Preparedness activities and research needs in addressing emerging infectious animal and zoonotic diseases.

Emerging infectious animal and zoonotic diseases can inflict significant losses on animal production and public health, and threaten the safety and security of the food system. Threat analysis (forecasting), which monitors the measurable risk indicators of disease emergence, should be in place before the emergence of any threat. Animal and public health authorities develop and regularly re-evaluate disease preparedness, response and recovery plans, based on the 'One Health' principle. These plans should include surveillance, biosecurity measures, communication channels and training for personnel. Scenarios for outbreaks of natural emerging infectious disease or bioterrorist events should be prepared and practised. National and international legislation should be regularly updated to provide a robust legal basis to manage outbreaks. Reference laboratories should have reliable and validated diagnostic tools for rapid, high-throughput testing. Strict biosafety, biocontainment and biosecurity control measures must be implemented in laboratories in order to prevent the accidental or malicious release of pathogens. The pharmaceutical industry should be incentivised to develop vaccines and/or antiviral drugs against disease outbreaks. Conventions between public authorities and the pharmaceutical industry should guarantee adequate stockpiling of the pharmaceuticals needed to control large-scale outbreaks. In the early phase of disease emergence (early warning), veterinarians and stakeholders play an important role in early detection at the farm level. Upon notification, veterinary authorities must take rapid response measures to limit disease spread. National and international short- and medium-term strategic research agendas should be developed, based on a comprehensive gap analysis and horizon scan. This planning will help to guide funding agencies and non-governmental organisations in their quest to support relevant research.

Les maladies animales infectieuses et les zoonoses émergentes ont un coût élevé pour la santé animale et la santé publique, en plus d'entraîner d'importantes pertes de production dans les élevages et de menacer la sécurité des systèmes de production alimentaire. Une analyse des menaces (anticipation), grâce au suivi d'indicateurs mesurables du risque d'émergence des maladies animales, devrait être en place avant que ces menaces n'émergent. Les autorités en charge de la santé animale et de la santé publique développent et réévaluent régulièrement des plans de préparation, de réponse et de récupération vis-à-vis de maladies, sur la base du principe « Une seule santé ¼. Ces plans doivent inclure des mesures de surveillance et de biosécurité, en plus de se doter de moyens de communication et de formation du personnel. Il convient d'élaborer et de mettre en pratique des scénarios d'émergence de maladies infectieuses, que celle-ci soit d'origine naturelle ou d'origine bioterroriste. Les législations nationales et internationales en la matière doivent être actualisées régulièrement afin de fournir un fondement juridique solide à la gestion des émergences. Les laboratoires de référence doivent disposer d'outils diagnostiques fiables et validés permettant la réalisation de tests rapides et à haut débit. Des mesures strictes de contrôle de la biosécurité, du bioconfinement et de la biosûreté doivent être appliquées dans les laboratoires pour prévenir toute libération accidentelle ou malintentionnée d'agents pathogènes. L'industrie pharmaceutique doit être incitée au développement de vaccins et d'antiviraux pour maîtriser les maladies émergentes. Les conventions entre les autorités publiques et l'industrie pharmaceutique doivent permettre de garantir la constitution de stocks suffisants de produits pharmaceutiques pour maîtriser les émergences de grande ampleur. Lors des premières phases d'émergence d'un foyer (alerte précoce), les vétérinaires et autres acteurs de terrain jouent un rôle important dans la détection précoce au niveau des élevages. Dès la notification d'un foyer, les autorités vétérinaires doivent réagir rapidement afin d'en limiter la propagation. Il convient de développer des programmes nationaux et internationaux de recherche stratégique à court et moyen terme, basés sur un examen exhaustif des lacunes et sur une analyse prospective complète. Cette planification contribuera à fournir aux agences de financement et aux organisations non gouvernementales des orientations leur permettant de déterminer quel soutien apporter à la recherche.

Las enfermedades animales infecciosas y las zoonosis emergentes pueden causar pérdidas cuantiosas en los ámbitos de la producción animal y la salud pública, además de amenazar la higiene y la seguridad de los sistemas alimentarios. El análisis (pronóstico) de amenazas, que consiste en seguir de cerca indicadores cuantificables del riesgo de aparición de enfermedades animales, es algo que debería estar implantado antes de que surja toda amenaza. Las autoridades sanitarias y zoosanitarias definen y periódicamente reevalúan planes de preparación, respuesta y recuperación frente a enfermedades, basándose para ello en el principio de «Una sola salud¼. Estos planes deben incluir labores de vigilancia y medidas de seguridad biológica, además de prever cauces de comunicación y actividades de formación del personal. También hay que elaborar y aplicar planes para hipotéticos brotes infecciosos, ya sean de origen natural u obra de bioterroristas. Asimismo, a fin de contar con sólidas bases jurídicas para combatir la aparición de enfermedades, es preciso actualizar periódicamente la legislación nacional e internacional. Los laboratorios de referencia deben contar con herramientas de diagnóstico fiables y validadas que permitan efectuar pruebas rápidas y de alto rendimiento. Es preciso implantar en los laboratorios estrictas medidas de control de la protección, la contención y la seguridad biológicas para evitar toda liberación accidental o malintencionada de patógenos. Hay que incentivar asimismo a la industria farmacéutica para que desarrolle vacunas y fármacos antivirales contra las enfermedades emergentes. Por otra parte, las autoridades públicas deben suscribir con el sector farmacéutico convenios que garanticen la constitución de reservas suficientes de los productos farmacéuticos requeridos para hacer frente a la aparición de brotes de grandes dimensiones. En las primeras fases de la aparición de un foco (alerta rápida), los veterinarios y otros interlocutores cumplen una importante función para detectar con prontitud la patología dentro de las explotaciones. Al recibir notificación, las autoridades veterinarias deben reaccionar con rapidez para poner coto a la propagación de la enfermedad. Por último, a partir de un análisis exhaustivo de las carencias existentes y de un estudio prospectivo completo, es preciso elaborar planes nacionales e internacionales de investigación estratégica a corto y medio plazo. Tal planificación ayudará a orientar a los organismos de financiación y las organizaciones no gubernamentales (ONG) en su labor de apoyo a las investigaciones de interés.

Tapia's syndrome in the intensive care unit: a rare cause of combined cranial nerve palsy following intubation.

Tapia's syndrome is characterized by unilateral paralysis of the tongue and vocal cord, and is caused by a concurrent lesion of both the recurrent laryngeal and hypoglossal nerves. The proposed mechanism in most patients is compression or stretching of these nerves on their extracranial course due to airway manipulation under general anaesthesia. As Tapia's syndrome is a rare and possibly devastating condition, recognition of the presence of concurrent paralyses is an important step in diagnosis and treatment. We report two cases of Tapia's syndrome as a complication of intubation in the intensive care unit.

Defining European preparedness and research needs regarding emerging infectious animal diseases: results from a Delphi expert consultation.

Emerging and major infectious animal diseases can have significant international impact on social, economic and environmental level, and are being driven by various factors. Prevention and control measures should be prepared at both national and international level to mitigate these disease risks. Research to support such policy development is mostly carried out at national level and dedicated transnational research programmes are still in its infancy. This research reports on part of a process to develop a common strategic research agenda on emerging and major infectious diseases of livestock in Europe, covering a 5-15-year time span. A two round online Delphi study was conducted to explore the views of experts on issues relating to research needs on emerging infectious diseases of livestock in Europe. Drivers that may influence the incidence of emerging infectious animal diseases in both the short (next 5 years) and medium term (10-15 years) were identified. Drivers related to regulatory measures and biological science developments were thought to decrease the incidence, and socio-economic factors to increase the incidence of emerging infectious animal diseases. From the first round a list of threats to animal health was compiled and participants combined these threats with relevant drivers in the second round. Next to identifying threats to animal health, also possible mitigatory actions to reduce the negative impact of these threats were identified. Participants emphasised that interdisciplinary research is needed to understand drivers of emerging infectious animal diseases, as well as to develop prevention and control measures which are both socio-economic and technical. From this it can be concluded that interdisciplinary research combining both natural and social research themes is required. Some of the European member states research budget needs to be allocated so that effective prevention and mitigation strategies can be developed.

Nucleoside analogs induce proteasomal down-regulation of p21 in chronic lymphocytic leukemia cell lines.

Nucleoside analogs (NAs) represent an important class of anticancer agents that induce cell death after conversion to triphosphate derivatives. One of their most important mechanisms of action is the activation of p53, leading to apoptosis through the intrinsic pathway. Classically, the activation of p53 also induces p21 accumulation, which leads to cell cycle arrest at the G1/S transition. In previous work, we observed that 2-chloro-2'-deoxyadenosine (CdA), a NA with high activity in lymphoid disorders, including chronic lymphocytic leukemia (CLL), promotes the G1/S transition in the CLL cell line EHEB at cytotoxic concentrations. This finding led us to investigate the p21 response to NAs in these cells. We show here that CdA, but also fludarabine, gemcitabine, and cytarabine, strongly reduced the p21 protein level in EHEB cells as well as in JVM-2 cells, another CLL cell line. This p21 depletion occurred despite induction of p53 and increase of p21 mRNA and was prevented by proteasome inhibitors. Increase of proteasomal degradation caused by NAs appeared to be ubiquitin-independent. Also, NAs induced in these cells an increase of cyclin-dependent kinase (Cdk2) activity and a monoubiquitination of cell proliferating nuclear antigen (PCNA), two processes that are negatively regulated by p21. These changes were not observed with other p53 activators, like etoposide and nutlin-3a that increased the p21 protein level. In conclusion, our study reveals that NAs can induce an alternative pattern of cellular response in some cell models.

New evidences for a regulation of deoxycytidine kinase activity by reversible phosphorylation.

Recent studies indicate that deoxycytidine kinase (dCK), which activates various nucleoside analogues used in antileukemic therapy, can be regulated by post-translational modification, most probably through reversible phosphorylation. To further unravel its regulation, dCK was overexpressed in HEK-293 cells as a His-tag fusion protein. Western blot analysis showed that purified overexpressed dCK appears as doublet protein bands. The slower band disappeared after treatment with protein phosphatase lambda (PP lambda) in parallel with a decrease of dCK activity, providing additional arguments in favor of both phosphorylated and unphosphorylated forms of dCK.

Effects of 2-chloro-2'-deoxyadenosine on the cell cycle in the human leukemia EHEB cell line.

To explain why 2-chloro-2'-deoxyadenosine (CdA) is unable to block DNA synthesis and cell cycle progression, and paradoxically enhances progression from G1 into S phase in the CdA-resistant leukemia EHEB cell line, we studied its metabolism and effects on proteins regulating the transition from G1 to S phase. A low deoxycytidine kinase activity and CdATP accumulation, and a lack of p21 induction despite p53 phosphorylation and accumulation may account for the inability of CdA to block the cell cycle. An alternative pathway involving pRb phosphorylation seems implicated in the CdA-induced increase in G1 to S phase progression.

2-Chloro-2'-deoxyadenosine inhibits DNA repair synthesis and potentiates UVC cytotoxicity in chronic lymphocytic leukemia B lymphocytes.

2-Chloro-2'-deoxyadenosine (CdA) is a deoxyadenosine analogue which targets enzymes involved in DNA synthesis, and hence might interfere with the resynthesis step of DNA repair. We tested this hypothesis in resting B cell chronic lymphocytic leukemia (B-CLL) lymphocytes, after firstly characterizing unscheduled DNA synthesis occurring in these cells. We observed that the spontaneous incorporation of [methyl-3H]thymidine (dThd) into DNA of B-CLL cells was not completely inhibitable by hydroxyurea (HU) which blocks DNA replication. In addition, in the presence of HU, dThd incorporation could be upregulated by UVC radiation or DNA alkylation, without re-entry of the cells into S phase. CdA was found to inhibit both spontaneous and upregulated DNA synthesis in B-CLL cells. Phosphorylation of CdA was essential to exert this effect. We finally observed a strong synergistic cytotoxicity between UV light and CdA, which was correlated with activation of caspase-3 and high molecular weight DNA fragmentation, two markers of apoptosis. Taken together, these observations indicate that in B-CLL cells CdA inhibits unscheduled DNA synthesis which represents the polymerizing step of a repair process responsive to DNA aggression. Inhibition of this process by CdA, together with a combined activation of the apoptotic proteolytic cascade by CdA and UV, may explain their synergistic cytotoxicity.

Resistance to 2-chloro-2'-deoxyadenosine of the human B-cell leukemia cell line EHEB.

The effects of 2-chloro-2'-deoxyadenosine (CdA, cladribine), an adenosine deaminase-resistant analogue toxic for both proliferating and resting lymphoid cells, were investigated in the human leukemia cell line EHEB, which was derived from a patient with B-cell chronic lymphocytic leukemia. These cells were found to be less sensitive to CdA than B-cell chronic lymphocytic leukemia lymphocytes (approximately 25-fold) and other human lymphoblastic cell lines (10-1000-fold). Phosphorylation of CdA by deoxycytidine kinase and intracellular accumulation of 2-chloro-2'-deoxyadenosine triphosphate (CdATP) were similar in EHEB cells and in other CdA-sensitive cell lines. In contrast, the inhibitory effect of CdA on ribonucleotide reductase activity, which was investigated in situ by the conversion of cytidine into deoxyribonucleotides and its incorporation into DNA, was much less pronounced in EHEB cells than in other human lymphoblastic cells. Accordingly, concentrations of deoxynucleoside triphosphates did not decrease and even tended to rise. Unexpectedly, incorporation of thymidine and deoxycytidine into DNA was increased severalfold after a 24-h incubation with CdA. CdA also increased the activities of deoxycytidine kinase and thymidine kinase approximately 4-fold. Analysis of the cell cycle by flow cytometry showed that after 24 h, CdA provoked an increase in the proportion of cells in S phase, synthesizing DNA. We conclude that the EHEB cell line is resistant to the cytotoxic action of CdA not only because of a lack of inhibition of ribonucleotide reduction but also because CdA, in contrast with its known effects, provokes in this cell line an increase in the proportion of cells replicating their DNA. Unraveling of the mechanism of this effect may shed light on clinical resistance to CdA.

Metabolism and cytotoxic effects of 2-chloroadenine, the major catabolite of 2-chloro-2'-deoxyadenosine.

EHEB cells, a continuous cell line derived from a patient with B cell chronic lymphocytic leukemia (B-CLL), synthesized, when incubated with tritiated 2-chloro-2'-deoxyadenosine (CdA), labeled mono-, di-, and triphosphate ribonucleosides at a much higher rate than CdA deoxyribonucleotides. Further analysis revealed that these ribonucleotides were formed from labeled 2-chloroadenine (CAde), which contaminated commercial tritiated CdA at a proportion of 2-3%. Since CAde is the major catabolite of CdA measured in plasma after oral or intravenous administration of CdA to patients, its metabolism and in particular its potential cytotoxicity were investigated both in EHEB cells and in B-CLL lymphocytes. Phosphorylation of CAde was inhibited by adenine, indicating that its initial metabolism most probably proceeds via adenine phosphoribosyltransferase (EC 2.4.2.7). In both cell types, chloro-ATP was the major metabolite formed from CAde and its concentration increased proportionally at least up to 50 microM CAde. At high concentration, CAde metabolism was accompanied by a decrease in intracellular ATP. Cytotoxicity of CAde, evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, showed an IC(50) of 16 microM in EHEB cells and 5 microM in B-CLL lymphocytes. At cytotoxic concentrations, apopain/caspase-3 activation and high molecular weight DNA fragmentation were observed, indicating that CAde cytotoxicity results from induction of apoptosis. However, since CAde cytotoxicity requires higher concentrations than CdA, it probably does not play a role in the therapeutic effect of CdA in the treatment of hematologic malignancies.

Potentiation of antitumor effects of cyclophosphamide derivatives in B-chronic lymphocytic leukemia cells by 2-chloro-2'-deoxyadenosine.

Because 2-chloro-2'-deoxyadenosine (CdA) is active in B-chronic lymphocytic leukemia (B-CLL), and may interfere with DNA repair, we investigated the potentiating effect of CdA on the cytotoxicity induced in vitro in B-CLL lymphocytes by cyclophosphamide (CP) derivatives, which induce DNA damage by DNA cross-linking. Exposure to CdA at clinically achievable concentrations for 2 h, followed by mafosfamide (MAF) or 4-hydroxycyclophosphamide (4HC) for 22 h, resulted in synergistic cytotoxicity in the majority of B-CLL samples tested. Synergy between CdA and MAF was observed in cell samples of sensitive/untreated patients, as well as in cells of resistant/pretreated patients, particularly at the highest concentrations of MAF. In the cells treated with CdA and MAF, we observed loss in ATP and hallmarks of apoptosis, as evidenced by cellular morphology and high molecular weight DNA fragmentation. The synergy could be explained neither by an influence of MAF on the phosphorylation of CdA, nor by an increase in the incorporation of CdA into DNA in the presence of MAF. The in vitro synergy between CdA and CP derivatives provides a rationale for the use of this association in B-CLL patients.