Effects of extrusion, lipid concentration and purity on physico-chemical and biological properties of cationic liposomes for gene vaccine applications

We developed cationic liposomes containing DNA through a conventional process involving steps of (i) preformation of liposomes, (ii) extrusion, (iii) drying and rehydration and (iv) DNA complexation. Owing to its high prophylactic potentiality against tuberculosis, which had already been demonstrated in preclinical assays, we introduced modifications into the conventional process towards getting a simpler and more economical process for further scale-up. Elimination of the extrusion step, increasing the lipid concentration (from 16 to 64 mM) of the preformed liposomes and using good manufacturing practice bulk lipids (96-98% purity) instead of analytical grade purity lipids (99.9-100%) were the modifications studied. The differences in the physico-chemical properties, such as average diameter, zeta potential, melting point and morphology of the liposomes prepared through the modified process, were not as significant for the biological properties, such as DNA loading on the cationic liposomes, and effective immune response in mice after immunisation as the control liposomes prepared through the conventional process. Beneficially, the modified process increased productivity by 22% and reduced the cost of raw material by 75%.

CHARACTERIZATION OF REGOSOLS IN THE SEMIARID REGION OF PERNAMBUCO, BRAZIL

CHARACTERIZATION OF REGOSOLS IN THE SEMIARID REGION OF PERNAMBUCO, BRAZIL Studies on soil characterization in unexplored regions, besides the generation of data banks for the soil classes of the country, also produce scientific information about soil properties, important for the development of good management practices and sustainable land use. One of the main soil classes in the semiarid region of Pernambuco State, the Regosols, cover about 27 % of the state area, and are used mainly for family agriculture. Due to different geological and climatic aspects Regosols with different chemical, physical and mineralogical properties are found in Pernambuco, which were characterized for the semiarid region of the State. Five Regosol profiles were selected in different regions of the State (P1=Sao Caetano; P2=Lagoa do Ouro; P3=Caetes; P4=Sao Joao; P5=Parnamirim). The soils were morphologically characterized and samples collected from all horizons and the bedrock. Routine physical and chemical analyses were carried out for soil classification of all samples and mineralogical analyses of the coarse fractions (gravel and sand) by optical microscopy and of the silt and clay fractions by X ray diffraction (XRD), as well as petrographic analyses of the rock samples. The results showed similarities between the soils, with a low degree of pedogenetic development, varying from medium to very deep, with the horizon sequence A-AC-C-Cr and a sandy to sandy loam texture. In the deeper layers of two profiles (P1 and P5), a solodic character was observed. Organic matter and available phosphorus content were low in all studied soils. Despite the low levels of exchangeable cations, all soil profiles showed high base saturation. The mineralogical composition of gravel, sand and silt fractions consisted, essentially, of quartz, followed by feldspars and mica, supporting the results of the petrographic analysis of the bedrock. Kaolinite was the main clay mineral in all studied profiles and horizons, indicating an important monosialitization process in autochthonous soils of a typical semiarid region. In soil profile P2, at a lower landscape position, smectite minerals were observed, with mixing phases of montmorillonite, beidelite or nontronite, indentified by the Greene-Kelly test in the DRX analysis.

Nitrogen fertilizer (15N) leaching in a central pivot fertigated coffee crop

Nitrogen has a complex dynamics in the soil-plant-atmosphere system. N fertilizers are subject to chemical and microbial transformations in soils that can result in significant losses. Considering the cost of fertilizers, the adoption of good management practices like fertigation could improve the N use efficiency by crops. Water balances (WB) were applied to evaluate fertilizer N leaching using 15N labeled urea in west Bahia, Brazil. Three scenarios (2008/2009) were established: i) rainfall + irrigation the full year, ii) rainfall only; and iii) rainfall + irrigation only in the dry season. The water excess was considered equal to the deep drainage for the very flat area (runoff = 0) with a water table located several meters below soil surface (capillary rise = 0). The control volume for water balance calculations was the 0 - 1 m soil layer, considering that it involves the active root system. The water drained below 1 m was used to estimate fertilizer N leaching losses. WB calculations used the mathematic model of Penman-Monteith for evapotranspiration, considering the crop coefficient equal to unity. The high N application rate associated to the high rainfall plus irrigation was found to be the main cause for leaching, which values were 14.7 and 104.5 kg ha-1 for the rates 400 and 800 kg ha-1 of N, corresponding to 3.7 and 13.1 % of the applied fertilizer, respectively.

Monitoring and determination of fungi and mycotoxins in stored Brazil nuts

Brazil nut (Bertholletia excelsa) is an important commodity from the Brazilian Amazon, and approximately 37,000 tons (3.36 × 10⁷ kg) of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs, with subsequent production of mycotoxins. In this context, the objective of the present investigation was to evaluate the presence of fungi and mycotoxins (aflatoxins and cyclopiazonic acid) in 110 stored samples of cultivated Brazil nut (55 samples of nuts and 55 samples of shells) collected monthly for 11 months in Itacoatiara, State of Amazonas, Brazil. The samples were inoculated in duplicate onto Aspergillus flavus and Aspergillus parasiticus agar and potato dextrose agar for the detection of fungi, and the presence of mycotoxins was determined by high-performance liquid chromatography. The most prevalent fungi in nuts and shells were Aspergillus spp., Fusarium spp., and Penicillium spp. A polyphasic approach was used for identification of Aspergillus species. Aflatoxins and cyclopiazonic acid were not detected in any of the samples analyzed. The low water activity of the substrate was a determinant factor for the presence of fungi and the absence of aflatoxin in Brazil nut samples. The high frequency of isolation of aflatoxigenic Aspergillus section Flavi strains, mainly A. flavus, and their persistence during storage increase the chances of aflatoxin production on these substrates and indicates the need for good management practices to prevent mycotoxin contamination in Brazil nuts.

Mycobiota, aflatoxins and cyclopiazonic acid in stored peanut cultivars

This study evaluated the presence of fungi and mycotoxins [aflatoxins (AFs), cyclopiazonic acid (CPA), and aspergillic acid] in stored samples of peanut cultivar Runner IAC Caiapó and cultivar Runner IAC 886 during 6 months. A total of 70 pod and 70 kernel samples were directly seeded onto Aspergillus flavus and Aspergillus parasiticus agar for fungi isolation and aspergillic acid detection, and AFs and CPA were analyzed by high-performance liquid chromatography. The results showed the predominance of Aspergillus section Flavi strains, Aspergillus section Nigri strains, Fusarium spp., Penicillium spp. and Rhizopus spp. from both peanut cultivars. AFs were detected in 11.4% of kernel samples of the two cultivars and in 5.7% and 8.6% of pod samples of the Caiapó and 886 cultivars, respectively. CPA was detected in 60.0% and 74.3% of kernel samples of the Caiapó and 886 cultivars, respectively. Co-occurrence of both mycotoxins was observed in 11.4% of kernel samples of the two cultivars. These results indicate a potential risk of aflatoxin production if good storage practices are not applied. In addition, the large number of samples contaminated with CPA and the simultaneous detection of AFs and CPA highlight the need to investigate factors related to the control and co-occurrence of these toxins in peanuts.

Elaboración de un mapa de lixiviación de nitratos mediante una metodología de acople SIG-modelo de simulación : aplicación al acuífero de La Aldea (Gran Canaria)

[ES] La contaminación difusa por nitrato constituye una de las mayores amenazas actuales para la calidad de las aguas subterráneas. De hecho, varias directivas europeas, nacionales y regionales se han legislado con el fin de minimizar el efecto de las prácticas agrarias en la contaminación de los acuíferos por nitratos. El acuífero de La Aldea (Gran Canaria, España) se ha declarado como vulnerable a la contaminación por nitrato según dichas normas. En este estudio se presenta una metodología para desarrollar el acople de un sistema de información geográfica-SIG con el modelo de simulación de nitrato GLEAMS. Esta herramienta permite calcular la cantidad de nitrato lixiviado procedente de los cultivos de tomate bajo invernadero y da la oportunidad de simular otros rangos de fertilización para minimizar el riesgo de contaminación de las aguas subterráneas. Se comprueba que la pérdida de nitrato por lixiviación en la zona a partir de dichos cultivos podía llegar a los 500 kg N/ha, casi un 62% del aportado como fertilizante mineral en un manejo tradicional. Por ello, se aconseja la aplicación de las recomendaciones de abonado incluidas en el código de buenas prácticas agrarias de Canarias o cualquier otro sistema de recomendación de abonado mineral para reducir estas pérdidas, minimizando de esta forma el riesgo de contaminación de las aguas subterráneas. ABSTRACT: Nitrate diffuse pollution is one of the main risks that affect the groundwater quality. Several european directives, national and regional guidelines have been enacted to protect the aquifers against the effect of the agricultural management practices. The “La Aldea” aquifer was declared nitrate vulnerable area following these laws. In this study a methodology was developed to link a Geographical Information System (GIS) with a nitrogen simulation model (GLEAMS) in this area. This tool allows to assess the amount of nitrate leaching that coming from the traditional nitrogen fertilization rates in greenhouses tomato crops, and gives the opportunity to simulate other fertilization rates to reduce the risk of groundwater pollution. The nitrate leaching reached to 500 kg N/ha in several zones of the study area, that represent the 62% of the nitrogen fertiliser apply in a traditional management. It was recommended the application of the Code of Good Management Practices or other recommendation system to decrease the nitrate leaching, in order to reduce the risk of groundwater pollution.

Aplicación web cuaderno de campo

[ES] El cuaderno de campo agrícola es un documento donde los productores agrariosregistran determinadas operaciones llevadas a cabo en su explotación. Esta herramienta debe ser supervisada por un técnico agrícola y sintetizar todos los requerimientos existentes en la legislación europea, nacional y autonómica en las siguientes materias: seguridad alimentaria; trazabilidad; seguridad en la aplicación de productos fitosanitarios; protección de aguas y suelos frente a la contaminación; protección de hábitats naturales; salud pública; condicionalidad. De esta manera, se garantiza que se llevan a cabo buenas prácticas agrícolas en laproducción hortofrutícola, respetando el medio ambiente y aportando confianza a los consumidores finales. Los modelos de cuaderno de campo agrícola existentes consisten en varias hojas con diferentes tablas que se cumplimentan en papel, con la dificultad que implica tanto para el agricultor en el registro de operaciones como para ser supervisado por los técnicos. El objetivo del presente trabajo es realizar una aplicación web que facilite al agricultor y a los técnicos agrícolas la gestión y supervisión del cuaderno de campo.

In vitro generation and characterization of acute myeloid leukemia-reactive CD8 + cytotoxic T-lymphocyte clones from healthy donors

Donor-derived CD8+ cytotoxic T lymphocytes (CTLs) eliminating host leukemic cells mediate curative graft-versus-leukemia (GVL) reactions after allogeneic hematopoietic stem cell transplantation (HSCT). The leukemia-reactive CTLs recognize hematopoiesis-restricted or broadly expressed minor histocompatibility and leukemia-associated peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on recipient cells. The development of allogeneic CTL therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of current techniques for generating leukemia-reactive CTLs from unprimed healthy donors in vitro. In this work, a novel allogeneic mini-mixed lymphocyte/leukemia culture (mini-MLLC) approach was established by stimulating CD8+ T cells isolated from peripheral blood of healthy donors at comparably low numbers (i.e. 10e4/well) with HLA class I-matched primary AML blasts in 96-well microtiter plates. Before culture, CD8+ T cells were immunomagnetically separated into CD62L(high)+ and CD62L(low)+/neg subsets enriched for naive/central memory and effector memory cells, respectively. The application of 96-well microtiter plates aimed at creating multiple different responder-stimulator cell compositions in order to provide for the growth of leukemia-reactive CTLs optimized culture conditions by chance. The culture medium was supplemented with interleukin (IL)-7, IL-12, and IL-15. On day 14, IL-12 was replaced by IL-2. In eight different related and unrelated donor/AML pairs with complete HLA class I match, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs recognized neither lymphoblastoid B cell lines of donor and patient origin nor primary B cell leukemias expressing the corresponding HLA restriction element. CTLs expressed T cell receptors of single V-beta chain families, indicating their clonality. The vast majority of CTL clones were obtained from mini-MLLCs initiated with CD8+ CD62L(high)+ cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10e8-10e10 cells within six to eight weeks. The capability of mini-MLLC derived AML-reactive CTL clones to inhibit the engraftment of human primary AML blasts was investigated in the immunodeficient nonobese diabetic/severe combined immune deficient IL-2 receptor common γ-chain deficient (NOD/SCID IL2Rγnull) mouse model. The leukemic engraftment in NOD/SCID IL2Rγnull was specifically prevented if inoculated AML blasts had been pre-incubated in vitro with AML-reactive CTLs, but not with anti-melanoma control CTLs. These results demonstrate that myeloid leukemia-specific CTL clones capable of preventing AML engraftment in mice can be rapidly isolated from CD8+ CD62L(high)+ T cells of healthy donors in vitro. The efficient generation and expansion of these CTLs by the newly established mini-MLLC approach opens the door for several potential applications. First, CTLs can be used within T cell-driven antigen identification strategies to extend the panel of molecularly defined AML antigens that are recognizable by T cells of healthy donors. Second, because these CTLs can be isolated from the stem cell donor by mini-MLLC prior to transplantation, they could be infused into AML patients as a part of the stem cell allograft, or early after transplantation when the leukemia burden is low. The capability of these T cells to expand and function in vivo might require the simultaneous administration of AML-reactive CD4+ T cells generated by a similar in vitro strategy or, less complex, the co-transfer of CD8-depleted donor lymphocytes. To prepare clinical testing, the mini-MLLC approach should now be translated into a protocol that is compatible with good manufacturing practice guidelines.

Se il lavoro è l’unica risposta. Studio comparato sulle pratiche di accompagnamento al lavoro di fasce svantaggiate nelle città di Bologna e Valencia

Scopo del presente lavoro di ricerca è quello di comparare due contesti metropolitani, valenciano e bolognese, sulle pratiche di accompagnamento al lavoro rivolte a fasce svantaggiate, in particolare persone con problemi di dipendenza da sostanze psicotrope. L’indagine propone un confronto su alcune tematiche trasversali (tipologia di azioni messe in campo, organizzazione territoriale e governance, profilo degli utenti, inserimento sociale, coinvolgimento del mondo produttivo) e pone in evidenza gli elementi che ci consentono di individuare e segnalare sia delle buone pratiche trasferibili sia delle linee progettuali, partendo dunque dal presupposto che capacitare una persona significa innanzitutto offrirle congrue opportunità di scelta, nel senso seniano e come spiegato dalla stessa Nussbaum, ma soprattutto accompagnarla e sostenerla nel percorso di inserimento lavorativo e, in parallelo, sociale. Il bisogno raccolto è quello di un sostegno, motivazionale e orientativo, che segua un approccio socio educativo capace di fornire, alla persona, una risposta integrata, di unicità, capace dunque di agire sull’autonomia, sull’autostima, sull’elaborazione delle proprie esperienze di vita e lavorative, nonché su elementi anche di contesto quali la casa, le reti amicali e familiari, spesso compromesse. L’elemento distintivo che consente di agire in questa direzione è il lavoro di collaborazione tra i diversi servizi e la co-progettazione del percorso con l’utente stesso. Il tema degli inserimenti lavorativi è un argomento molto complesso che chiama in causa diversi aspetti: i mutamenti sociali e le trasformazioni del lavoro; l’emergere di nuove fasce deboli e il rischio di aggravamento delle condizioni di esclusione per le fasce deboli “tradizionali”; l’importanza del lavoro per la costruzione di percorsi identitari e di riconoscimento; l’impatto delle politiche attive sulle fasce svantaggiate e i concetti di capitazione e attivazione; il ruolo del capitale sociale e l’emergere di nuovi welfare; la rete degli attori coinvolti dal processo di inserimento e il tema della governace territoriale.

Toward cell therapy using placenta-derived cells: disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table

Among the many cell types that may prove useful to regenerative medicine, mounting evidence suggests that human term placenta-derived cells will join the list of significant contributors. In making new cell therapy-based strategies a clinical reality, it is fundamental that no a priori claims are made regarding which cell source is preferable for a particular therapeutic application. Rather, ongoing comparisons of the potentiality and characteristics of cells from different sources should be made to promote constant improvement in cell therapies, and such comparisons will likely show that individually tailored cells can address disease-specific clinical needs. The principle underlying such an approach is resistance to the notion that comprehensive characterization of any cell type has been achieved, neither in terms of phenotype nor risks-to-benefits ratio. Tailoring cell therapy approaches to specific conditions also requires an understanding of basic disease mechanisms and close collaboration between translational researchers and clinicians, to identify current needs and shortcomings in existing treatments. To this end, the international workshop entitled "Placenta-derived stem cells for treatment of inflammatory diseases: moving toward clinical application" was held in Brescia, Italy, in March 2009, and aimed to harness an understanding of basic inflammatory mechanisms inherent in human diseases with updated findings regarding biological and therapeutic properties of human placenta-derived cells, with particular emphasis on their potential for treating inflammatory diseases. Finally, steps required to allow their future clinical application according to regulatory aspects including good manufacturing practice (GMP) were also considered. In September 2009, the International Placenta Stem Cell Society (IPLASS) was founded to help strengthen the research network in this field.

Understanding the underlying mechanisms of recent Jatropha curcas L. adoption by smallholders in Kenya: A rural livelihood assessment in Bondo, Kibwezi, and Kwale districts

Between 2004 and 2007, NGOs, community based organisations and private investors promoted jatropha in Kenya with the aim of generating additional income and producing biofuel for rural development. By 2008 it became gradually evident that jatropha plantations (both mono- and intercropping) are uneconomical and risky due to competition for land and labour with food crops. Cultivation of jatropha hedges was found to have better chances of economic success and to present only little risks for the adopting farmers. Still, after 2008 a number of farmers went on adopting jatropha in plots rather than as hedges. It is hypothesised that lack of awareness about the low economic prospects of jatropha plantations was the main reason for continued adoption, and that smallholder farmers with higher resource endowments mainly ventured into its cultivation. In this study we provide an empirical basis for understanding the role of households' capital assets in taking up new livelihood strategies by smallholder farmers in three rural districts in Kenya. For that purpose, we assess the motivation and enabling factors that led to the adoption of jatropha as a new livelihood strategy, as well as the context in which promotion and adoption took place. A household survey was conducted in 2010, using a structured questionnaire, to collect information on household characteristics and capital asset endowment. Data were analysed using descriptive statistics and non-parametric statistical tests. We established that access to additional income and own energy supply were the main motivation for adoption of jatropha, and that financial capital assets do not necessarily have a positive influence on adoption as hypothesised. Further, we found that the main challenges that adopting farmers faced were lack of access to information on good management practices and lack of a reliable market. We conclude that continued adoption of on-farm jatropha after 2008 is a result of lacking awareness about the low economic value of this production type. We recommend abandoning on-farm production of jatropha until improved seed material and locally adapted agronomic knowledge about jatropha cultivation becomes available and its production becomes economically competitive.

The cyclotron laboratory and the RFQ accelerator in Bern

Two proton accelerators have been recently put in operation in Bern: an 18 MeV cyclotron and a 2 MeV RFQ linac. The commercial IBA 18/18 cyclotron, equipped with a specifically conceived 6 m long external beam line ending in a separate bunker, will provide beams for routine 18-F and other PET radioisotope production as well as for novel detector, radiation biophysics, radioprotection, radiochemistry and radiopharmacy developments. The accelerator is embedded into a complex building hosting two physics laboratories and four Good Manufacturing Practice (GMP) laboratories. This project is the result of a successful collaboration between the Inselspital, the University of Bern and private investors, aiming at the constitution of a combined medical and research centre able to provide the most cutting-edge technologies in medical imaging and cancer radiation therapy. The cyclotron is complemented by the RFQ with the primary goals of elemental analysis via Particle Induced Gamma Emission (PIGE), and the detection of potentially dangerous materials with high nitrogen content using the Gamma-Resonant Nuclear Absorption (GRNA) technique. In this context, beam instrumentation devices have been developed, in particular an innovative beam profile monitor based on doped silica fibres and a setup for emittance measurements using the pepper-pot technique. On this basis, the establishment of a proton therapy centre on the campus of the Inselspital is in the phase of advanced study.

On the Dark Side of Therapies with Immunoglobulin Concentrates: The Adverse Events

Therapy by human immunoglobulin G (IgG) concentrates is a success story ongoing for decades with an ever increasing demand for this plasma product. The success of IgG concentrates on a clinical level is documented by the slowly increasing number of registered indication and the more rapid increase of the off-label uses, a topic dealt with in another contribution to this special issue of Frontiers in Immunology. A part of the success is the adverse event (AE) profile of IgG concentrates which is, even at life-long need for therapy, excellent. Transmission of pathogens in the last decade could be entirely controlled through the antecedent introduction by authorities of a regulatory network and installing quality standards by the plasma fractionation industry. The cornerstone of the regulatory network is current good manufacturing practice. Non-infectious AEs occur rarely and mainly are mild to moderate. However, in recent times, the increase in frequency of hemolytic and thrombotic AEs raised worrying questions on the possible background for these AEs. Below, we review elements of non-infectious AEs, and particularly focus on hemolysis and thrombosis. We discuss how the introduction of plasma fractionation by ion-exchange chromatography and polishing by immunoaffinity chromatographic steps might alter repertoire of specificities and influence AE profiles and efficacy of IgG concentrates.

Clinical trials, informed consent, & emergency medicine: A systematic literature review

Background: In the United States, the Food and Drug Administration (FDA) regulates clinical trials. These regulations address good clinical practices as well as human subject protection (FDA, 2012). One of the most important legal and ethical concerns in clinical trials is informed consent. 21 CFR 50 governs human subjects research. Part 50.24 provides an emergency research exception to the informed consent requirement. Research was conducted to determine the appropriateness of this exception, whether the benefit justifies the exception, and its public health significance.^ Methods: A systematic literature review was conducted and articles were identified from peer-reviewed journals.^ Results: There is some variance in opinions regarding the appropriateness of the exception, but the literature reviewed found the study results of these trials justified the waiver.^ Conclusion: The exception to the informed consent requirement is likely appropriate and justified in emergency research when implemented within the specified guidelines.^

Estrategias para el diseño de un sistema de gestión ambiental en una bodega : (Mendoza, Argentina)

En los últimos tiempos se ha fomentado, a través de distintas normas, la protección del medio ambiente como parte integrante de las diferentes actividades y políticas tanto del sector público como privado, con el propósito de lograr un desarrollo equilibrado y sostenible, compatible con el actual modelo económico. En función de ello, en una bodega emplazada en Maipú (Mendoza, Argentina) se realizó un diagnóstico (Revisión Inicial Ambiental - RIA) para conocer la situación actual del establecimiento con respecto a su relación con el medio ambiente. Una vez recabada la información y concluido el diagnóstico, se planteó un plan de medidas de gestión ambiental. Dichas medidas, que incorporaron los nuevos conceptos de producción limpia, ecoeficiencia y buenas prácticas ambientales, fueron documentadas a través de registros y permitieron conocer el desempeño ambiental a partir de los indicadores propuestos para cada uno de los Puntos Críticos ambientales (PCA). La propuesta inicial del presente trabajo fue lograr que la implementación de las medidas de acción, junto con los registros e indicadores de desempeño ambiental, sirvieran de soporte decisorio y brindaran los lineamientos básicos para diseñar un Sistema de Gestión Ambiental (SGA) que, junto con las Auditorías Ambientales (AA), formaran parte de la propuesta final.