Conjunctival vaccination against Brucella ovis in mice with mannosylated nanoparticles

The use of sub-unit vaccines can solve some drawbacks associated with traditional attenuated or inactivated ones. However, in order to improve their immunogenicity, these vaccines needs to be associated to an appropriate adjuvant which, adequately selected, may also offer an alternative pathway for administration. The aim of this work was to evaluate the protection offered by the hot saline complex extracted from Brucella ovis (HS) encapsulated in mannosylated nanoparticles (MAN-NP-HS) when instilled conjunctivally in mice. Nanoparticles displayed a size of 300 nm and the antigen loading was close to 30 μg per mg nanoparticle. Importantly, encapsulated HS maintained its protein profile, structural integrity and antigenicity during and after the preparative process of nanoparticles. The ocular immunization was performed on BALB/c mice. Eight weeks after vaccination animals were challenged with B. ovis, and 3 weeks later, were slaughtered for bacteriological examinations. Animals immunized with MAN-NP-HS displayed a 3-log reduction in spleen CFU compared with unvaccinated animals. This degree of protection was significantly higher than that observed for the commercial vaccine (Rev1) subcutaneously administered. Interestingly, the mucosal IgA response induced by MAN-NP-HS was found to be much more intense than that offered by Rev1 and prolonged in time. Furthermore, the elicited IL-2, IL-4 and γ-IFN levels showed good correlation with the degree of protection. On the other hand, biodistribution studies in animals were performed with nanoparticles labelled with either 99mtechnetium or rhodamine B isothiocyanate. The biodistribution revealed that, after instillation, MAN-NP-HS moved from the palpebral area to the nasal region and, the gastrointestinal tract. This profile of distribution was different to that observed for free 99mTcO4− colloids, which remained for at least 24 h in the site of administration. In summary, mannosylated nanoparticles appear to be a safe and suitable adjuvant for conjunctival vaccination

Radiomarcaje y estudios de biodistribución de nanopartículas poliméricas como adyuvantes para la vacunación oftálmica frente a la brucelosis

Objetivos: Optimizar el radiomarcaje con 99mTc de nanopartículas de Gantrez® manosiladas y cargadas con el antígeno de Brucella Ovis (Man-NP-HS) y llevar a cabo estudios de biodistribución en ratón tras la administración de las nanopartículas por vía ocular. Metodología: Las Man-NP-HS se obtuvieron por el método de desplazamiento de disolvente. Se purificaron, liofilizaron y caracterizaron. A continuación, se marcaron con 74 MBq de 99mTcO4 - previamente reducido con una disolución ácida de cloruro de estaño, trabajando en ausencia de oxígeno y con un pH final de 4. El rendimiento del marcaje se evaluó mediante TLC. Los estudios de biodistribución se llevaron a cabo en ratones tras la administración oftálmica de la formulación y de un control de 99mTcO4 - libre. Para ello, se sacrificaron los animales a las 2 y a las 24 horas tras la administración ocular y se contaron los órganos en un contador gamma. Resultados: Se obtuvo un rendimiento de marcaje superior al 90%. Los estudios de biodistribución de 99mTc-Man-NP-HS permitieron detectar la actividad concentrada en mucosa nasal y ocular y tracto gastrointestinal tanto a las 2 como a las 24 horas, frente a la biodistribución de 99mTcO4 - libre que permaneció concentrado en la piel alrededor del ojo y en tracto gastrointestinal. Conclusión: Los estudios de biodistribución de 99mTc-Man-NP-HS tras administración oftálmica han permitido demostrar su biodistribución en mucosas y tracto gastrointestinal, característica indispensable como sistema de liberación de antígenos a través de mucosa ocular. Esto, junto con su elevada respuesta inmune, efectiva protección y no virulencia, convierte a estas nanopartículas en una vacuna ideal anti Brucelosis

PET tracers for clinical imaging of breast cancer

Molecular imaging of breast cancer has undoubtedly permitted a substantial development of the overall diagnostic accuracy of this malignancy in the last years. Accurate tumour staging, design of individually suited therapies, response evaluation, early detection of recurrence and distant lesions have also evolved in parallel with the development of novel molecular imaging approaches. In this context, positron emission tomography (PET) can be probably seen as the most interesting molecular imaging technology with straightforward clinical application for such purposes. Dozens of radiotracers for PET imaging of breast cancer have been tested in laboratory animals. However, in this review we shall focus mainly in the smaller group of PET radiopharmaceuticals that have lead through into the clinical setting. PET imaging can be used to target general metabolic phenomena related to tumoural transformation, including glucose metabolism and cell proliferation, but can also be directed to specific hormone receptors that are characteristic of the breast cancer cell. Many other receptors and transport molecules present in the tumour cells could also be of interest for imaging. Furthermore, molecules related with the tumour microenvironment, tumour induced angiogenesis or even hypoxia could also be used as molecular biomarkers for breast cancer imaging

Dissecting the role of the NADPH oxidase NOX4 in TGF-beta signaling in hepatocellular carcinoma

The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-I3) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-I3-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-I3. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-I3, whereas non-canonical signals, such as the phos-phorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-I3-mediated in-hibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-I3 in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-I31-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-I3 that may contribute to tumor progression, we found that NOX4 is also required for TGF-I3-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-I3-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-I3-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-I3 signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-I3 pathway

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Radiofármacos PET

Dada la tasa actual de evolución de la biología molecular de los receptores del SNC, la diferencia entre el número de radiotrazadores disponibles y las drogas potenciales va creciendo rápidamente99. Debemos por tanto insistir en que la utilidad de la tecnología PET en la búsqueda de nuevos agentes terapéuticos descansa sin duda en la obtención de compuestos marcados, por lo que la potenciación de la radioquímica y la radiofarmacia van a ser puntos clave en el desarrollo de este apasionante campo

Radiomarcaje y estudios de biodistribución de nanopartículas poliméricas como adyuvantes para vacunación oftálmica frente a brucelosis

Objetivos Optimizar el radiomarcaje con 99mTc de nanopartículas de Gantrez® manosiladas y cargadas con el antígeno de Brucella Ovis (Man-NP-HS) y llevar a cabo estudios de biodistribución en ratón tras la administración de las nanopartículas por vía ocular. Metodología Las Man-NP-HS se obtuvieron por el método de desplazamiento de disolvente. Se purificaron, liofilizaron y caracterizaron. A continuación, se marcaron con 74 MBq de 99mTcO4− previamente reducido con una disolución ácida de cloruro de estaño, trabajando en ausencia de oxígeno y con un pH final de 4. El rendimiento del marcaje se evaluó mediante TLC. Los estudios de biodistribución se llevaron a cabo en ratones tras la administración oftálmica de la formulación y de un control de 99mTcO4− libre. Para ello, se sacrificaron los animales a las 2 y a las 24 horas tras la administración ocular y se contaron los órganos en un contador gamma. Resultados Se obtuvo un rendimiento de marcaje superior al 90%. Los estudios de biodistribución de 99mTc-Man-NP-HS permitieron detectar la actividad concentrada en mucosa nasal y ocular y tracto gastrointestinal tanto a las 2 como a las 24 horas, frente a la biodistribución de 99mTcO4− libre que permaneció concentrado en la piel alrededor del ojo y en tracto gastrointestinal. Conclusión Los estudios de biodistribución de 99mTc-Man-NP-HS tras administración oftálmica han permitido demostrar su biodistribución en mucosas y tracto gastrointestinal, característica indispensable como sistema de liberación de antígenos a través de mucosa ocular. Esto, junto con su elevada respuesta inmune, efectiva protección y no virulencia, convierte a estas nanopartículas en una vacuna ideal anti Brucelosis

Effects of nursery shading on seedling quality and post-planting performance in two Mediterranean species with contrasting shade tolerance

In Mediterranean climates, seedlings are frequently shaded in the nursery to avoid heat damage and save water. However, the impact of this shading on the seedling quality and transplanting performance of Mediterranean species is not well known. We studied the effect of nursery shading on pre-planting features and post-planting performance of two Mediterranean tree species: the shade-intolerant pioneer Pinus halepensis and the shade-tolerant late-successional Quercus ilex. We grew one-year-old seedlings of both species under 100, 40 and 5% full sunlight. Shade had a low impact on the morphology and physiology of Q. ilex seedlings. In pines, only the deep shade treatment produced low quality seedlings with poor root development. In both species, transference to high light at planting in autumn did not impose any additional stress than that caused by frosts, but initial root growth was impaired in the two shaded treatments in pine. Post-planting growth and survival of oak seedlings showed no difference between treatments. Pine seedlings grown in deep shade showed higher mortality and lower growth after planting than those grown in full sun and intermediate light treatments, while intermediate light only reduced growth. For the nursery culture of Q. ilex seedlings, we advise using low light levels during summer to save water without impairing field performance. In P. halepensis, seedlings should be cultured under full sunlight conditions to maximize post-planting growth, but they can be cultured under intermediate light without impairing survival