Publication and non-publication of clinical trials: longitudinal study of applications submitted to a research ethics committee

BACKGROUND: Not all clinical trials are published, which may distort the evidence that is available in the literature. We studied the publication rate of a cohort of clinical trials and identified factors associated with publication and nonpublication of results. METHODS: We analysed the protocols of randomized clinical trials of drug interventions submitted to the research ethics committee of University Hospital (Inselspital) Bern, Switzerland from 1988 to 1998. We identified full articles published up to 2006 by searching the Cochrane CENTRAL database (issue 02/2006) and by contacting investigators. We analyzed factors associated with the publication of trials using descriptive statistics and logistic regression models. RESULTS: 451 study protocols and 375 corresponding articles were analyzed. 233 protocols resulted in at least one publication, a publication rate of 52%. A total of 366 (81%) trials were commercially funded, 47 (10%) had non-commercial funding. 346 trials (77%) were multi-centre studies and 272 of these (79%) were international collaborations. In the adjusted logistic regression model non-commercial funding (Odds Ratio [OR] 2.42, 95% CI 1.14-5.17), multi-centre status (OR 2.09, 95% CI 1.03-4.24), international collaboration (OR 1.87, 95% CI 0.99-3.55) and a sample size above the median of 236 participants (OR 2.04, 95% CI 1.23-3.39) were associated with full publication. CONCLUSIONS: In this cohort of applications to an ethics committee in Switzerland, only about half of clinical drug trials were published. Large multi-centre trials with non-commercial funding were more likely to be published than other trials, but most trials were funded by industry.

Nonviolent factors: Variables associated with ethnic violence, and factors which are associated with nonviolent response to grievances toward members of other ethnic groups

Ethnic violence appears to be the major source of violence in the world. Ethnic hostilities are potentially all-pervasive because most countries in the world are multi-ethnic. Public health's focus on violence documents its increasing role in this issue.^ The present study is based on a secondary analysis of a dataset of responses by 272 individuals from four ethnic groups (Anglo, African, Mexican, and Vietnamese Americans) who answered questions regarding variables related to ethnic violence from a general questionnaire which was distributed to ethnically diverse purposive, nonprobability, self-selected groups of individuals in Houston, Texas, in 1993.^ One goal was psychometric: learning about issues in analysis of datasets with modest numbers, comparison of two approaches to dealing with missing observations not missing at random (conducting analysis on two datasets), transformation analysis of continuous variables for logistic regression, and logistic regression diagnostics.^ Regarding the psychometric goal, it was concluded that measurement model analysis was not possible with a relatively small dataset with nonnormal variables, such as Likert-scaled variables; therefore, exploratory factor analysis was used. The two approaches to dealing with missing values resulted in comparable findings. Transformation analysis suggested that the continuous variables were in the correct scale, and diagnostics that the model fit was adequate.^ The substantive portion of the analysis included the testing of four hypotheses. Hypothesis One proposed that attitudes/efficacy regarding alternative approaches to resolving grievances from the general questionnaire represented underlying factors: nonpunitive social norms and strategies for addressing grievances--using the political system, organizing protests, using the system to punish offenders, and personal mediation. Evidence was found to support all but one factor, nonpunitive social norms.^ Hypothesis Two proposed that the factor variables and the other independent variables--jail, grievance, male, young, and membership in a particular ethnic group--were associated with (non)violence. Jail, grievance, and not using the political system to address grievances were associated with a greater likelihood of intergroup violence.^ No evidence was found to support Hypotheses Three and Four, which proposed that grievance and ethnic group membership would interact with other variables (i.e., age, gender, etc.) to produce variant levels of subgroup (non)violence.^ The generalizability of the results of this study are constrained by the purposive self-selected nature of the sample and small sample size (n = 272).^ Suggestions for future research include incorporating other possible variables or factors predictive of intergroup violence in models of the kind tested here, and the development and evaluation of interventions that promote electoral and nonelectoral political participation as means of reducing interethnic conflict. ^

Confirmatory factor analysis and psychometric properties of the Anxiety Sensitivity Index-Revised in clinical and normative populations

The present investigation aimed to critically examine the factor structure and psychometric properties of the Anxiety Sensitivity Index - Revised (ASI-R). Confirmatory factor analysis using a clinical sample of adults (N = 248) revealed that the ASI-R could be improved substantially through the removal of 15 problematic items in order to account for the most robust dimensions of anxiety sensitivity. This modified scale was renamed the 21-item Anxiety Sensitivity Index (21-item ASI) and reanalyzed with a large sample of normative adults (N = 435), revealing configural and metric invariance across groups. Further comparisons with other alternative models, using multi-sample analysis, indicated the 21-item ASI to be the best fitting model for both groups. There was also evidence of internal consistency, test-retest reliability, and construct validity for both samples suggesting that the 21-item ASI is a useful assessment device for investigating the construct of anxiety sensitivity in both clinical and normative populations.

Development and evaluation of an ion induced x-ray emission system for the analysis of light and medium weight elements

The present work describes the development of a proton induced X-ray emission (PIXE) analysis system, especially designed and builtfor routine quantitative multi-elemental analysis of a large number of samples. The historical and general developments of the analytical technique and the physical processes involved are discussed. The philosophy, design, constructional details and evaluation of a versatile vacuum chamber, an automatic multi-sample changer, an on-demand beam pulsing system and ion beam current monitoring facility are described.The system calibration using thin standard foils of Si, P, S,Cl, K, Ca, Ti, V, Fe, Cu, Ga, Ge, Rb, Y and Mo was undertaken at proton beam energies of 1 to 3 MeV in steps of 0.5 MeV energy and compared with theoretical calculations. An independent calibration check using bovine liver Standard Reference Material was performed.  The minimum detectable limits have been experimentally determined at detector positions of 90° and 135° with respect to the incident beam for the above range of proton energies as a function of atomic number Z. The system has detection limits of typically 10- 7 to 10- 9 g for elements 14and calculations of areal density of thin foils using Rutherford backscattering data.  Amniotic fluid samples supplied by South Sefton Health Authority were successfully analysed for their low base line elemental concentrations. In conclusion the findings of this work are discussed with suggestions for further work .

Pryolytic and kinetic study of Chlorella Vulgaris under isothermal and non-isothermal conditions

Algae are a new potential biomass for energy production but there is limited information on their pyrolysis and kinetics. The main aim of this thesis is to investigate the pyrolytic behaviour and kinetics of Chlorella vulgaris, a green microalga. Under pyrolysis conditions, these microalgae show their comparable capabilities to terrestrial biomass for energy and chemicals production. Also, the evidence from a preliminary pyrolysis by the intermediate pilot-scale reactor supports the applicability of these microalgae in the existing pyrolysis reactor. Thermal decomposition of Chlorella vulgaris occurs in a wide range of temperature (200-550°C) with multi-step reactions. To evaluate the kinetic parameters of their pyrolysis process, two approaches which are isothermal and non-isothermal experiments are applied in this work. New developed Pyrolysis-Mass Spectrometry (Py-MS) technique has the potential for isothermal measurements with a short run time and small sample size requirement. The equipment and procedure are assessed by the kinetic evaluation of thermal decomposition of polyethylene and lignocellulosic derived materials (cellulose, hemicellulose, and lignin). In the case of non-isothermal experiment, Thermogravimetry- Mass Spectrometry (TG-MS) technique is used in this work. Evolved gas analysis provides the information on the evolution of volatiles and these data lead to a multi-component model. Triplet kinetic values (apparent activation energy, pre-exponential factor, and apparent reaction order) from isothermal experiment are 57 (kJ/mol), 5.32 (logA, min-1), 1.21-1.45; 9 (kJ/mol), 1.75 (logA, min-1), 1.45 and 40 (kJ/mol), 3.88 (logA, min-1), 1.45- 1.15 for low, middle and high temperature region, respectively. The kinetic parameters from non-isothermal experiment are varied depending on the different fractions in algal biomass when the range of apparent activation energies are 73-207 (kJ/mol); pre-exponential factor are 5-16 (logA, min-1); and apparent reaction orders are 1.32–2.00. The kinetic procedures reported in this thesis are able to be applied to other kinds of biomass and algae for future works.

Real-time biosensor for the assessment of nanotoxicity and cancer electrotherapy

Knowledge of cell electronics has led to their integration to medicine either by physically interfacing electronic devices with biological systems or by using electronics for both detection and characterization of biological materials. In this dissertation, an electrical impedance sensor (EIS) was used to measure the electrode surface impedance changes from cell samples of human and environmental toxicity of nanoscale materials in 2D and 3D cell culture models. The impedimetric response of human lung fibroblasts and rainbow trout gill epithelial cells when exposed to various nanomaterials was tested to determine their kinetic effects towards the cells and to demonstrate the biosensor's ability to monitor nanotoxicity in real-time. Further, the EIS allowed rapid, real-time and multi-sample analysis creating a versatile, noninvasive tool that is able to provide quantitative information with respect to alteration in cellular function. We then extended the application of the unique capabilities of the EIS to do real-time analysis of cancer cell response to externally applied alternating electric fields at different intermediate frequencies and low-intensity. Decreases in the growth profiles of the ovarian and breast cancer cells were observed with the application of 200 and 100 kHz, respectively, indicating specific inhibitory effects on dividing cells in culture in contrast to the non-cancerous HUVECs and mammary epithelial cells. We then sought to enhance the effects of the electric field by altering the cancer cell's electronegative membrane properties with HER2 antibody functionalized nanoparticles. An Annexin V/EthD-III assay and zeta potential were performed to determine the cell death mechanism indicating apoptosis and a decrease in zeta potential with the incorporation of the nanoparticles. With more negatively charged HER2-AuNPs attached to the cancer cell membrane, the decrease in membrane potential would thus leave the cells more vulnerable to the detrimental effects of the applied electric field due to the decrease in surface charge. Therefore, by altering the cell membrane potential, one could possibly control the fate of the cell. This whole cell-based biosensor will enhance our understanding of the responsiveness of cancer cells to electric field therapy and demonstrate potential therapeutic opportunities for electric field therapy in the treatment of cancer.

Real-time Biosensor for the Assessment of Nanotoxicity and Cancer Electrotherapy

Knowledge of cell electronics has led to their integration to medicine either by physically interfacing electronic devices with biological systems or by using electronics for both detection and characterization of biological materials. In this dissertation, an electrical impedance sensor (EIS) was used to measure the electrode surface impedance changes from cell samples of human and environmental toxicity of nanoscale materials in 2D and 3D cell culture models. The impedimetric response of human lung fibroblasts and rainbow trout gill epithelial cells when exposed to various nanomaterials was tested to determine their kinetic effects towards the cells and to demonstrate the biosensor’s ability to monitor nanotoxicity in real-time. Further, the EIS allowed rapid, real-time and multi-sample analysis creating a versatile, noninvasive tool that is able to provide quantitative information with respect to alteration in cellular function. We then extended the application of the unique capabilities of the EIS to do real-time analysis of cancer cell response to externally applied alternating electric fields at different intermediate frequencies and low-intensity. Decreases in the growth profiles of the ovarian and breast cancer cells were observed with the application of 200 and 100 kHz, respectively, indicating specific inhibitory effects on dividing cells in culture in contrast to the non-cancerous HUVECs and mammary epithelial cells. We then sought to enhance the effects of the electric field by altering the cancer cell’s electronegative membrane properties with HER2 antibody functionalized nanoparticles. An Annexin V/EthD-III assay and zeta potential were performed to determine the cell death mechanism indicating apoptosis and a decrease in zeta potential with the incorporation of the nanoparticles. With more negatively charged HER2-AuNPs attached to the cancer cell membrane, the decrease in membrane potential would thus leave the cells more vulnerable to the detrimental effects of the applied electric field due to the decrease in surface charge. Therefore, by altering the cell membrane potential, one could possibly control the fate of the cell. This whole cell-based biosensor will enhance our understanding of the responsiveness of cancer cells to electric field therapy and demonstrate potential therapeutic opportunities for electric field therapy in the treatment of cancer.

Advanced cell culture platforms: methods for drug testing with microfluidics and microstructured devices

Advanced cell cultures are developing rapidly in biomedical research. Nowadays, various approaches and technologies are being used, however, these culturing systems present limitations from increasing complexity, requiring high costs, and not easily customization. We present two versatile and cost-effective methods for developing culturing systems that integrate 3D cell culture and microfluidic platforms. Firstly, for drug screening applications, many high-quality cell spheres of homogeneous size and shape are required. Conventional approaches usually have a dearth of control over the size and geometry of cell spheres and require sample collection and manipulation. To overcome this difficulty, in this study, hundreds of spheroids of several cell lines were generated using multi-well plates that housed our microdevices. Tumor spheroids grow at a uniform rate (in scaffolded or scaffold-free environments) and can be harvested at will. Microscopy imaging are done in real time during or after the culture. After in situ immunostaining, fluorescence imaging can be conducted while keeping the spatial distribution of spheroids in the microwells. Drug effects were successfully observed through viability, growth, and morphologic investigations. Also, we fabricated a microfluidic device suitable for directed and selective cell culture treatments. The microfluidic device was used to reproduce and confirm in vitro investigations carried out using normal culture methods, using a microglia cell line. The device layout and the syringe pump system, entirely designed in our lab, successfully allowed culture growth and medium flow regulation. Solution flows can be finely controlled, allowing treatments and immunofluorescence in one single chamber selectively. To conclude, we propose the development of two culturing platforms (microstructured well devices and in-flow microfluidic chip), which are the result of separate scientific investigations but have the primary goal of performing treatments in a reproducible manner. Our devices shall improve future studies on drug exposure testing, representing adjustable and versatile cell culture systems.

Mapping the quality of life experience in Alfama: a case study in Lisbon, Portugal.

Dissertation submitted in partial fulfilment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Observation and measurement of Higgs boson decays to WW∗ with the ATLAS detector

We report the observation of Higgs boson decays to WW∗ based on an excess over background of 6.1 standard deviations in the dilepton final state, where the Standard Model expectation is 5.8 standard deviations. Evidence for the vector-boson fusion (VBF) production process is obtained with a significance of 3.2 standard deviations. The results are obtained from a data sample corresponding to an integrated luminosity of 25 pb−1 from s√=7 and 8 TeV pp collisions recorded by the ATLAS detector at the LHC. For a Higgs boson mass of 125.36 GeV, the ratio of the measured value to the expected value of the total production cross section times branching fraction is 1.09+0.16−0.15 (stat.)+0.17−0.14 (syst.). The corresponding ratios for the gluon fusion and vector-boson fusion production mechanisms are 1.02±0.19 (stat.)+0.22−0.18 (syst.) and 1.27+0.44−0.40 (stat.)+0.30−0.21 (syst.), respectively. At s√=8 TeV, the total production cross sections are measured to be σ(gg→ H→WW∗)=4.6±0.9(stat.)+0.8−0.7(syst.)pb and σ(VBF H→WW∗)=0.51+0.17−0.15(stat.)+0.13−0.08(syst.)pb. The fiducial cross section is determined for the gluon-fusion process in exclusive final states with zero or one associated jet.

Study of (W/Z)H production and Higgs boson couplings using H -> WW* decays with the ATLAS detector

A search for Higgs boson production in association with a W or Z boson, in the H→ W W ∗ decay channel, is performed with a data sample collected with the ATLAS detector at the LHC in proton-proton collisions at centre-of-mass energies s√=7 TeV and 8 TeV, corresponding to integrated luminosities of 4.5 fb−1 and 20.3 fb−1, respectively. The WH production mode is studied in two-lepton and three-lepton final states, while two- lepton and four-lepton final states are used to search for the ZH production mode. The observed significance, for the combined W H and ZH production, is 2.5 standard deviations while a significance of 0.9 standard deviations is expected in the Standard Model Higgs boson hypothesis. The ratio of the combined W H and ZH signal yield to the Standard Model expectation, μ V H , is found to be μ V H  = 3.0 − 1.1 + 1.3 (stat.) − 0.7 + 1.0 (sys.) for the Higgs boson mass of 125.36 GeV. The W H and ZH production modes are also combined with the gluon fusion and vector boson fusion production modes studied in the H → W W ∗ → ℓνℓν decay channel, resulting in an overall observed significance of 6.5 standard deviations and μ ggF + VBF + VH = 1. 16 − 0.15 + 0.16 (stat.) − 0.15 + 0.18 (sys.). The results are interpreted in terms of scaling factors of the Higgs boson couplings to vector bosons (κ V ) and fermions (κ F ); the combined results are: |κ V | = 1.06 − 0.10 + 0.10 , |κ F | = 0. 85 − 0.20 + 0.26 .

HARNESSING iNKT CELLS WITH RECOMBINANT CD1d FUSION PROTEINS TO REDIRECT INNATE AND ADAPTIVE IMMUNITY TO THE TUMOR

Les thérapies du cancer, comme la radiothérapie et la chimiothérapie, sont couramment utilisées mais ont de nombreux effets secondaires. Ces thérapies invasives pour le patient nécessitent d'être améliorées et de nombreuses avancées ont été faites afin d'adapter et de personnaliser le traitement du cancer. L'immunothérapie a pour but de renforcer le système immunitaire du patient et de le rediriger de manière spécifique contre la tumeur. Dans notre projet, nous activons les lymphocytes Invariant Natural Killer T (iNKT) afin de mettre en place une immunothérapie innovatrice contre le cancer. Les cellules iNKT sont une unique sous-population de lymphocytes T qui ont la particularité de réunir les propriétés de l'immunité innée ainsi qu'adaptative. En effet, les cellules iNKT expriment à leur surface des molécules présentes aussi sur les cellules tueuses NK, caractéristique de l'immunité innée, ainsi qu'un récepteur de cellules T (TCR) qui représente l'immunité adaptative. Les cellules iNKT reconnaissent avec leur TCR des antigènes présentés par la molécule CD1d. Les antigènes sont des protéines, des polysaccharides ou des lipides reconnus par les cellules du système immunitaire ou les anticorps pour engendrer une réponse immunitaire. Dans le cas des cellules iNKT, l'alpha-galactosylceramide (αGC) est un antigène lipidique fréquemment utilisé dans les études cliniques comme puissant activateur. Après l'activation des cellules iNKT avec l'αGC, celles-ci produisent abondamment et rapidement des cytokines. Ces cytokines sont des molécules agissant comme des signaux activateurs d'autres cellules du système immunitaire telles que les cellules NK et les lymphocytes T. Cependant, les cellules iNKT deviennent anergiques après un seul traitement avec l'αGC c'est à dire qu'elles ne peuvent plus être réactivées, ce qui limite leur utilisation dans l'immunothérapie du cancer. Dans notre groupe, Stirnemann et al ont publié une molécule recombinante innovante, composée de la molécule CD1d soluble et chargée avec le ligand αGC (αGC/sCD1d). Cette protéine est capable d'activer les cellules iNKT tout en évitant l'anergie. Dans le système immunitaire, les anticorps sont indispensables pour combattre une infection bactérienne ou virale. En effet, les anticorps ont la capacité de reconnaître et lier spécifiquement un antigène et permettent l'élimination de la cellule qui exprime cet antigène. Dans le domaine de l'immunothérapie, les anticorps sont utilisés afin de cibler des antigènes présentés seulement par la tumeur. Ce procédé permet de réduire efficacement les effets secondaires lors du traitement du cancer. Nous avons donc fusionné la protéine recombinante αGC/CD1d à un fragment d'anticorps qui reconnaît un antigène spécifique des cellules tumorales. Dans une étude préclinique, nous avons démontré que la protéine αGC/sCD1d avec un fragment d'anticorps dirigé contre la tumeur engendre une meilleure activation des cellules iNKT et entraîne un effet anti-tumeur prolongé. Cet effet anti-tumeur est augmenté comparé à une protéine αGC/CD1d qui ne cible pas la tumeur. Nous avons aussi montré que l'activation des cellules iNKT avec la protéine αGC/sCD1d-anti-tumeur améliore l'effet anti- tumoral d'un vaccin pour le cancer. Lors d'expériences in vitro, la protéine αGC/sCD1d-anti- tumeur permet aussi d'activer les cellules humaines iNKT et ainsi tuer spécifiquement les cellules tumorales humaines. La protéine αGC/sCD1d-anti-tumeur représente une alternative thérapeutique prometteuse dans l'immunothérapie du cancer. - Les cellules Invariant Natural Killer T (iNKT), dont les effets anti-tumoraux ont été démontrés, sont de puissants activateurs des cellules Natural Killer (NK), des cellules dendritiques (DC) et des lymphocytes T. Cependant, une seule injection du ligand de haute affinité alpha-galactosylceramide (αGC) n'induit une forte activation des cellules iNKT que durant une courte période. Celle-ci est alors suivie d'une longue phase d'anergie, limitant ainsi leur utilisation pour la thérapie. Comme alternative prometteuse, nous avons montré que des injections répétées d'αGC chargé sur une protéine recombinante de CD1d soluble (αGC/sCD1d) chez la souris entraînent une activation prolongée des cellules iNKT, associée à une production continue de cytokine. De plus, le maintien de la réactivité des cellules iNKT permet de prolonger l'activité anti-tumorale lorsque la protéine αGC/sCD1d est fusionnée à un fragment d'anticorps (scFv) dirigé contre la tumeur. L'inhibition de la croissance tumorale n'est optimale que lorsque les souris sont traitées avec la protéine αGC/sCD1d-scFv ciblant la tumeur, la protéine αGC/sCD1d-scFv non-appropriée étant moins efficace. Dans le système humain, les protéines recombinantes αGC/sCD1d-anti-HER2 et anti-CEA sont capables d'activer et de faire proliférer des cellules iNKT à partir de PBMCs issues de donneurs sains. De plus, la protéine αGC/sCD1d-scFv a la capacité d'activer directement des clones iNKT humains en l'absence de cellules présentatrices d'antigènes (CPA), contrairement au ligand αGC libre. Mais surtout, la lyse des cellules tumorales par les iNKT humaines n'est obtenue que lorsqu'elles sont incubées avec la protéine αGC/sCD1d-scFv anti- tumeur. En outre, la redirection de la cytotoxicité des cellules iNKT vers la tumeur est supérieure à celle obtenue avec une stimulation par des CPA chargées avec l'αGC. Afin d'augmenter les effets anti-tumoraux, nous avons exploité la capacité des cellules iNKT à activer l'immunité adaptive. Pour ce faire, nous avons combiné l'immunothérapie NKT/CD1d avec un vaccin anti-tumoral composé d'un peptide OVA. Des effets synergiques ont été obtenus lorsque les traitements avec la protéine αGC/sCD1d-anti-HER2 étaient associés avec le CpG ODN comme adjuvant pour la vaccination avec le peptide OVA. Ces effets ont été observés à travers l'activation de nombreux lymphocytes T CD8+ spécifique de la tumeur, ainsi que par la forte expansion des cellules NK. Les réponses, innée et adaptive, élevées après le traitement avec la protéine αGC/sCD1d-anti-HER2 combinée au vaccin OVA/CpG ODN étaient associées à un fort ralentissement de la croissance des tumeurs B16- OVA-HER2. Cet effet anti-tumoral corrèle avec l'enrichissement des lymphocytes T CD8+ spécifiques observé à la tumeur. Afin d'étendre l'application des protéines αGC/sCD1d et d'améliorer leur efficacité, nous avons développé des fusions CD1d alternatives. Premièrement, une protéine αGC/sCD1d dimérique, qui permet d'augmenter l'avidité de la molécule CD1d pour les cellules iNKT. Dans un deuxième temps, nous avons fusionné la protéine αGC/sCD1d avec un scFv dirigé contre le récepteur 3 du facteur de croissance pour l'endothélium vasculaire (VEGFR-3), afin de cibler l'environnement de la tumeur. Dans l'ensemble, ces résultats démontrent que la thérapie médiée par la protéine recombinante αGC/sCD1d-scFv est une approche prometteuse pour rediriger l'immunité innée et adaptive vers le site tumoral. - Invariant Natural Killer T cells (iNKT) are potent activators of Natural Killer (NK), dendritic cells (DC) and T lymphocytes, and their anti-tumor activities have been well demonstrated. However, a single injection of the high affinity CD1d ligand alpha-galactosylceramide (αGC) leads to a strong but short-lived iNKT cell activation followed by a phase of long-term anergy, limiting the therapeutic use of this ligand. As a promising alternative, we have demonstrated that when αGC is loaded on recombinant soluble CD1d molecules (αGC/sCD1d), repeated injections in mice led to the sustained iNKT cell activation associated with continued cytokine secretion. Importantly, the retained reactivity of iNKT cell led to prolonged antitumor activity when the αGC/sCD1d was fused to an anti-tumor scFv fragments. Optimal inhibition of tumor growth was obtained only when mice were treated with the tumor-targeted αGC/CD1d-scFv fusion, whereas the irrelevant αGC/CD1d-scFv fusion was less efficient. When tested in a human system, the recombinant αGC/sCD1d-anti-HER2 and -anti-CEA fusion proteins were able to expand iNKT cells from PBMCs of healthy donors. Furthermore, the αGC/sCD1d-scFv fusion had the capacity to directly activate human iNKT cells clones without the presence of antigen-presenting cells (APCs), in contrast to the free αGC ligand. Most importantly, tumor cell killing by human iNKT cells was obtained only when co- incubated with the tumor targeted sCD1d-antitumor scFv, and their direct tumor cytotoxicity was superior to the bystander killing obtained with αGC-loaded APCs stimulation. To further enhance the anti-tumor effects, we exploited the ability of iNKT cells to transactivate the adaptive immunity, by combining the NKT/CD1d immunotherapy with a peptide cancer vaccine. Interestingly, synergistic effects were obtained when the αGC/sCD1d- anti-HER2 fusion treatment was combined with CpG ODN as adjuvant for the OVA peptide vaccine, as seen by higher numbers of activated antigen-specific CD8 T cells and NK cells, as compared to each regimen alone. The increased innate and adaptive immune responses upon combined tumor targeted sCD1d-scFv treatment and OVA/CpG vaccine were associated with a strong delay in B16-OVA-HER2 melanoma tumor growth, which correlated with an enrichment of antigen-specific CD8 cells at the tumor site. In order to extend the application of the CD1d fusion, we designed alternative CD1d fusion proteins. First, a dimeric αGC/sCD1d-Fc fusion, which permits to augment the avidity of the CD1d for iNKT cells and second, an αGC/sCD1d fused to an anti vascular endothelial growth factor receptor-3 (VEGFR-3) scFv, in order to target tumor stroma environment. Altogether, these results demonstrate that the iNKT-mediated immunotherapy via recombinant αGC/sCD1d-scFv fusion is a promising approach to redirect the innate and adaptive antitumor immune response to the tumor site.

Effectiveness of cladribine therapy in patients with pulmonary Langerhans cell histiocytosis.

BackgroundPulmonary Langerhans cell histiocytosis (PLCH) is a rare disorder characterised by granulomatous proliferation of CD1a-positive histiocytes forming granulomas within lung parenchyma, in strong association with tobacco smoking, and which may result in chronic respiratory failure. Smoking cessation is considered to be critical in management, but has variable effects on outcome. No drug therapy has been validated. Cladribine (chlorodeoxyadenosine, 2-CDA) down-regulates histiocyte proliferation and has been successful in curbing multi-system Langerhans cell histiocytosis and isolated PLCH.Methods and patientsWe retrospectively studied 5 patients (aged 37¿55 years, 3 females) with PLCH who received 3 to 4 courses of cladribine therapy as a single agent (0.1 mg/kg per day for 5 consecutive days at monthly intervals). One patient was treated twice because of relapse at 1 year. Progressive pulmonary disease with obstructive ventilatory pattern despite smoking cessation and/or corticosteroid therapy were indications for treatment. Patients were administered oral trimethoprim/sulfamethoxazole and valaciclovir to prevent opportunistic infections. They gave written consent to receive off-label cladribine in the absence of validated treatment.ResultsFunctional class dyspnea improved with cladribine therapy in 4 out of 5 cases, and forced expiratory volume in 1 second (FEV1) increased in all cases by a mean of 387 ml (100¿920 ml), contrasting with a steady decline prior to treatment. Chest high-resolution computed tomography (HRCT) features improved with cladribine therapy in 4 patients. Hemodynamic improvement was observed in 1 patient with pre-capillary pulmonary hypertension. The results suggested a greater treatment effect in subjects with nodular lung lesions and/or thick-walled cysts on chest HRCT, with diffuse hypermetabolism of lung lesions on positron emission tomography (PET)-scan, and with progressive disease despite smoking cessation. Infectious pneumonia developed in 1 patient, with later grade 4 neutrocytopenia but without infection.DiscussionData interpretation was limited by the retrospective, uncontrolled study design and small sample size.ConclusionCladribine as a single agent may be effective therapy in patients with progressive PLCH.

Wide resection of the lateral malleolus and adjacent tibial, talar and calcanear bones with ankle fusion and allograft reconstruction for an osteosarcoma.

Introduction: Primary bone sarcomas around the ankle are rare. Due to the proximity of neurovascular structures and limited soft tissue reserves, limb salvage is often not possible. Case report: A 19 yo male presented with pain and a progressive swelling of his ankle. X-rays revealed cortical erosions and an extensive periosteal reaction (sunburst) of the distal fibula. MRI showed a large mass of the fibula invading adjacent soft tissue. The lesion appeared close to the ankle joint, but with the articular cartilage as a barrier and without joint effusion. Core-needle biopsy revealed a high-grade chondroblastic osteosarcoma. No metastases were detected. After presentation at our multidisciplinary sarcoma board, the patient was subjected to neo-adjuvant chemotherapy (AOST 03-331). Without any sign of intra-articular contamination of the ankle joint, surgical treatment consisted of wide resection of the lateral malleolus including a large skin patch, the distal third of the fibula, the lateral surfaces of the tibia and talus as well as the insertion of the lateral ligament on the calcaneus. The distal parts of the anterior, peroneal, and posterior muscular compartments were resected en bloc with the tumor. The defect was reconstructed with tibio-talar and talo-calcanear fusion, bony allograft and a plate. Soft-tissue coverage was achieved with a free fascio-cutaneous flap from the controlateral thigh. Histological analysis revealed clear margins and 50% of tumor necrosis. The oncologic treatment was completed with adjuvant chemotherapy. Conclusion: Wide resection and reconstruction of the lateral malleolus is technically demanding but possible in selected cases. Despite some important functional loss, limb salvage is superior to an amputation.

JARID2 is a direct target of the PAX3-FOXO1 fusion protein and inhibits myogenic differentiation of rhabdomyosarcoma cells.

Rhabdomyosarcomas (RMS) are the most frequent soft-tissue sarcoma in children and characteristically show features of developing skeletal muscle. The alveolar subtype is frequently associated with a PAX3-FOXO1 fusion protein that is known to contribute to the undifferentiated myogenic phenotype of RMS cells. Histone methylation of lysine residues controls developmental processes in both normal and malignant cell contexts. Here we show that JARID2, which encodes a protein known to recruit various complexes with histone-methylating activity to their target genes, is significantly overexpressed in RMS with PAX3-FOXO1 compared with the fusion gene-negative RMS (t-test; P < 0.0001). Multivariate analyses showed that higher JARID2 levels are also associated with metastases at diagnosis, independent of fusion gene status and RMS subtype (n = 120; P = 0.039). JARID2 levels were altered by silencing or overexpressing PAX3-FOXO1 in RMS cell lines with and without the fusion gene, respectively. Consistent with this, we demonstrated that JARID2 is a direct transcriptional target of the PAX3-FOXO1 fusion protein. Silencing JARID2 resulted in reduced cell proliferation coupled with myogenic differentiation, including increased expression of Myogenin (MYOG) and Myosin Light Chain (MYL1) in RMS cell lines representative of both the alveolar and embryonal subtypes. Induced myogenic differentiation was associated with a decrease in JARID2 levels and this phenotype could be rescued by overexpressing JARID2. Furthermore, we that showed JARID2 binds to and alters the methylation status of histone H3 lysine 27 in the promoter regions of MYOG and MYL1 and that the interaction of JARID2 at these promoters is dependent on EED, a core component of the polycomb repressive complex 2 (PRC2). Therefore, JARID2 is a downstream effector of PAX3-FOXO1 that maintains an undifferentiated myogenic phenotype that is characteristic of RMS. JARID2 and other components of PRC2 may represent novel therapeutic targets for treating RMS patients.