Preclinical safety and tolerability of a repeatedly administered human leishmaniasis DNA vaccine.

The leishmaniases are a complex of vector-borne diseases caused by protozoan parasites of the genus Leishmania. LEISHDNAVAX is a multi-antigen, T-cell epitope-enriched DNA vaccine candidate against human leishmaniasis. The vaccine candidate has been proven immunogenic and showed prophylactic efficacy in preclinical studies. Here, we describe the safety testing of LEISHDNAVAX in naive mice and rats, complemented by the demonstration of tolerability in Leishmania-infected mice. Biodistribution and persistence were examined following single and repeated intradermal (i.d.) administration to rats. DNA vectors were distributed systemically but did not accumulate upon repeated injections. Although vector DNA was cleared from most other tissues within 60 days after the last injection, it persisted in skin at the site of injection and in draining lymph nodes. Evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after i.d. administration to naive, non-infected mice did not reveal any safety concerns. LEISHDNAVAX was also well tolerated in Leishmania-infected mice. Taken together, our results substantiate a favorable safety profile of LEISHDNAVAX in both naive and infected animals and thus, support the initiation of clinical trials for both preventive and therapeutic applications of the vaccine

A vaccine based on recombinant modified Vaccinia Ankara containing the nucleoprotein from Lassa virus protects against disease progression in a guinea pig model.

Lassa fever remains the most imported viral haemorrhagic fever in Europe and is responsible for 5000 deaths per year throughout Western Africa. There is no vaccine and treatment is often ineffective. We have developed a vaccine based on modified Vaccinia Ankara expressing the nucleoprotein from Lassa virus (MVALassaNP). This study investigated the immunogenicity (in mice) and efficacy (in guinea pigs) of the MVALassaNP vaccine as a prime/boost or single vaccination regime. ELISA and ELISpot assays confirmed humoral and T-cell immunity following both a prime and prime/boost vaccination, with the prime/boost regime producing a statistically increased response compared to a prime only vaccine (P < 0.0001). The vaccine offered protection in guinea pigs against disease manifestations after challenge with virulent Lassa virus. Clinical signs, weight loss and temperature increases were observed in all animals receiving a control MVA vaccine, after challenge with Lassa virus. In contrast, no clinical signs, fever or weight loss were observed in any of the MVALassaNP vaccinated animals demonstrating that both a single immunisation, and prime/boost regime confer protection against disease progression. In conclusion, the MVALassaNP vaccine candidate elicits an immune response, demonstrates efficacy against Lassa virus disease and is suitable for further preclinical and clinical development

Merkel Cell Polyomavirus Small T Antigen Drives Cell Motility via Rho-GTPase-Induced Filopodium Formation

Cell motility and migration is a complex, multistep, and multicomponent process intrinsic to progression and metastasis. Motility is dependent on the activities of integrin receptors and Rho family GTPases, resulting in the remodeling of the actin cytoskeleton and formation of various motile actin-based protrusions. Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high likelihood of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is associated with the majority of MCC cases, and MCPyV-induced tumorigenesis largely depends on the expression of the small tumor antigen (ST). Since the discovery of MCPyV, a number of mechanisms have been suggested to account for replication and tumorigenesis, but to date, little is known about potential links between MCPyV T antigen expression and the metastatic nature of MCC. Previously, we described the action of MCPyV ST on the microtubule network and how it impacts cell motility and migration. Here, we demonstrate that MCPyV ST affects the actin cytoskeleton to promote the formation of filopodia through a mechanism involving the catalytic subunit of protein phosphatase 4 (PP4C). We also show that MCPyV ST-induced cell motility is dependent upon the activities of the Rho family GTPases Cdc42 and RhoA. In addition, our results indicate that the MCPyV ST-PP4C interaction results in the dephosphorylation of β1 integrin, likely driving the cell motility pathway. These findings describe a novel mechanism by which a tumor virus induces cell motility, which may ultimately lead to cancer metastasis, and provides opportunities and strategies for targeted interventions for disseminated MCC

Protección de la Heparina a las células B- pancreáticas frente a radicales libres de Oxígeno.

La diabetes autoinmune tipo 1 se caracteriza por la invasión de células mononucleares en los islotes pancreáticos, destruyendo así las células beta productoras de insulina. Se ha visto que in vivo la destrucción autoinmune de los islotes se asocia con la producción de heparanasa, la cual degrada heparán sulfato (molécula imprescindible para la supervivencia de los islotes) y se permite la entrada de las células inmunitarias que atacarán los islotes beta pancreáticos. Se han obtenido resultados mediante la adición de concentraciones conocidas de heparina, la cual confiere una protección extra frente a radicales libres de oxígeno y como consecuencia hay una disminución de la mortalidad celular.A través de tres líneas celulares distintas se ha llevado a cabo el experimento. Primeramente con Rinm5F productora de insulina y somatostatina, con células Ins capaces de responder al estímulo de glucosa produciendo y secretando insulina y finalmente con fibroblastos. El experimento se basa en dejar crecer las cepas celulares en un número determinado de flacs según el tratamiento. Se añade heparina a una concentración conocida y establecida anteriormente y al día siguiente con una concentración exacta de agua oxigenada (aporta los radicales libres de oxígeno) se la añadimos al cultivo. Recogemos las células y gracias al ioduro de propidio con concentración 1 mg/ml marcamos las células muertas para así poder comprobar el porcentaje de supervivencia celular que le ha conferido la heparina a cada línea celular. Procedemos a realizar el contaje con el citómetro que indica el % de muerte celular, ya que el ioduro de propidio es un agente intercalante que se une a los ácidos nucleicos. Esta molécula fluorescente se utiliza para evaluar la viabilidad celular o el contenido de ADN en las células. Se puede utilizar para diferenciar células necróticas, apoptóticas o vivas.Los resultados obtenidos con las líneas celulares Rinm5F y las Ins nos muestran que a bajas concentraciones de agua oxigenada y con heparina, efectivamente hay protección ya que la muerte celular se reduce de un 10 a un 15%. En cambio con los fibroblastos no vemos protección a ninguna de las concentraciones establecidas, resultado que ya esperábamos en nuestra hipótesis inicial. Estos resultados sólo son el inicio de un largo estudio, ya que  primero se ajustan las concentraciones a trabajar con radicales libres de oxígeno, pero en un  futuro el estudio se realizará también con radicales de nitrógeno

Relación entre muerte celular y mantenimiento de la pluripotencialidad de células mES en protocolos de diferenciación con óxido nítrico

En este proyecto se pretende estudiar la relación entre la muerte celular y la diferenciación durante el desarrollo usando como modelo experimental células madre embrionarias de ratón (mESC). Se ha descrito que las altas concentraciones de DETA-NO (500uM) provocan la diferenciación de estas mESC hacia endodermo (Mora-Castilla et al., 2010), pero en estos protocolos no todas consiguen sobrevivir y un alto porcentaje de células muere. Es por ello que se quiere analizar la relación entre ambos eventos. Se estudiará, por un lado, si el bloqueo de la apoptosis con inhibidores de caspasas tiene efectos sobre la diferenciación; y por otro lado si es probable que exista una liberación de moléculas al medio por parte de las células apoptóticas que contribuya a la pérdida de la pluripotencialidad de sus vecinas. Para ello se ha analizado el mantenimiento del estado pluripotente en dos tipos de ensayos. Por un lado se ha estudiado el efecto de inhibidores de caspasas en tratamientos con DETA-NO en dos líneas de mESC (D3 y R1/E), y por otro lado se ha evaluado el efecto del reciclaje de medios de cultivo en la línea celular D3. Para medir el mantenimiento de la pluripotencialidad se han empleado distintas técnicas de biología molecular (extracción de RNA, PCR, qPCR, Western Blotting…) y técnicas de microscopía. Con ello se han buscado diferencias de expresión de los marcadores de pluripotencia (Nanog y Oct4) y marcadores de endodermo (Pdx1, Cxcr4), mesodermo (Brachyury) y ectodermo (Zic1). Se ha demostrado que el uso de inhibidores de caspasas en tratamientos con DETA-NO bloquea la regulación a la baja de Nanog, tanto en niveles de RNAm como de proteína, y disminuye la expresión de los marcadores de diferenciación. Por otro lado, el uso de medios de cultivo reciclados procedentes de tratamientos anteriores con y sin DETA-NO, suplementados y con el factor inhibidor de la diferenciación LIF (Leukemia Inhibitory Factor) estimula la diferenciación de las células. Los resultados obtenidos apuntan a que podría existir una relación entre la muerte celular y la diferenciación, ya que al inhibir la muerte por apoptosis se favorece el mantenimiento del estado pluripotente . Además, el uso de medios reciclados ayuda a la diferenciación de las células incluso en presencia de LIF. Por ello se cree que células apoptóticas podrían estar secretando sustancias al medio que las células vecinas estarían utilizando como señales para diferenciarse

Injecting Domain Knowledge in Electronic Medical Records to Improve Hospitalization Prediction

International audienceElectronic medical records (EMR) contain key information about the different symptomatic episodes that a patient went through. They carry a great potential in order to improve the well-being of patients and therefore represent a very valuable input for artificial intelligence approaches. However, the explicit knowledge directly available through these records remains limited, the extracted features to be used by machine learning algorithms do not contain all the implicit knowledge of medical expert. In order to evaluate the impact of domain knowledge when processing EMRs, we augment the features extracted from EMRs with ontological resources before turning them into vectors used by machine learning algorithms. We evaluate these augmentations with several machine learning algorithms to predict hospitalization. Our approach was experimented on data from the PRIMEGE PACA database that contains more than 350,000 consultations carried out by 16 general practitioners (GPs)

Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells

Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin.This study was supported by grants Group BIO 157 from the Technology and Innovation Council of the Andalucian regional government and AGL2006-12210-C03-02/ALI, SAF2005-01627, ISCIII-RTICC (RD06/0020/0046) from the Spanish government and European Union FEDER funds

In Vivo Dioxin Favors Interleukin-22 Production by Human CD4+ T Cells in an Aryl Hydrocarbon Receptor (AhR)-Dependent Manner

The transcription factor aryl hydrocarbon receptor (AhR) mediates the effects of a group of chemicals known as dioxins, ubiquitously present in our environment. However, it is poorly known how the in vivo exposure to these chemicals affects in humans the adaptive immune response. We therefore assessed the functional phenotype of T cells from an individual who developed a severe cutaneous and systemic syndrome after having been exposed to an extremely high dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).T cells of the TCDD-exposed individual were studied for their capacity to produce cytokines in response to polyclonal and superantigenic stimulation, and for the expression of chemokine receptors involved in skin homing. The supernatants from T cells of the exposed individual contained a substantially increased amount of interleukin (IL)-22 but not of IL-17A, interferon (IFN)-γ or IL-10 when compared to nine healthy controls. In vitro experiments confirmed a direct, AhR-dependent, enhancing effect of TCDD on IL-22 production by CD4+ T cells. The increased production of IL-22 was not dependent on AhR occupancy by residual TCDD molecules, as demonstrated in competition experiments with the specific AhR antagonist CH-223191. In contrast, it was due to an increased frequency of IL-22 single producing cells accompanied by an increased percentage of cells expressing the skin-homing chemokine receptors CCR6 and CCR4, identified through a multiparameter flow cytometry approach. Of interest, the frequency of CD4+CD25(hi)FoxP3+ T regulatory cells was similar in the TCDD-exposed and healthy individuals.This case strongly supports the contention that human exposure to persistent AhR ligands in vivo induce a long-lasting effect on the human adaptive immune system and specifically polarizes CD4+ T cells to produce IL-22 and not other T cell cytokines with no effect on T regulatory cells

Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs

Toxicogenomic analysis of exposure to TCDD, PCB126 and PCB153: identification of genomic biomarkers of exposure to AhR ligands

<p>Abstract</p> <p>Background</p> <p>Two year cancer bioassays conducted by the National Toxicology Program have shown chronic exposure to dioxin-like compounds (DLCs) to lead to the development of both neoplastic and non-neoplastic lesions in the hepatic tissue of female Sprague Dawley rats. Most, if not all, of the hepatotoxic effects induced by DLC's are believed to involve the binding and activation of the transcription factor, the aryl hydrocarbon receptor (AhR). Toxicogenomics was implemented to identify genomic responses that may be contributing to the development of hepatotoxicity in rats.</p> <p>Results</p> <p>Through comparative analysis of time-course microarray data, unique hepatic gene expression signatures were identified for the DLCs, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (100 ng/kg/day) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) (1000 ng/kg/day) and the non-DLC 2,2',4,4',5,5',-hexachlorobiphenyl (PCB153) (1000 μg/kg/day). A common time independent signature of 41 AhR genomic biomarkers was identified which exhibited at least a 2-fold change in expression following subchronic (13-wk) and chronic (52-wk) p.o. exposure to TCDD and PCB126, but not the non DLC, PCB153. Real time qPCR analysis validated that 30 of these genes also exhibited at least a 2-fold change in hepatic expression at 24 hr following a single exposure to TCDD (5 μg/kg, po). Phenotypic anchoring was conducted which identified forty-six genes that were differently expressed both following chronic p.o. exposure to DLCs and in previously reported studies of cholangiocarcinoma or hepatocellular adenoma.</p> <p>Conclusions</p> <p>Together these analyses provide a comprehensive description of the genomic responses which occur in rat hepatic tissue with exposure to AhR ligands and will help to isolate those genomic responses which are contributing to the hepatotoxicity observed with exposure to DLCs. In addition, the time independent gene expression signature of the AhR ligands may assist in identifying other agents with the potential to elicit dioxin-like hepatotoxic responses.</p