and the microshear tests, correlated well with the clinical findings. As regards the data from a single-test institute, the correlation was not statistically significant. Conclusion: Macrotensile and microtensile bond strength tests showed an adequate correlation with the retention rate of cervical restorations after two years. Bond strength tests should be carried out by different operators and/or research institutes to determine the reliability and technique sensitivity of the material under investigation.

Study of chemical modification of alkaline lignin by the glyoxalation reaction

In this study, glyoxalated alkaline lignins with a non-volatile and non-toxic aldehyde, which can be obtained from several natural resources, namely glyoxal, were prepared and characterized for its use in wood adhesives. The preparation method consisted of the reaction of lignin with glyoxal under an alkaline medium. The influence of reaction conditions such as the molar ratio of sodium hydroxide-to-lignin and reaction time were studied relative to the properties of the prepared adducts. The analytical techniques used were FTIR and 1H-NMR spectroscopies, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Results from both the FTIR and 1H-NMR spectroscopies showed that the amount of introduced aliphatic hydroxyl groups onto the lignin molecule increased with increasing reaction time and reached a maximum value at 10 h, and after they began to decrease. The molecular weights remained unchanged until 10 h of reaction time, and then started to increase, possibly due to the repolymerization reactions. DSC analysis showed that the glass transition temperature (Tg) decreased with the introduction of glyoxal onto the lignin molecule due to the increase in free volume of the lignin molecules. TGA analysis showed that the thermal stability of glyoxalated lignin is not influenced and remained suitable for wood adhesives. Compared to the original lignin, the improved lignin is reactive and a suitable raw material for adhesive formula

Régulation du processus métastatique tumoral par l'oxyde nitrique (NO), le TGF-β el les cellules de l'hôte

RESUME Nous avons étudié le rôle de deux molécules, le Transfon-ning Growth Factor (TGF-β) et l'oxyde nitrique (NO), dans le processus métastatique. Deux clones tumoraux ont été sélectionnés à partir d'un carcinome du côlon pour leur différence de potentiel tumorigénique dans des rats syngéniques. La croissance tumorale du clone progressif PROb a été corrélée à sa capacité à sécréter le TGF-β actif Cependant, la transfection du clone régressif REGb, sécrétant du TGF-β latent, par une vecteur codant pour le TGF-β bio-actif n'a pas permis d'induire le développement tumoral. Les deux clones tumoraux présentent des activités des protéases MMP-2, APN et DPPIV identiques et qui ne semblent pas modifiées par le TGF-β. L'interaction des cellules tumorales avec l'endothélium et l'activité de la NO synthase (iNOS) responsable de la synthèse de NO sont impliqués dans la progression de nombreux cancers. Le clone PROb, mais pas le clone REGb, inhibe l'activation de la iNOS des cellules endothéliales par sa sécrétion de TGF-β actif Les deux clones montrent cependant des propriétés d'adhésion identiques aux cellules endothéliales et sont capables d'inhiber par contact cellulaire direct l'activation de la iNOS endothéliale. Ceci suggère que ces contacts directs pourraient créer un micro-environnement favorable à la conversion du TGF-β latent en TGF-β actif ou à d'autres interactions moléculaires pouvant réguler l'activation endothéliale. Par ailleurs, les deux clones activent des macrophages du système nerveux central, organe où ils ne forment pas de métastases, mais pas les macrophages circulants, illustrant des mécanismes différentiels et spécifiques dans l'activation de différents types de cellules immunitaires. Afin de mieux comprendre le rôle du NO dans la dissémination métastatique, deux clones cellulaires différant par le taux d'activité de la iNOS ont été sélectionnés à partir de la lignée murine parentale de carcinome du sein EMT-6. Bien que le NO soit un inhibiteur potentiel de la prolifération cellulaire, les deux clones montrent des propriétés prolifératives identiques in vitro. Les cellules EMT-6H qui produisent peu de NO in vitro forment de nombreux nodules tumoraux pulmonaires in vivo corrélés à une mortalité significative des souris syngéniques injectées. Les cellules EMT-6J qui présentent une expression élevée de iNOS et de NO induisent de rares nodules tumoraux pulmonaires et peu de mortalité. Dans ce modèle, l'expression tumorale de NO semble donc défavoriser la croissance tumorale. Les deux clones cellulaires ont des propriétés identiques d'adhésion et de prolifération mesurées in vitro sur des cellules endothéliales primaires isolées de différents organes et in vivo par une colocalisation identique dans les poumons de souris syngéniques 48h après leur injection. Les cellules EMT-6H présentent une activité MMP-2 plus élevée alors que les activités des protéases APN et DPPIV sont identiques dans les deux clones cellulaires. Le TGF-β soluble ainsi que les fibroblastes primaires bloquent la prolifération des deux clones cellulaires. Cependant, l'activation préalable des fibroblastes par du TGF-β restaure partiellement la prolifération du clone EMT-6H mais pas celle du clone EMT-6J. Ces résultats montrent que le rôle de molécules telles que le TGF-β et le NO tumoral dans la progression tumorale doit être considéré dans un contexte d'interactions des cellules tumorales avec les différentes types cellulaires de l'hôte: en particulier, notre travail souligne que les macrophages et les fibroblastes sont déterminants dans la progression métastatique des carcinomes du côlon ou du sein. RESUME DESTINE A UN LARGE PUBLIC Les métastases tumorales, disséminées et intraitables par chirurgie, représentent un problème majeur dans le traitement clinique du cancer. Elles sont dues à des cellules tumorales qui ont migré de leur site tumoral primaire, circulé et survécu dans le système vasculaire de l'hôte, échappé au système immunitaire, adhéré à et survécu sur l'endothélium des vaisseaux, et envahi le tissu sous-jacent où elles ont proliféré. Cette capacité à former des métastases implique de nombreux facteurs dont certains ont été identifiés mais dont le rôle reste controversé dans les différentes études. Nous nous sommes intéressés au rôle de l'oxyde nitrique (NO) et du facteur de croissance et de transformation cellulaire TGF-β. Dans les carcinomes du sein, l'expression des enzymes responsables de la synthèse de NO a été corrélée avec l'invasion tumorale mais aussi avec un pronostic favorable selon les études. Deux clones cellulaires ont été isolés à partir de la tumeur mammaire EMT-6 chez la souris. Le clone EMT-6H sécrète peu de NO et forme de nombreuses tumeurs dans les poumons des souris *entraînant leur décès. Le clone EMT-6J sécrète beaucoup de NO et ne se développe que peu dans les poumons. Dans ce modèle expérimental, le NO semble donc défavoriser la croissance tumorale. L'analyse des interactions avec les cellules de l'hôte rencontrées lors de la formation de métastases pulmonaires a montré que les deux clones cellulaires adhérent et prolifèrent de manière similaire sur les cellules endothéliales tapissant l'intérieur des vaisseaux sanguins. L'arrêt des cellules tumorales dans les poumons ne permet donc pas d'expliquer la différence de croissance tumorale. Cependant, le clone agressif EMT-6H présente une activité élevée d'une protéase (MMP-2) qui lui permettrait par la suite d'envahir le tissu pulmonaire. Par ailleurs, l'activation des fibroblastes du tissu pulmonaire par le TGF-β, une molécule observée dans des conditions inflammatoires, permet au clone agressif EMT-6H de proliférer mais inhibe la croissance du clone EMT-6J. Dans un modèle expérimental de carcinome du côlon, le TGF-β est considéré favorable à la croissance tumorale. Isolées à partir de la même tumeur initiale, deux lignées de cellules ont des comportements opposés lorsqu'elles sont injectées sous la peau des rats. La capacité de la lignée PROb à former des tumeurs a été corrélée à la sécrétion de TGF-β actif L'introduction du gène codant pour le TGF-β actif dans la lignée REGb, qui ne sécrète pas de TGF-β actif et ne forme pas de tumeurs chez le rat, ne restaure pas leur potentiel tumorigénique. Dans ce modèle, l'expression de TGF-β actif ne semble donc pas suffisante à la croissance tumorale. Les interactions avec différents types cellulaires de l'hôte ont été étudiées. Les deux lignées tumorales adhérent de manière similaire sur les cellules endothéliales et sont capables d'inhiber leur activation, un mécanisme qui pourrait participer à la destruction. Les deux lignées activent les cellules immunitaires du système nerveux central, un organe où elles ne forment pas de métastase. Ces résultats suggèrent que la sélection des cellules métastatiques ne s'effectue pas sur l'endothélium des vaisseaux sanguins mais à des étapes ultérieures dans le micro- environnement cellulaire du nouvel organe colonisé. SUMMARY Metastasis results from the migration of tumor cells from their primary tumor, circulation through the bloodstream, attachment to the endothelium, and invasion of the surrounding tissue where they create a microenvironnement favoring their growth. This multistep process implies various cellular interactions and molecules. Among those, we were interested in the role of the Transforming Growth Factor beta (TGF-β) and the nitric oxide (NO). Two cell lines were isolated from a rat colon tumor and assessed for their metastatic potential in vivo. The PROb cell line that expresses active TGF-β formed subcutaneous tumors in rats while the REGb cell line that expresses only latent TGF-β did not. Transfection of REGb cells with a plasmid encoding for the active form of TGF-β failed to restore their metastatic ability. Thus TGF-β secretion is not sufficient to induce colon carcinoma progression. Activities of various proteases such as APN, DPPIV and MMP were similar in both cell lines and were not regulated by TGF-β. Interactions with the endothelium as well as NO synthase activity (iNOS) and local NO concentrations are believed to be crucial steps in cancer metastasis. Coculture of the two clones with endothelial cells inhibited the cytokine-triggered activation of the iNOS enzyme in primary rat endothelial cells but only PROb cells were capable of increasing the expression of IL-6, a protumoral interleukin that may participate in the impairment of the anti-tumoral immune response of the host. Both cell lines exhibited potential to activate microglial cells but not bone marrow-derived macrophages, pointing to a differential regulation of specialized immune cells. To better understand the conflicting role of NO in breast cancer progression, two cell clones were selected from the murine tumorigenic cell line EMT-6 based on their iNOS activity and NO secretion. Although NO has been shown to inhibit cell proliferation, the two cell clones exhibited similar proliferation rates in vitro. The EMT-6H cells expressed little NO and grew actively in the lungs of syngenic mice, leading to their death. Opposite results were observed with the EMT-6J cells. In these in vivo conditions, NO seems to impair tumor growth. Both clones exhibited similar in vitro adhesive properties to primary endothelial cells isolated from various mouse organs and similar localization in the lungs of mice 48 hours after injection. Sustained metalloproteinase MMP-2 activity was detected in the tumorigenic EMT-6H clone, but not in the EMT-6J cells while other proteases such as APN and DPPIV showed no difference. These results suggested that the two clones differed in invasion steps following adhesion to the endothelium and that NO did not participate in previous steps. Consistent with this, both soluble TGF-β and supernatants of cultures of mouse primary lung fibroblasts inhibited the growth of the two clones. However, previous activation of these fibroblasts with TGF-β restored the growth of the tumorigenic EMT-6H cells, but not of EMT-6J cells. Altogether, these results indicate that the role of a given molecule, such as NO or TGF-β, must be considered in a context of interaction of tumor cells with host cells. They further imply that interaction of tumor cells with specialized immune cells and with stromal cells of the colonized organ, rather than with the endothelium, are critical in regulating metastasis.

Outcome after surgical treatment of symptomatic delayed unions and nonunions of midshaft clavicle fractures

Introduction: Nonoperative treatment of displaced midshaft clavicle fractures is associated with higher nonunion rate than previously reported. Moreover, its occurrence can compromise shoulder function. The aim of this study was to evaluate the outcome of surgical treatment of symptomatic clavicle midshaft delayed and nonunion. Methods: Between 1999 and 2008, 19 clavicle delayed unions and nonunions were treated by open reduction and reconstructive plate fixation with augmentation by autologous bone graft. Iliac bone graft was used in 15 atrophic cases, and graft from the callus was used in 4 hypertrophic nonunions. There were 14 men and 5 women, with an average age of 41 years (range, 19 to 59 years) at time of surgery. No patient had undergone a previous surgery and all complained of shoulder pain. Delayed unions and nonunions were defined as non-healing after 3 and 6 months respectively. The mean time to surgery was 8 months (range, 4 to 23 months). All patients were pre and postoperatively clinically evaluated and imaged with standard radiographs until complete healing. Results: After a mean time of 3 months (range, 2 to 7 months) all fractures were completely healed. All patients reported full range of motion at time of last follow-up. Nine patients (47%) reported slight shoulder pain but all returned to their previous professional activities after a mean time of 3 months (range, 1 to 8 months). We reported 12 (63%) minor complications. There were 6 (32%) plate-related discomforts which resolved after hardware removal, two (11%) scar numbness, two (11%) adhesive capsulitis with spontaneous complete recovery, and two (11%) AC-joint pain treated successfully with local corticosteroids injection. Conclusion: Surgical treatment of delayed unions and nonunions of midshaft clavicle fractures yields satisfactory results and a high union rate. However, 50% of the patients may still complain of slight residual shoulder pain.

Evalution of Hot Mix Asphalt Moisture Sensitivity Using the Nottingham Asphalt Test Equipment, July 2005

Moisture sensitivity of Hot Mix Asphalt (HMA) mixtures, generally called stripping, is a major form of distress in asphalt concrete pavement. It is characterized by the loss of adhesive bond between the asphalt binder and the aggregate (a failure of the bonding of the binder to the aggregate) or by a softening of the cohesive bonds within the asphalt binder (a failure within the binder itself), both of which are due to the action of loading under traffic in the presence of moisture. The evaluation of HMA moisture sensitivity has been divided into two categories: visual inspection test and mechanical test. However, most of them have been developed in pre-Superpave mix design. This research was undertaken to develop a protocol for evaluating the moisture sensitivity potential of HMA mixtures using the Nottingham Asphalt Tester (NAT). The mechanisms of HMA moisture sensitivity were reviewed and the test protocols using the NAT were developed. Different types of blends as moisture-sensitive groups and non-moisture-sensitive groups were used to evaluate the potential of the proposed test. The test results were analyzed with three parameters based on performance character: the retained flow number depending on critical permanent deformation failure (RFNP), the retained flow number depending on cohesion failure (RFNC), and energy ratio (ER). Analysis based on energy ratio of elastic strain (EREE ) at flow number of cohesion failure (FNC) has higher potential to evaluate the HMA moisture sensitivity than other parameters. If the measurement error in data-acquisition process is removed, analyses based on RFNP and RFNC would also have high potential to evaluate the HMA moisture sensitivity. The vacuum pressure saturation used in AASHTO T 283 and proposed test has a risk to damage specimen before the load applying.

Determination of bacterial quantity by sonication in vacum-assisted closure (VAC) foams used for treatment of chronic wounds

Background: Negative pressure wound treatment is increasingly used through a Vacuum-Assisted Closure (VAC) device in complex wound situations. For this purpose, sterile polyurethane (PU) and polyvinyl alcohol (PVA) foam dressings are fitted to the wound size and covered with an adhesive drape to create an airtight seal. Little information exists about the type and quantity of microorganisms within the foams. Therefore, we investigated VAC foams after removal from the wound using a validated method (sonication) to detect the bacterial bioburden in the foam consisting as microbial biofilms.Methods: We prospectively included VAC foams (PU and PVA, KCI, Rümlamg, Switzerland) without antibacterial additions (e.g. silver), which were removed from wounds in patients with chronic ulcers from January 2007 through December 2008. Excluded were patients with acute wound infection, necrotizing fasciitis, underlying osteomyelitis or implant. Removed foams from regular changes of dressing were aseptically placed in a container with 100 ml sterile Ringer's solution. Within 4 hours after removal, foams were sonicated for 5 min at 40 kHz (as described in NEJM 2007;357:654). The resulting sonication fluid was cultured at 37°C on aerobic blood agar plates for 5 days. Microbes were quantified as No. of colony-forming units (CFU)/ml sonication fluid and identified to the species level.Results: A total of 68 foams (38 PU and 30 PVA) from 55 patients were included in the study (median age 71 years; range 33-88 years, 57% were man). Foams were removed from the following anatomic sites: sacrum (n=29), ischium (n=18), heel (n=13), calves (n=6) and ankle (n=2). The median duration of being in place was 3 days (range, 1-8 days). In all 68 foams, bacteria were found in large quantities (median 105 CFU/ml, range 102-7 CFU/ml sonication fluid. No differences were found between PU and PVA foams. One type of organisms was found in 11 (16%), two in 17 (24%) and 3 or more in 40 (60%) foams. Gram-negative rods (Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa) were isolated in 70%, followed by Staphylococcus aureus (20%), koagulase-negative staphylococci, streptococci (8%), and enterococci (2%).Conclusion: With sonication, a high density of bacteria present in VAC foams was demonstrated after a median of 3 days. Future studies are needed to investigate whether antimicrobial-impregnated foams can reduce the bacterial load in foams and potentially improve wound healing.

The effects of arterial flow on platelet activation, thrombus growth, and stabilization.

Injury of an arterial vessel wall acutely triggers a multifaceted process of thrombus formation, which is dictated by the high-shear flow conditions in the artery. In this overview, we describe how the classical concept of arterial thrombus formation and vascular occlusion, driven by platelet activation and fibrin formation, can be extended and fine-tuned. This has become possible because of recent insight into the mechanisms of: (i) platelet-vessel wall and platelet-platelet communication, (ii) autocrine platelet activation, and (iii) platelet-coagulation interactions, in relation to blood flow dynamics. We list over 40 studies with genetically modified mice showing a role of platelet and plasma proteins in the control of thrombus stability after vascular injury. These include multiple platelet adhesive receptors and other junctional molecules, components of the ADP receptor signalling cascade to integrin activation, proteins controlling platelet shape, and autocrine activation processes, as well as multiple plasma proteins binding to platelets and proteins of the intrinsic coagulation cascade. Regulatory roles herein of the endothelium and other blood cells are recapitulated as well. Patient studies support the contribution of platelet- and coagulation activation in the regulation of thrombus stability. Analysis of the factors determining flow-dependent thrombus stabilization and embolus formation in mice will help to understand the regulation of this process in human arterial disease.

Efficiency of recombinant human TNF in human cancer therapy.

Recombinant human tumour necrosis factor (TNF) has a selective effect on angiogenic vessels in tumours. Given that it induces vasoplegia, its clinical use has been limited to administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs. When combined with the alkylating agent melphalan, a single ILP produces a very high objective response rate. In melanoma, the complete response (CR) rate is around 80% and the overall objective response rate greater than 90%. In soft tissue sarcomas that are inextirpable, ILP is a neoadjuvant treatment resulting in limb salvage in 80% of the cases. The CR rate averages 20% and the objective response rate is around 80%. The mode of action of TNF-based ILP involves two distinct and successive effects on the tumour-associated vasculature: first, an increase in endothelium permeability leading to improved chemotherapy penetration within the tumour tissue, and second, a selective killing of angiogenic endothelial cells resulting in tumour vessel destruction. The mechanism whereby these events occur involves rapid (of the order of minutes) perturbation of cell-cell adhesive junctions and inhibition of alphavbeta3 integrin signalling in tumour-associated vessels, followed by massive death of endothelial cells and tumour vascular collapse 24 hours later. New, promising approaches for the systemic use of TNF in cancer therapy include TNF targeting by means of single chain antibodies or endothelial cell ligands, or combined administration with drugs perturbing integrin-dependent signalling and sensitizing angiogenic endothelial cells to TNF-induced death.

Engineered aprotinin for improved stability of fibrin biomaterials.

Fibrin has been long used clinically for hemostasis and sealing, yet extension of use in other applications has been limited due to its relatively rapid resorption in vivo, even with addition of aprotinin or other protease inhibitors. We report an engineered aprotinin variant that can be immobilized within fibrin and thus provide extended longevity. When recombinantly fused to a transglutaminase substrate domain from α(2)-plasmin inhibitor (α(2)PI(1-8)), the resulting variant, aprotinin-α(2)PI(1-8), was covalently crosslinked into fibrin matrices during normal thrombin/factor XIIIa-mediated polymerization. Challenge with physiological plasmin concentrations revealed that aprotinin-α(2)PI(1-8)-containing matrices retained 78% of their mass after 3 wk, whereas matrices containing wild type (WT) aprotinin degraded completely within 1 wk. Plasmin challenge of commercial sealants Omrixil and Tisseel, supplemented with aprotinin-α(2)PI(1-8) or WT aprotinin, showed extended longevity as well. When seeded with human dermal fibroblasts, aprotinin-α(2)PI(1-8)-supplemented matrices supported cell growth for at least 33% longer than those containing WT aprotinin. Subcutaneously implanted matrices containing aprotinin-α(2)PI(1-8) were detectable in mice for more than twice as long as those containing WT aprotinin. We conclude that our engineered recombinant aprotinin variant can confer extended longevity to fibrin matrices more effectively than WT aprotinin in vitro and in vivo.

Handling sticky resin by stingless bees (Hymenoptera, Apidae)

For their nest defense, stingless bees (Meliponini) collect plant resins which they stick on intruders like ants or cleptobiotic robber bees causing their immobilization. The aim of this article is to identify all parts of stingless bee workers contacting these sticky resins. Of special interest are those body parts with anti-adhesive properties to resin, where it can be removed without residues. For that, extensive behavioral observations during foraging flight, handling and application of the resin have been carried out. When handling the resin, all tarsi touch the resin while walking above it. For transportation from plants to the nest during foraging flight, the resin is packed to the corbicula via tarsi and basitarsi of front and middle legs. Once stuck to the resin or after the corbicula had been unloaded, the bee's legs have to be cleaned thoroughly. Only the tips of the mandibles, that form, cut and apply the sticky resin, seem to have at least temporarily resin-rejecting properties.

New insights into the molecular basis of desmoplakin- and desmin-related cardiomyopathies.

Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.

Sensitization of endothelial cells to TNFα-induced death

RESUME L'angiogénèse tumorale est un processus essentiel au développement des tumeurs. Les intégrines, molécules d'adhésions transmembranaires, sont d'importants effecteurs de l'angiogenèse. En permettant l'adhésion à la matrice extra-cellulaire, les intégrines transmettant des signaux de survie, de migration, et de prolifération. Le facteur de nécrose tumorale α (TNFα) est utilisé pour le traitement régional de cancers chez l'homme. II agit en détruisant sélectivement les vaisseaux angiogéniques. Cependant, son administration systémique chez l'homme est limitée par les réactions de vaso-dilatation sévères qu'il provoque. Le but de mon travail fut de rechercher des conditions permettant la sensibilisation des cellules endothéliales au TNFα et qui pourraient être applicables en clinique, ceci afin d'accroître l'efficacité de cette molécule. Nous avons testé la possibilité d'interférer avec les signaux de survie provenant des intégrines. Pour cela, des cellules endothéliales furent cultivées dans des conditions d'adhésion ou en suspension, ou alors exposées dans des conditions d'adhésion au zoledronate (biphosphonate contenant du nitrogène). Dans ces conditions, les effets du TNFα sur les cellules endothéliales furent étudiés, en particulier l'induction de la mort cellulaire. Dans ce travail, nous montrons que le zoledronate sensibilise les cellules endothéliales à la nécrose induite par TNFα. Cet effet s'accompagne de l'inhibition de la phosphorylation de FAK, PKB, et JNK, ainsi que de l'inhibition de la prénylation des protéines. En revanche, l'activation de NF-kB et p38 n'est pas perturbée. La restoration de la prénylation des protéines empêche la mort des HUVEC traitées par zoledronate et TNFα, et rétablit la phosphorylation de FAK, PKB, et JNK. Des essais d'angiogénèse in vivo montrent que le zoledronate inhibe l'angiogénèse induite par FGF-2. Le zoledronate encapsulé dans des liposomes permet de ralentir la croissance tumorale et synergise avec le TNFα en l'inhibant. L'inihibtion de la prénylation des protéines est un des mécanismes de sensibilisation du zoledronate au TNFα. In vivo, la synergie de leur association sur la croissance tumorale est efficace. Ces résultats encouragent la poursuite de l'étude des effets de ces deux drogues sur la croissance tumorale. SUMMARY The formation of tumor-associated vessels is essential for tumor progression. Cell adhesion molecules of the integrin family are important mediators of angiogenesis, by providing adhesive signals necessary for endothelial cell migration, proliferation and survival. Anti-angiogenic therapies are currently considered as highly promising in the treatment of human cancer. Tumor Necrosis Factor α (TNFα) is used for the regional treatment of human cancer, whose mechanisms of action involved selective disruption of angiogenic tumor vessels. Systemic administration of TNFα in humans, however, induces a severe inflammatory condition that prevents its use far the treatments of tumors localized outside of limbs. The aim of my work was to find strategies to sensitize angiogenic endothelial cells to TNFα-induced death, which could be potentially translated into clinical setting to improve the therapeutic efficacy of TNFα. We specifically tested the hypothesis whether interference with integrin-mediated adhesion and signaling may sensitize endothelial cells to TNFα-induced death. To test this hypothesis we cultured endothelial cells (EC) under conditions of cell-matrix or cell-cell adhesion or exposed matrix-adherent EC to the nitrogen-containing bisphosphonate zoledronate, and characterized the effect on TNFα-mediated signaling events and cell death. We show that zoledronate sensitizes HUVEC to TNFα-induced necrosis-like programmed cell death. This effect was associated with suppression of sustained phosphorylation of PKB and JNK and decreased protein prenylation, whereas TNFα-induced activation of NF-kB and p38 were not inhibited. Restoration of protein prenylation rescued HUVEC from zoledronate and TNFα-induced death, and restored FAK, PKB and JNK phosphorylation. By using in vivo angiogenesis assay we showed that zoledronate suppressed FGF-2-induced angiogenesis. Liposome-encapulated zoledronate partially inhibited tumor growth and synergized with TNFα to fully suppress tumor growth. Taken together, this work has identified protein prenylation as a mechanisms by which zoledronate sensitizes endothelial cells to TNFα-induced death in vitro and provides initial evidence that zoledronate synergizes with TNFα in vivo resulting in improved anti-tumor activity. These results warrant further study of the anti-tumor effects of zoledronate and TNFα and should be further studies in view of their clinical relevance.

Micropatterning of single endothelial cell shape reveals a tight coupling between nuclear volume in G1 and proliferation

Shape-dependent local differentials in cell proliferation are considered to be a major driving mechanism of structuring processes in vivo, such as embryogenesis, wound healing, and angiogenesis. However, the specific biophysical signaling by which changes in cell shape contribute to cell cycle regulation remains poorly understood. Here, we describe our study of the roles of nuclear volume and cytoskeletal mechanics in mediating shape control of proliferation in single endothelial cells. Micropatterned adhesive islands were used to independently control cell spreading and elongation. We show that, irrespective of elongation, nuclear volume and apparent chromatin decondensation of cells in G1 systematically increased with cell spreading and highly correlated with DNA synthesis (percent of cells in the S phase). In contrast, cell elongation dramatically affected the organization of the actin cytoskeleton, markedly reduced both cytoskeletal stiffness (measured dorsally with atomic force microscopy) and contractility (measured ventrally with traction microscopy), and increased mechanical anisotropy, without affecting either DNA synthesis or nuclear volume. Our results reveal that the nuclear volume in G1 is predictive of the proliferative status of single endothelial cells within a population, whereas cell stiffness and contractility are not. These findings show that the effects of cell mechanics in shape control of proliferation are far more complex than a linear or straightforward relationship. Our data are consistent with a mechanism by which spreading of cells in G1 partially enhances proliferation by inducing nuclear swelling and decreasing chromatin condensation, thereby rendering DNA more accessible to the replication machinery.

Thermal modeling and management in ultrathin chip stack technology

This paper presents a thermal modeling for power management of a new three-dimensional (3-D) thinned dies stacking process. Besides the high concentration of power dissipating sources, which is the direct consequence of the very interesting integration efficiency increase, this new ultra-compact packaging technology can suffer of the poor thermal conductivity (about 700 times smaller than silicon one) of the benzocyclobutene (BCB) used as both adhesive and planarization layers in each level of the stack. Thermal simulation was conducted using three-dimensional (3-D) FEM tool to analyze the specific behaviors in such stacked structure and to optimize the design rules. This study first describes the heat transfer limitation through the vertical path by examining particularly the case of the high dissipating sources under small area. First results of characterization in transient regime by means of dedicated test device mounted in single level structure are presented. For the design optimization, the thermal draining capabilities of a copper grid or full copper plate embedded in the intermediate layer of stacked structure are evaluated as a function of the technological parameters and the physical properties. It is shown an interest for the transverse heat extraction under the buffer devices dissipating most the power and generally localized in the peripheral zone, and for the temperature uniformization, by heat spreading mechanism, in the localized regions where the attachment of the thin die is altered. Finally, all conclusions of this analysis are used for the quantitative projections of the thermal performance of a first demonstrator based on a three-levels stacking structure for space application.