Análisis por técnicas morfológicas y secuenciación de ADN del polen atmosférico de la Comunidad de Madrid: estudios preliminares. Morphological analysis and DNA sequencing of atmospheric pollen in Madrid region: preliminary study

Hasta el momento, el estudio de las partículas biológicas en el aire que respiramos, se ha dirigido, principalmente, al conocimiento y control del polen y esporas, aeroalérgenos cuyo impacto en salud es bien conocido. Recientemente la comunidad científica ha sugerido que el aire es un ecosistema en sí mismo, que tendría su propia “aerobiota”, compuesta principalmente por virus, bacterias, esporas de hongos y polen. Para estudiar en conjunto toda esta biodiversidad en el aire urbano en la Comunidad de Madrid, surge el consorcio pluridisciplinar AIRBIOTA-CM, que integra a cinco grupos de investigación de áreas muy diferentes, que pretenden obtener una visión conjunta sobre la composición y dinámica de las partículas biológicas del aire, optimizando los sistemas de muestreo y análisis. Las propuestas más novedosas de las investigaciones iniciadas por este consorcio, son la utilización de técnicas innovadoras de génetica molecular como la secuenciación masiva aplicada en metagenómica (“Next Generation Sequencing”, NGS) y el uso de nuevas estrategias de captación, como el empleo de aeronaves no tripuladas, para muestrear a diferentes alturas y en localizaciones geográficas urbanas que a priori puedan tener una composición diferente de la biota y tengan una actividad humana relevante. El proyecto se inicia en otoño de 2014, y los resultados preliminares que presentamos son los obtenidos mediante el análisis morfológico tradicional y el análisis del ADN del polen de una misma muestra procedente de un captador Burkard. Estos resultados evidencian que los captadores tipo Hirst utilizados por las redes aerobiológicas pueden emplearse también en los estudios de metagenómica, y que los datos obtenidos mediante la aplicación de ambos métodos de análisis coinciden a grandes rasgos, lo que revela que esta nueva metodología constituye una buena aproximación y posible alternativa al análisis morfológico, aunque se necesitan más estudios comparativos para adaptar bien esta tecnología. So far, the study of the biological particles in the air we breathe has been mainly directed at knowing and controlling pollen and spores, aeroallergens with a well-known health impact., It has been recently suggested that the air is an ecosystem in itself, and that it probably has its own biota, which would be composed mainly of viruses, bacteria, fungal spores, and pollen. The main objective of the AIRBIOTA-CM project is to study this diverse set of biological particles present in the urban air in the Community of Madrid using a multidisciplinary, innovative and integrative approach. The project is collaboration between five research groups in very different fields, which aim is to get an overview on the composition and dynamics of biological particles in the air to optimize the methods of sampling and analysis. As a methodological innovation, there is an attempt to apply the breakthroughs in metagenomics to the study of bioaerosols. In addition, new collection strategies have been used, such as the use of unmanned aerial vehicles by designing or adapting new samplers for these vehicles, to sample at different altitudes and in urban geographic locations that might presumably have a different composition of the biota and relevant human activity. The project started in autumn 2014. The preliminary results presented here refer to the comparison of results obtained by means of traditional (light microscopy) and metagenomics methods on atmospheric pollen in the Community of Madrid. The data obtained by both analyses coincide broadly, revealing that the molecular methodology is a good and possible alternative approach to morphological analysis, although more comparative studies to adapt well this technology are needed

Monitoring of airborne biological particles in outdoor atmosphere. Part 1: Importance, variability and ratios

The first part of this review ("Monitoring of airborne biological particles in outdoor atmosphere. Part 1: Importance, variability and ratios") describes the current knowledge on the major biological particles present in the air regarding their global distribution, concentrations, ratios and influence of meteorological factors in an attempt to provide a framework for monitoring their biodiversity and variability in such a singular environment as the atmosphere. Viruses, bacteria, fungi, pollen and fragments thereof are the most abundant microscopic biological particles in the air outdoors. Some of them can cause allergy and severe diseases in humans, other animals and plants, with the subsequent economic impact. Despite the harsh conditions, they can be found from land and sea surfaces to beyond the troposphere and have been proposed to play a role also in weather conditions and climate change by acting as nucleation particles and inducing water vapour condensation. In regards to their global distribution, marine environments act mostly as a source for bacteria while continents additionally provide fungal and pollen elements. Within terrestrial environments, their abundances and diversity seem to be influenced by the land-use type (rural, urban, coastal) and their particularities. Temporal variability has been observed for all these organisms, mostly triggered by global changes in temperature, relative humidity, et cetera. Local fluctuations in meteorological factors may also result in pronounced changes in the airbiota. Although biological particles can be transported several hundreds of meters from the original source, and even intercontinentally, the time and final distance travelled are strongly influenced by factors such as wind speed and direction

Monitoring of airborne biological particles in outdoor atmosphere. Part 2: Metagenomics applied to urban environments

The air we breathe contains microscopic biological particles such as viruses, bacteria, fungi and pollen, some of them with relevant clinic importance. These organisms and/or their propagules have been traditionally studied by different disciplines and diverse methodologies like culture and microscopy. These techniques require time, expertise and also have some important biases. As a consequence, our knowledge on the total diversity and the relationships between the different biological entities present in the air is far from being complete. Currently, metagenomics and next-generation sequencing (NGS) may resolve this shortage of information and have been recently applied to metropolitan areas. Although the procedures and methods are not totally standardized yet, the first studies from urban air samples confirm the previous results obtained by culture and microscopy regarding abundance and variation of these biological particles. However, DNA-sequence analyses call into question some preceding ideas and also provide new interesting insights into diversity and their spatial distribution inside the cities. Here, we review the procedures, results and perspectives of the recent works that apply NGS to study the main biological particles present in the air of urban environments

Cytotoxic cell populations developed during treatment with tyrosine kinase inhibitors protect autologous CD4+ T cells from HIV-1 infection

Factor de impacto: 5,858 Q1Tyrosine kinase inhibitors (TKIs) are successfully used in clinic to treat chronic myeloid leukemia (CML). Our group previously described that CD4+ T cells from patients with CML on treatment with TKIs such as dasatinib were resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral activity was primarily based on the inhibition of SAMHD1 phosphorylation, which preserves the activity against HIV-1 of this innate immune factor. Approximately 50% CML patients who achieved a deep molecular response (DMR) may safely withdraw TKI treatment without molecular recurrence. Therefore, it has been speculated that TKIs may induce a potent antileukemic response that is maintained in most patients even one year after treatment interruption (TI). Subsequent to in vitro T-cell activation, we observed that SAMHD1 was phosphorylated in CD4+ T cells from CML patients who withdrew TKI treatment more than one year earlier, which indicated that these cells were now susceptible to HIV-1 infection. Importantly, these patients were seronegative for HIV-1 and seropositive for cytomegalovirus (CMV), but without CMV viremia. Although activated CD4+ T cells from CML patients on TI were apparently permissive to HIV-1 infection ex vivo, the frequency of proviral integration was reduced more than 12-fold on average when these cells were infected ex vivo in comparison with cells isolated from untreated, healthy donors. This reduced susceptibility to infection could be related to an enhanced NK-dependent cytotoxic activity, which was increased 8-fold on average when CD4+ T cells were infected ex vivo with HIV-1 in the presence of autologous NK cells. Enhanced cytotoxic activity was also observed in CD8 + T cells from these patients, which showed 8-fold increased expression of TCRγδ and more than 18-fold increased production of IFNγ upon activation with CMV peptides. In conclusion, treatment with TKIs induced a potent antileukemic response that may also have antiviral effects against HIV-1 and CMV, suggesting that transient use of TKIs in HIV-infected patients could develop a sustained antiviral response that would potentially interfere with HIV-1 reservoir dynamics.This work was supported by NIH grant R01AI143567; the Spanish Ministry of Economy and Competitiveness (SAF2016-78480-R); the Spanish AIDS Research Network RD16CIII/0002/0001 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2016-2020, Instituto de Salud Carlos III, European Region Development Fund (ERDF). The work of María Rosa López-Huertas and Sara Rodríguez-Mora is financed by NIH grant R01AI143567. The work of Lorena Vigón is supported by a pre-doctoral grant from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER). The work of Elena Mateos is supported by the Spanish Ministry of Economy and Competitiveness SAF2016-78480-R.S

Strong Humoral but Not Cellular Immune Responses against SARS-CoV-2 in Individuals with Oncohematological Disease Who Were Treated with Rituximab before Receiving a Vaccine Booster

The humoral immune response developed after receiving the full vaccination schedule against COVID-19 is impaired in individuals who received anti-CD20 therapy 6-9 months before vaccination. However, there is little information about the cellular immune responses elicited in these individuals. In this study, we analyzed the humoral and cellular immune responses in 18 individuals with hematological disease who received the last dose of rituximab 13.8 months (IQR 9.4-19) before the booster dose. One month after receiving the booster dose, the seroconversion rate in the rituximab-treated cohort increased from 83.3% to 88.9% and titers of specific IgGs against SARS-CoV-2 increased 1.53-fold (p = 0.0098), while the levels of neutralizing antibodies increased 3.03-fold (p = 0.0381). However, the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) from rituximab-treated individuals remained unchanged, and both antibody-dependent cellular cytotoxicity (ADCC) and direct cellular cytotoxicity (CDD) were reduced 1.7-fold (p = 0.0047) and 2.0-fold (p = 0.0086), respectively, in comparison with healthy donors. Breakthrough infections rate was higher in our cohort of rituximab-treated individuals (33.33%), although most of the infected patients (83.4%) developed a mild form of COVID-19. In conclusion, our findings confirm a benefit in the humoral, but not in the cellular, immune response in rituximab-treated individuals after receiving a booster dose of an mRNA-based vaccine against COVID-19.This work was supported by the Strategic Action in Health 2017–2020 of the Instituto de Salud Carlos III (PI21/00877), by the Coordinated Research Activities at the National Center of Microbiology (CNM, Instituto de Salud Carlos III) (COV20_00679) to promote an integrated response against SARS-CoV-2 in Spain (Spanish Ministry of Science and Innovation) that is coordinated by Dr Inmaculada Casas (WHO National Influenza Center of the CNM), and by a generous donation provided by Chiesi España, S.A.U. (Barcelona, Spain). The work of Montserrat Torres is financed by the Hematology and Hemotherapy Service, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Hospital Universitario Ramón y Cajal (Madrid, Spain). The work of Sara Rodríguez-Mora is financed by NIH grant R01AI143567. The work of Guiomar Casado is financed by CIBERINFEC, co-financed by the European Regional Development Fund (FEDER) “A way to make Europe”. The work of Fernando Ramos-Martín is financed by the Spanish Ministry of Science and Innovation (PID2019-110275RB-I00).S

Early Cellular and Humoral Responses Developed in Oncohematological Patients after Vaccination with One Dose against COVID-19

Individuals with oncohematological diseases (OHD) may develop an impaired immune response against vaccines due to the characteristics of the disease or to its treatment. Humoral response against SARS-CoV-2 has been described to be suboptimal in these patients, but the quality and efficiency of the cellular immune response has not been yet completely characterized. In this study, we analyzed the early humoral and cellular immune responses in individuals with different OHD after receiving one dose of an authorized vaccine against SARS-CoV-2. Humoral response, determined by antibodies titers and neutralizing capacity, was overall impaired in individuals with OHD, except for the cohort of chronic myeloid leukemia (CML), which showed higher levels of specific IgGs than healthy donors. Conversely, the specific direct cytotoxic cellular immunity response (DCC) against SARS-CoV-2, appeared to be enhanced, especially in individuals with CML and chronic lymphocytic leukemia (CLL). This increased cellular immune response, developed earlier than in healthy donors, showed a modest cytotoxic activity that was compensated by significantly increased numbers, likely due to the disease or its treatment. The analysis of the immune response through subsequent vaccine doses will help establish the real efficacy of COVID-19 vaccines in individuals with OHD.This work was supported by the Strategic Action in Health 2017–2020 of the Instituto de Salud Carlos III (PI21/00877); the Coordinated Research Activities at the National Center of Microbiology (CNM, Instituto de Salud Carlos III) (COV20_00679) to promote an integrated response against SARS-CoV-2 in Spain (Spanish Ministry of Science and Innovation) that is coordinated by Dr Inmaculada Casas (WHO National Influenza Center of the CNM); a generous donation provided by Chiesi España, S.A.U. (Barcelona, Spain). The work of Sara Rodríguez-Mora is financed by NIH grant R01AI143567. The work of Montserrat Torres is financed by the Hematology and Hemotherapy Service of the Hospital Universitario Ramón y Cajal. The work of Fernando Ramos-Martín is financed by the Spanish Ministry of Science and Innovation (PID2019-110275RB-I00). The work of Lorena Vigón is supported by a pre-doctoral grant from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER). The work of Mario Manzanares is supported by a pre-doctoral grant from Instituto de Salud Carlos III (ISCIII-PFIS FI20CIII/00021).S

Cellular and humoral functional responses after BNT162b2 mRNA vaccination differ longitudinally between naive and subjects recovered from COVID-19

We have analyzed BNT162b2 vaccine-induced immune responses in naive subjects and individuals recovered from coronavirus disease 2019 (COVID-19), both soon after (14 days) and later after (almost 8 months) vaccination. Plasma spike (S)-specific immunoglobulins peak after one vaccine shot in individuals recovered from COVID-19, while a second dose is needed in naive subjects, although the latter group shows reduced levels all along the analyzed period. Despite how the neutralization capacity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mirrors this behavior early after vaccination, both groups show comparable neutralizing antibodies and S-specific B cell levels late post-vaccination. When studying cellular responses, naive individuals exhibit higher SARS-CoV-2-specific cytokine production, CD4+ T cell activation, and proliferation than do individuals recovered from COVID-19, with patent inverse correlations between humoral and cellular variables early post-vaccination. However, almost 8 months post-vaccination, SARS-CoV-2-specific responses are comparable between both groups. Our data indicate that a previous history of COVID-19 differentially determines the functional T and B cell-mediated responses to BNT162b2 vaccination over time.C.d.F., J.G.-P., and J.A. are supported by Instituto de Salud Carlos III (ISCII). We thank JM Ligos and Cytek Biosciences for their technical support. Research in E.L.-C.’s lab was supported by Fundación Familia Alonso, Santander Bank, Real Seguros, Fundación Mutua Madrileña, Fundación Uria, Fundación La Caixa, and Ayuntamiento de Madrid.S

Provirus reactivation is impaired in HIV-1 infected individuals on treatment with dasatinib and antiretroviral therapy

The latent viral reservoir formed by HIV-1, mainly in CD4 + T cells, is responsible for the failure of antiretroviral therapy (ART) to achieve a complete elimination of the virus in infected individuals. We previously determined that CD4 + T cells from individuals with chronic myeloid leukemia (CML) on treatment with dasatinib are resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral effect is the preservation of SAMHD1 activity. In this study, we aimed to evaluate the impact of dasatinib on the viral reservoir of HIV-infected individuals with CML who were on simultaneous treatment with ART and dasatinib. Due to the low estimated incidence of HIV-1 infection and CML (1:65,000), three male individuals were recruited in Spain and Germany. These individuals had been on treatment with standard ART and dasatinib for median 1.3 years (IQR 1.3-5.3 years). Reservoir size and composition in PBMCs from these individuals was analyzed in comparison with HIV-infected individuals on triple ART regimen and undetectable viremia. The frequency of latently infected cells was reduced more than 5-fold in these individuals. The reactivation of proviruses from these cells was reduced more than 4-fold and, upon activation, SAMHD1 phosphorylation was reduced 40-fold. Plasma levels of the homeostatic cytokine IL-7 and CD4 effector subpopulations TEM and TEMRA in peripheral blood were also reduced. Therefore, treatment of HIV-infected individuals with dasatinib as adjuvant of ART could disturb the reservoir reactivation and reseeding, which might have a beneficial impact to reduce its size

Valoración del bagazo de cerveza en la alimentación del ganado ovino

La creciente concienciación social con la protección del medio ambiente ha provocado un aumento de iniciativas para aumentar el uso de los subproductos y residuos de la industria agroalimentaria en la alimentación animal. En el sector cervecero, España es el undécimo productor a nivel mundial y el tercero de la Unión Europea. El bagazo de cerveza (BC) es el subproducto más abundante en la elaboración de la cerveza, produciéndose en España unas 800000 toneladas al año. Este trabajo se centra en la valoración nutritiva del BC y su posible inclusión en dietas para rumiantes de leche y de cebo. Se usaron un total de 22 muestras de BC (14 de BC fresco, 5 de BC seco y 3 de BC seco y granulado) proporcionadas por distintas empresas cerveceras y granjas de España, a las que se analizó la composición química, la producción de gas y los parámetros fermentativos mediante incubaciones in vitro con líquido ruminal de ovejas. Los resultados indicaron que tanto la composición química como los parámetros de producción de gas y fermentativos presentan una alta variabilidad entre BC. El secado a baja temperatura del BC no provocó cambios importantes en su fermentación ruminal, pero aumentó (P=0,040) la producción total de ácidos grasos volátiles (AGV) mientras que el secado y posterior granulado redujo la degradabilidad de la proteína (P=0,028) y aumentó la producción total de AGV (P=0,011). También se formularon dietas isoproteicas e isoenergéticas para rumiantes de leche y cebo que contenían cantidades crecientes de BC seco. Las dietas de leche contenían 0, 6, 12 y 18 % de BC (en materia fresca) en sustitución parcial de heno de alfalfa, harina de soja y DDGS de maíz. A partir de la inclusión de un 12% de BC se observó una disminución lineal en la producción de gas (P = 0,003) y de la concentración NH3N tras 8 h de incubación (P=0,011). En las dietas de cebo se sustituyó parcialmente maíz y harina de soja por 10, 20 y 30% de BC seco (en materia fresca). Se observó una reducción lineal (P<0,001) en la producción de gas a partir de la inclusión de un 10% de BC, pero la máxima producción total de AGV se logró en las dietas con 10 y 20% de BC. A nivel económico, la conveniencia de la inclusión de BC dependerá del coste de secado y del precio de los ingredientes a los que sustituya. Hay que destacar la necesidad de analizar correctamente cada lote de BC antes de su utilización práctica, dada la alta variablidad observada ABSTRACT Growing social awareness of environmental protection has led to an increase in initiatives to increase the use of by-products and waste from the agri-food industry in animal feeding. In the brewing sector, Spain is the 11th largest producer worldwide and the third largest in the European Union. Brewers grains (BC) is the most common by-product generated in the brewing process, being produced in Spain around 800,000 tons per year. This work will focus on the nutritional assessment of BC samples generated in Spain and on the evaluation of its possible inclusion in diets for dairy and fattening ruminants. A total of 22 BC samples (14 fresh BC, 5 dried BC and 3 dried and granulated BC) provided by different brewing companies and livestock farms in Spain were used and analysed for chemical composition, gas production and fermentative parameters by in vitro incubations with sheep rumen fluid. The results indicated that chemical composition and both gas production and fermentative parameters show high variability between BC samples. Low temperature drying did not result in significant changes in rumen fermentation of BC, but increased total volatile fatty acids (VFA) production (P=0.040), whereas drying and pelleting resulted in reduced protein degradability (P=0.028) and increased total VFA production (P=0.011). Isoproteic and isoenergetic diets including increased amounts of BC were formulated for dairy and fattening ruminants. Dairy diets included 0, 6, 12 or 18% of dry BC (fresh matter basis) replacing partially alfalfa hay, soybean meal and corn DDGS. Including BC in dairy diets resulted in a linear decrease in gas production (P = 0.003) and NH3N concentration (P = 0.011) after 8 h of incubation, but did not affect AGV production. In the fattening diets, corn and soybean meal were partially replaced by 10, 20 and 30 % of dry BC (fresh matter basis). There was a linear reduction (P<0.001) in gas production with the inclusion of 10% of BC, but maximum total VFA production was achieved with the inclusion of 10 and 20% of BC. The economic convenience of the BC use will depend on the cost of drying and the price of the feedstuffs being replaced in the diet. However, it has to be highlighted the need of correctly analysing each batch of BC before its practical use because of the high variability observed

Concurrence of iridovirus, polyomavirus, and a unique member of a new group of fish papillomaviruses in lymphocystis disease-affected gilthead sea bream

Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought.Ministerio de Economía y Competitividad (RYC-2010-06300).Peer Reviewe