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This study was performed to assess the possibility to detect early osteoarthritic changes with ultrasound. A backscatter ultrasound microscope operating at 50 MHz (30 microns axial resolution) was used to image knee joints cartilage of rats in vitro. Acquisition of backscattered radio frequency signals was performed at a sampling rate of 400 MHz during the three dimensional (3D) scanning of the cartilage. Control knees and knees which have been injected with mono-iodo-acetate (MIA) were included in this study (MIA causes rapid cartilage degeneration). Patellas and tibial plateaux were obtained after sacrifice and dissection of the knee joints. As multiplanar parallel scans were performed to provide 3D information, the 3D cartilage surface (CS) could be reconstructed and displayed. The articular cartilage thickness could be measured accurately. It varies in different parts of the knee joint and ranges between 250 microns and 400 microns. We show images of cartilage lesions occurring at early stage of osteoarthritis (hypertrophic repair). Disruptions and irregularities of CS and cartilage bone interface caused by an advanced stage of the disease and corresponding to fibrillations and ulcerations are also visualized on both B-scans and 3D images. This study suggests that with further technologic improvements, intra-articular. Ultrasonography of cartilage may provide a valuable method for early defection of osteoarthritis

A single intra-articular (ia) injection of 2 mg zymosan on D0 led to the production of acute periarticular edema followed by subacute erosive synovitis. The development of the zymosan-induced arthritis was associated with an initial loss of running activity and with an initial decrease of proteoglycan synthesis. Febrile response was present only on D1. In addition, on D20 synovial pannus led to a marked depletion of the proteoglycan content in the articular cartilage. When injected ia, IL1 beta (1 microgram) provoked similar fever and similar changes in cartilage anabolism, but did not affect cartilage proteoglycan content (D20). These results suggest that zymosan-induced synovitis in the rat combines early prostaglandin-dependent processes (edema, pain, fever) with IL1-related effects on cartilage metabolism, thus allowing evaluation of chondroprotective drugs.

Elevated expression of phosphorylated mitogen activated protein kinase kinase (MEK) as a mechanism of resistance to the histone deacetylase inhibitor romidepsin in HUT 78 cutaneous T-cell lymphoma cells. Histone deacetylase inhibitors (HDIs) have shown promise in the treatment of T-cell lymphomas including cutaneous and peripheral T-cell lymphomas. However, resistance to romidepsin limits its activity in some patients. A detailed understanding of the mechanisms of resistance to HDIs may lead to strategies designed to increase clinical efficacy. To study mechanisms of resistance to the HDI romidepsin, the HUT78 cutaneous T-cell lymphoma cell line was exposed to increasing concentrations of romidepsin in the presence of the P-glycoprotein (P-gp) inhibitors verapamil or valspodar (PSC-833) to prevent the emergence of P-gp, a known resistance mechanism. The DpVp35 and DpVp50 sublines are maintained in 35 ng/ml and 50 ng/ml romidepsin, respectively, in the presence of 5 µg/ml verapamil while DpP75 is maintained in 75 ng/ml romidepsin and 3 µg/ml valspodar. In 4-day cytotoxicity assays, the sublines are approximately 55-fold resistant to romidepsin and are not cross resistant to the HDIs belinostat, panobinostat or vorinostat. Low but detectable levels of P-gp do not explain the resistance. We used a custom drug resistance gene expression array and found increased expression of insulin receptor (IR) in the resistant cells that was confirmed by immunoblot analysis. Elevated expression of phosphorylated mitogen activated protein kinase kinase (MEK), a downstream effector of the IR pathway, was also observed in the resistant cells compared to the parental cells. Interestingly, resistant cells were found to be exquisitely sensitive to MEK inhibition, as significant apoptosis was observed after 48 h in the presence of 5 nM of the MEK inhibitor PD0325901 and 10 nM of the MEK inhibitor AZD 6244 as measured by flow cytometry with annexin V and by immunoblot examining poly (ADP-ribose) polymerase (PARP) cleavage. No significant apoptosis was observed in parental cells at concentrations up to 500 nM. Resistant cells were not, however, sensitive to extracellular related kinase (ERK) inhibition or phosphatidylinositol 3-kinase (PI3K) inhibition in as determined by annexin V assay. In summary, we hypothesize that activated MEK can mediate resistance to romidepsin, but may also lead to collateral sensitivity to MEK inhibitors. The emerging role of activated MEK as a mechanism of resistance to romidepsin suggests combination of romidepsin with MEK inhibitors in future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2624. doi:10.1158/1538-7445.AM2011-2624

Abstract 2491 While much has been discovered in the laboratory regarding the mechanisms of multidrug resistance (MDR), based on phase III clinical trials of ATP-binding cassette (ABC) transporter modulators, these findings have not resulted in appreciable clinical benefit in acute myeloid leukemia (AML) patients. In this pilot study, we investigated the clinical relevance of 380 genes related to MDR in 31 samples from AML patients who had undergone conventional treatment, with 11 patients contributing samples at diagnosis and relapse, using TaqMan Low Density Arrays (TLDA). We and others have shown that the TaqMan-based qRT-PCR is superior to microarrays and SYBR-green-based qRT-PCR because of higher sensitivity and specificity in determining expression of highly homologous gene family members, such as the ABC transporters. 380 genes involved in metabolism, signal transduction, DNA repair, stress response, tumor suppressor activity, oncogenic transformation, apoptosis, and drug uptake or efflux were curated based on their potential role in MDR in the current literature. We found expression of four genes previously unreported in AML in both diagnostic and relapsed patient samples to correlate with duration of complete remission (CR) of induction chemotherapy: SLC7A8 and HSPE1 correlate negatively while GBP1 and ABCD2 correlate positively with CR duration (>2-fold change relative to the median, p2-fold change relative to the median, p2 fold change compared to diagnosis, p2 fold change compared to diagnosis, p Disclosures: No relevant conflicts of interest to declare.

Background: Cancer stem cells (CSCs) are presumed to possess the virtually unlimited ability to proliferate while preserving self-renewal. These cells are also highly resistant to chemotherapeutics, a feature which correlates with overexpression of ATP-binding cassette (ABC) transporters. No reports to our knowledge have shown that prolonged continuous selection of cells for drug resistance enriches cancer stem-like cells. Methods: We examined parental and drug-selected breast cancer cell lines to determine whether there is a relationship between drug resistance and the presence of cells with a CSC phenotype. MCF-7/ADR cells, generated from weakly tumorigenic parental MCF7 cells by continuous exposure to increasing concentrations of doxorubicin, were examined by microarray analysis, flow cytometry, invasion, in vivo tumorigenicity and other CSC characteristics. The putative CSC population was also examined for expression of multidrug resistance (MDR) genes using TaqMan Low Density Arrays. Results: Pathway analysis showed that MCF-7/ADR cells express ABCB1 as well as genes with patterns similar to breast CSCs. MCF-7/ADR cells grow in 3D cultures as mammospheres, are highly invasive and tumorigenic. In contrast to parental cells, over 30% of MCF-7/ADR cells are CD44+/CD24-. The CD44+/CD24- cells have the ability to self-renew and overexpress many MDR genes when compared to the CD44+/CD24+ population. No enrichment in CD44+/CD24- or CD133+ populations was detected in MCF-7/MDR or other cells with forced expression of the ABCB1 transporter or MDR genes. Conclusions: Cells with characteristics of CSCs increased in number following selection for doxorubicin resistance. Based on our findings, we speculate that prolonged drug treatment in vivo may result in expansion of a CSC-like population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-261.

We have been developing tools to reproducibly correlate the expression of multidrug-resistance (MDR)-associated genes with response to chemotherapy in primary cancers. This study reports the results of a novel MDR gene expression analysis of primary serous carcinoma of the ovary utilizing a TaqMan Low Density Array (TLDA) chip which includes 380 previously characterized multidrug resistance (MDR)-associated genes that were initially identified in cultured cancer cells. Primary tumor samples from 133 patients from 4 sites were studied. All patients were subsequently treated with standard chemotherapy and had known clinical outcome. A 13 gene signature was identified whose expression added statistical power to the risk status of the patients based on standard clinical parameters (age, CA125, and success of surgical debulking) (log-rank statistic p=0.02). When subsets of patients with defined clinical risk were studied, we found that a subset of clinically high risk patients that had low expression of the 13 gene signature had a better outcome than would be predicted by purely clinical criteria. Similarly, clinically low risk patients with high expression of the 13 gene signature had a poorer prognosis. Since the mechanism of action of the 13 MDR genes involved in the signature are well-understood, it might be possible to devise therapeutic strategies to some of these targets with the goal of improving clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-51.

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International audience; The effects of human recombinant interleukin-1 beta (HrIL-1 beta) were investigated in arthritic rats sensitized with type II collagen (CII) and muramyl dipeptide (MDP). When administered subcutaneously (sc) daily during established arthritis, low (0.02 micrograms) and medium (0.2 micrograms) HrIL-1 beta doses exerted paradoxical beneficial properties on paws with moderate and severe inflammation, respectively. In contrast, the highest dose (2 micrograms) had a pejorative effect on developing arthritis. In addition, HrIL-1 beta attenuated paw volume and deterioration of the joints as assessed radiologically. Hence, paw inflammation response to IL-1 exposure depended on the dosage and the severity of previous arthritis prior to the IL-1 challenge. Some of these paradoxical activities may be due to the capacity of IL-1 to induce its own inhibitors or feedback loops thus counterbalancing its phlogistic properties.

Sodium fluoride (NaF) is used in the treatment of axial osteoporosis and so is mostly given to old patients. Since its pharmacokinetics has not been studied in the elderly, the pharmacokinetics of an enteric-coated tablet containing 50 mg NaF has been investigated in 15 aged inpatients (aged 65 to 75 y) and 12 young healthy volunteers (aged 21 to 26 y). The serum AUC of fluoride was 1.7-time higher in older than in younger subjects. There was a strong inverse correlation between the AUC and either body surface area (BSA) or glomerular filtration rate (GFR), both of which were very much lower in the elderly. This concluded that if efficacy or safety are related to the bioavailability of fluoride, it may be valuable to adjust the dosage of fluoride accordingly to the GFR and BSA.

Les operations exterieures soumettent le corps du soldat a rude epreuve. Le danger, des tentations nouvelles, parfois l’inaction placent alors le maintien en condition physique du soldat au rang des principales preoccupations du chef. Celui-ci doit en effet fixer des regles claires, realistes et partagees en depit du relativisme moral ambiant. L’entrainement physique et operationnel consiste essentiellement a adapter les modalites pratiques aux caracteristiques de la mission. En revanche, lorsque la tentation et les risques de derives (femmes, alcool, drogue…) entrent en ligne de compte, le chef doit jouer a la fois sur la corde de la contrainte et sur celle de l’intelligence et du cœur. Le comportement des soldats a une influence directe sur la reussite de la mission. Ils l’acceptent d’autant mieux que les regles de vie correspondent a des choix moraux objectifs, exigeants et que les conditions de vie allient respect de l’intimite et un bon sens commun qui passe par la volonte d’ameliorer des le plus bas niveau les conditions de vie de l’unite.

We studied a French family in which 18 adult members had destructive arthropathy and enthesopathic changes. In 16 patients, the diagnosis was verified radiologically, and in the 2 other patients, the clinical history was consistent with familial arthropathy. The disease predominantly affects the wrists, fingers, shoulders, and peripheral entheses, and its onset occurs after age 18. Genetic transmission is autosomal dominant, with 100% penetrance, and is not related to the HLA system. The clinical and radiologic features are strikingly similar in all patients in successive generations and different branches of the genealogic tree, and this suggests monogenic transmission.

International audience; We investigated the influence of human recombinant interleukin-1 beta (hrIL-1 beta) on the time-course of collagen induced arthritis (CIA) when injected concomitantly with the arthritogenic emulsion. Three sensitizing procedures were compared. The control group received type II collagen only. The other groups differed by the adjunction of demonstrated (MDP) or potential (IL-1 beta) adjuvant. No adjuvant effect of IL-1 was observed as judged on clinical or radiological scores. On the contrary, MDP significantly worsened the lesions of the injected right hindpaw, and increased the incidence of CIA. Surprisingly, humoral response to type II collagen was decreased in the group receiving IL-1 beta. This might be explained by a non specific increase of antigen clearance.

A case is presented of a 55-year-old man with a MFH in a previous BI. The poor prognosis of such secondary MFH is related in part to the difficulty of its early diagnosis, its aggressive behavior, and its tendency to metastasize (mainly to bone sites and lungs). Nevertheless, although biopsy of asymptomatic bone marrow infarction is not indicated, MRI seems consistent in detecting sarcomatous transformation of symptomatic BIs and plays an important role in the patient's preoperative evaluation.

A micro-stratified 3D scaffold was designed by successive stacking of alginate gel layers (AGLs) and poly(l-lysine)-hyaluronic acid (PLL-HA) multilayer films. AGLs are obtained by complexation of alginate by Ca2+ ions. Alginate solutions are first sprayed onto a solid substrate inclined such that the excess of solution be removed by natural drainage. A CaCl2 solution is then either sprayed onto the substrate or the alginate covered substrate is dipped into a CaCl2 solution. The spraying of the CaCl2 solution leads to micro-porous AGLs, whereas the dipping in a CaCl2 aqueous solution leads to a more homogeneous gel layer without porosity. The second process also allows the formation of AGLs with a controlled thickness. With the goal of stacking different AGLs and PLL-HA films, the influence of a PLL-HA precursor film on the formation of AGLs is firstly investigated. It is found that when an alginate solution is sprayed on a PLL-HA multilayer built in the presence of CaCl2, the multilayer plays the role of reservoir of Ca2+ ions and of PLL chains, which both diffuse out of the multilayer film and complex alginate chains. This leads to the formation of a "pre-alginate gel". When this film is further dipped in the CaCl2 solution, an additional AGL forms, which is, however, free of PLL chains. Finally after the build-up of a PLL-HA film on the top of AGL, we succeeded in designing micro-stratified 3D scaffolds constituted by alternating strata of AGLs and PLL-HA films. This micro-stratified gel provides a new scaffold design with a perfectly controlled build-up: AGL aims to be a 3D scaffold for cell culture, and the PLL-HA multilayers should act as reservoirs for biologically active molecules.

Introduction L’IRM champs eleves 3 dimensions (3D) a haute resolution (HR) permettant d’optimiser la qualite des images est un outil de recherche interessant pour etudier le cartilage articulaire des petites articulations de facon non-invasive. A partir des donnees 3D de l’IRM, nous avons realise des mesures quantitatives de volumes et d’epaisseurs du cartilage qui furent confrontees aux donnees histologiques classiques. Materiel et Methodes 48 rats Wistar mâles ont subi une section du ligament croise anterieur du genou droit selon un protocole standardise, le genou gauche servant de temoin. Les genoux etaient explores par IRM in vivo a 7T (sequence 3D echo de gradient, suppression du signal de la graisse, antenne specifique a deux elements en reseau) a J7, J14, J28, J42 et J56. Apres l’acquisition, les animaux etaient sacrifies pour l’etude histologique. Des coupes histologiques sagittales colorees par l’hematoxyline-eosine-safran etaient realisees, correspondant aux zones articulaires portantes selectionnees en IRM. Pour les deux techniques d’imagerie, les cartilages femoraux et tibiaux etaient segmentes manuellement. Les masques binaires resultants permettaient de determiner les volumes et epaisseurs (confrontations IRM-histologie) du cartilage articulaire. Resultats Concernant l’epaisseur du cartilage, il existait une correlation significative p = 0,02 entre l’IRM et l’histologie, au niveau femoral et tibial. Les valeurs des scores histologiques (membrane synoviale et cartilage) sont significativement differentes entre les genoux operes et temoins. Le suivi de la progression arthrosique en IRM montre d’importantes lesions degeneratives avec pertes cartilagineuses predominant au niveau du compartiment femoral, le compartiment tibial etant plutot le siege de phenomenes inflammatoires et oedemateux. Une correlation significative existait entre les mesures d’epaisseur relevees en IRM et en histologie (femur : r = 0,45 ; p = 0,02 ; tibia : r = 0,47 ; p = 0,01). Conclusion L’IRM 3D HR a 7T permet l’analyse in vivo de la progression arthrosique chez le rat. Les valeurs quantitatives obtenues en IRM sont compatibles avec les variations d’epaisseurs relevees en histologie. Une correlation significative est etablie entre ces deux techniques. L’IRM 3D HR a 7T est un outil non-invasif prometteur pour l’evaluation et le suivi in vivo de reponses therapeutiques chez le rongeur arthrosique.

A major issue in the treatment of acute myeloid leukemia (AML) is the development of resistance to chemotherapeutic drugs. While more than 80% of children with AML may initially achieve complete remission with current therapeutic regimens, a large number of these patients relapse with resistant disease; even with aggressive therapy, the event free survival rate is only about 50%. Several mechanisms of drug resistance have been identified. One of these is the overexpression of ATP-binding-cassette (ABC)-transporters that function as drug efflux pumps. The best characterized member of this family is the P-glycoprotein (P-gp or MDR1 or ABCB1), which is an important cause of drug resistance in adult AML but not in children with AML. We could recently show that the multidrug resistance-associated protein 3 (MRP3 or ABCC3) and the breast cancer resistance protein (BCRP or ABCG2) are associated with a poor response to therapy in childhood AML. The aim of the present study was to identify other members of this family of proteins which are involved in drug resistance. We were particularly interested in ABC-transporters which were overexpressed in AML samples compared to healthy bone marrow because this might be an important prerequisite for using such proteins as therapeutic targets. A new microarray (DualChip human ABC) was developed for the simultaneous detection and quantification of 38 ABC transporter genes. Samples from 30 children with AML were analyzed with this microarray and the results were compared with normal bone marrow. The expression of five genes (ABCA2, ABCA3, ABCB2, ABCC1, and ABCC10) which were overexpressed in most patient samples was then analyzed by real time PCR in 40 selected samples from children with previously untreated AML. Half of the patients had achieved complete remission after the first course of chemotherapy. The other 20 patients had also undergone the full induction therapy but had failed to achieve remission at this stage. Only the expression of ABCA3 was significantly higher (p=0.015) in patients with a poor response to therapy. There was a 4-fold difference between the median expressions in both groups. The expression of ABCA3 was not significantly associated with the expression of P-gp, MRP3 or BCRP. Our results show that ABCA3 is overexpressed in childhood AML compared to healthy bone marrow and that the expression of ABCA3 is associated with a poor response to therapy. Interestingly, there are two ways in which ABCA3 might influence the prognosis of AML. Like other ABC-transporters, it could be involved in drug efflux. On the other hand, it was recently shown that ABCA3 plays a role in apoptosis and that many cancer cell lines show amplifications of the ABCA3 gene. Further studies are warranted to analyze the clinical and biological relevance of ABCA3 in leukemia.

A major problem in the treatment of tumors represents the development of resistance to chemotherapy. Many mechanisms are responsible for the failure of treatment, the main one being the activation of the ABC transporters. In the present investigation, we developed a low density DNA microarray which contains 38 ABC transporter genes. This tool has been validated with three different characterized multidrug-resistant sublines (CEM/ADR5000, HL60/AR, MCF7-CH1000) and their corresponding drug-sensitive parental cell lines (CCRF-CEM, HL60, MCF7). The multidrug-resistant cell lines used are known to overexpress either the MDR1, MRP1, or BCRP genes. Interestingly, we found not only the overexpression of these genes but also of other ABC transporter genes by using low density microarrays. These results were corroborated by quantitative real time RT-PCR. The microarray allowed the determination of the expression profile of ABC transporters in a single hybridization experiment and may, hence harbor the potential as diagnostic tool to detect drug resistance in the clinic.

An isocratic high-performance liquid chromatographic procedure is presented for the screening of plasma samples for the presence of sixteen non-steroidal anti-inflammatory drugs. Detection was achieved simultaneously at two wavelengths (254 and 370 nm) and the purity of the eluted peaks was tested using absorbance ratios at the two wavelengths; identification could thus be effective without interferences from substances of other pharmacological classes. The drugs were extracted simultaneously with diethyl ether after acidification and separated from each other on an octadecyl reversed-phase column using only one eluent, acetonitrile-0.3% acetic acid-tetrahydrofuran (36:63.1:0.9, v/v). The recovery, precision and reproducibility of the method were satisfactory as it allowed the determination of the drugs from infra- to supratherapeutic concentrations.

An analysis of the dermis and a study of collagen and lipid biosynthesis by fibroblast cultures were carried out in one case of epitheliogenesis imperfecta in a new born calf. This was found to be not only an epidermal disease with fissures and blisters within basal cells on the basement membrane but also a metabolic disease affecting the dermal fibroblasts. These fibroblasts showed a significant decrease in collagen biosynthesis with an increase in the percentage of type III collagen and also a decrease in the biosynthesis of lipids, especially glycerides and cholesterol.

Objective The present study aims at evaluating, in a rat model of cartilage defect, the potential of various polymers as filling and repair biomaterials. The macroscopic and histological observations are compared to biochemical parameters in order to appreciate the pertinence of the latter as suitable criteria in tissue engineering. Methods A hydrogel, consisting of hyaluronic acid (HA), covalently substituted by hydrophobic alkyl chains (HA12, HA18) and an alginate sponge, alone (Asp) or combined with HA (AHAsp) or combined with HA and chondrocytes (HYBsp) were evaluated. Cartilage lesions were drilled in femoral trochlea of rats. The analyses were performed on trochlea as well as on patella and condyles. Results Repairs achieved with hydrogels had a similar macroscopic appearance than those afforded by AHAsp and HYBsp. Best macroscopic and histological scores were obtained with HA18 and HYBsp in comparison with alginate group (P< 0.01 and P< 0.02 respectively). Biochemical evaluations confirmed the presence of similar amounts of proteoglycans in the repaired zones and in the controls, though with different ΔdiC4S/ΔdiC6S ratios and enhanced HA levels. Conclusions Hydrogels or sponges proved to be colonized by cells synthesizing a matrix with a high HA content. The matrix obtained eventually turns hyaline and takes over the scaffold. The addition of HA and/or chondrocytes to Asp significantly improves the macroscopic and histological scores (P< 0.05 and P< 0.02 respectively). However, biochemical parameters are significantly different of those evaluated in native cartilage. The present study shows that only biochemical parameters allow to discriminate between various biomaterials in tissue engineering and are essential informations which should be taken into account in addition to macroscopic and histological observations. Copyright 2003 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.

Objective The objective was to assess the relationship between maturation-related structural changes of articular cartilage and variations of acoustic parameters estimated using high frequency ultrasonography. Design Patellae taken from 48 immature Wistar male rats and divided into six age groups (from five to 11 weeks old) were explored in vitro using 50-MHz scanning acoustic microscopy, then assessed by histology for the analysis of the cartilage cell distribution and fibrillar collagen organization. The variation of cartilage proteoglycan and collagen content with age was evaluated. Thickness measurements performed on both B-scan images and histologic sections were compared. Ultrasonic radiofrequency signals reflected by the cartilage surface and backscattered from its internal matrix were processed to estimate the integrated reflection coefficient (IRC) and apparent integrated backscatter (AIB). Results One-way ANOVA indicated that acoustic parameters and thickness change significantly ( P 0.05) as the animal matures because of age-related changes in cartilage composition and morphology. A moderate correlation was found between IRC and the animal age. The parameter decreased slightly but significantly over time. However, a good correlation was observed between the rat age and the AIB, which decreased significantly over time. The parameter variation was mostly related to the changes in collagen fiber orientation, and/or to a change in cell size, density and organization. Conclusions Current results indicate that acoustic properties of cartilage are affected by maturation-related cartilage changes. This suggests that high frequency ultrasonography may serve as a useful means for the investigation of cartilage matrix structural changes occurring under various clinical circumstances, like those observed during osteoarthritis, and for the evaluation of the efficacy of specific therapeutics.

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International audience