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A comparative analysis of toxicities of both arsenic forms (arsenite and arsenate) in the model eukaryotic microorganism Tetrahymena thermophila (ciliate protozoa) has shown the presence of various detoxification mechanisms and cellular effects comparable to those of animal cells under arsenic stress. In the wild type strain SB1969 arsenate is almost 2.5 times more toxic than arsenite. According to the concentration addition model used in binary metallic mixtures their toxicities show an additive effect. Using fluorescent assays and flow cytometry, it has been detected that As(V) generates elevated levels of ROS/RNS compared to As(III). Both produce the same levels of superoxide anion, but As(V) also causes greater increases in hydrogen peroxide and peroxynitrite. The mitochondrial membrane potential is affected by both As(V) and As(III), and electron microscopy has also revealed that mitochondria are the main target of both arsenic ionic forms. Fusion/fission and swelling mitochondrial and mitophagy, together with macroautophagy, vacuolization and mucocyst extruction are mainly associated to As(V) toxicity, while As(III) induces an extensive lipid metabolism dysfunction (adipotropic effect). Quantitative RT-PCR analysis of some genes encoding antioxidant proteins or enzymes has shown that glutathione and thioredoxin metabolisms are involved in the response to arsenic stress. Likewise, the function of metallothioneins seems to be crucial in arsenic detoxification processes, after using both metallothionein knockout and knockdown strains and cells overexpressing metallothionein genes from this ciliate. The analysis of the differential toxicity of As(III) and As(V) shown in this study provides cytological and molecular tools to be used as biomarkers for each of the two arsenic ionic forms.

Bluetongue virus (BTV) is an arbovirus transmitted by the bite of infected Culicoides midges that affects domestic and wild ruminants producing great economic losses. The infection induces an IFN response, followed by an adaptive immune response that is essential in disease clearance. BTV can nonetheless impair IFN and humoral responses. The main goal of this study was to gain a more detailed understanding of BTV pathogenesis and its effects on immune cell populations. To this end, we combined flow cytometry and transcriptomic analyses of several immune cells at different times post-infection (pi). Four sheep were infected with BTV serotype 8 and blood samples collected at days 0, 3, 7 and 15pi to perform transcriptomic analysis of B-cell marker+, CD4+, CD8+, and CD14+ sorted peripheral mononuclear cells. The maximum number of differentially expressed genes occurred at day 7pi, which coincided with the peak of infection. KEGG pathway enrichment analysis indicated that genes belonging to virus sensing and immune response initiation pathways were enriched at day 3 and 7 pi in all 4 cell population analyzed. Transcriptomic analysis also showed that at day 7pi T cell exhaustion pathway was enriched in CD4+ cells, while CD8+ cells downregulated immune response initiation pathways. T cell functional studies demonstrated that BTV produced an acute inhibition of CD4+ and CD8+ T cell activation at the peak of replication. This coincided with PD-L1 upregulation on the surface of CD4+ and CD8+ T cells as well as monocytes. Taken together, these data indicate that BTV could exploit the PD1/PD-L1 immune checkpoint to impair T cell responses. These findings identify several mechanisms in the interaction between host and BTV, which could help develop better tools to combat the disease.

Arsenic (As) is quite an abundant metalloid, with ancient origin and ubiquitous distribution, which represents a severe environmental risk and a global problem for public health. Microbial exposure to As compounds in the environment has happened since the beginning of time. Selective pressure has induced the evolution of various genetic systems conferring useful capacities in many microorganisms to detoxify and even use arsenic, as an energy source. This review summarizes the microbial impact of the As biogeochemical cycle. Moreover, the poorly known adverse effects of this element on eukaryotic microbes, as well as the As uptake and detoxification mechanisms developed by yeast and protists, are discussed. Finally, an outlook of As microbial remediation makes evident the knowledge gaps and the necessity of new approaches to mitigate this environmental challenge.

SARS-CoV-2 vaccines currently in use have contributed to controlling the COVID-19 pandemic. Notwithstanding, the high mutation rate, fundamentally in the spike glycoprotein (S), is causing the emergence of new variants. Solely utilizing this antigen is a drawback that may reduce the efficacy of these vaccines. Herein we pre ent a DNA vaccine candidate that contains the genes encoding the S and the nucleocapsid (N) proteins implemented into the nonreplicative mammalian expression plasmid vector, pPAL. This plasmid lacks antibiotic resistance genes and contains an alternative selectable marker for production. The S gene sequence was modified to avoid furin cleavage (Sfs). Potent humoral and cellular immune responses were observed in C57BL/6J mice vaccinated with pPAL-Sfs + pPAL-N following a prime/boost regimen by the intramuscular route applying in vivo electroporation. The immunogen fully protected K18-hACE2 mice against a lethal dose (105 PFU) of SARS-CoV-2. Viral replication was completely controlled in the lungs, brain, and heart of vaccinated mice. Therefore, pPAL-Sfs + pPAL-N is a promising DNA vaccine candidate for protection from COVID-19., PTI-Salud Global (CSIC), Center for Technological and Industrial Development (CDTI), REACT-ANTICIPA-UCM (Comunidad de Madrid), European Research Council (Advanced Grant VERDI, ERC2015AdG grant number 694160), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

El virus de la peste de los pequeños rumiantes (PPRV) es un morbillivirus, familia Paramyxoviridae, estrechamente relacionado con los virus del sarampión y la peste bovina. Es el agente causal de la peste de los pequeños rumiantes (PPR), una enfermedad de grave impacto económico que afecta en especial a los países en vías de desarrollo con gran dependencia del ganado ovino y caprino. Las células dendríticas (DC) son células presentadoras de antígeno profesionales, que actúan como nexo entre la inmunidad innata y la inmunidad adaptativa. Son responsables del desarrollo de una correcta respuesta inmune frente a los patógenos y, a la vez, diana del virus. Nuestro objetivo fue evaluar si la infección con PPRV podía alterar la funcionalidad de las DC, obstaculizando el desarrollo de una respuesta inmune adaptativa eficaz frente al virus. Las DC pueden encontrarse principalmente en dos estados funcionales generales, inmaduro y maduro, por lo que se estudió el efecto de la infección por PPRV en ambas etapas. Las células CD14+ (monocitos) seleccionadas positivamente a partir de células mononucleares de sangre periférica (PBMC) se diferenciaron a DC derivadas de monocito inmaduras (iMoDC) con GM-CSF e IL-4. Las iMoDC se maduraron a DC derivadas de monocito maduras (mMoDC) mediante la transfección de Poly I:C. La infección con PPRV modificó la expresión de marcadores clave en las DC, tales como MHC-I, MHC-II, CD80,CD86, CD14, CD209, CD11b y CD11c, tanto en iMoDC como en mMoDC. La funcionalidad de las MoDC también se vio alterada por PPRV: la capacidad fagocítica de las iMoDC infectadas estaba reducida, mientras que las mMoDC infectadas eran incapaces de estimular linfocitos T...

El virus de la peste de los pequeños rumiantes (PPRV) es un morbillivirus, familia Paramyxoviridae, estrechamente relacionado con los virus del sarampión y la peste bovina. Es el agente causal de la peste de los pequeños rumiantes (PPR), una enfermedad de grave impacto económico que afecta en especial a los países en vías de desarrollo con gran dependencia del ganado ovino y caprino. Las células dendríticas (DC) son células presentadoras de antígeno profesionales, que actúan como nexo entre la inmunidad innata y la inmunidad adaptativa. Son responsables del desarrollo de una correcta respuesta inmune frente a los patógenos y, a la vez, diana del virus. Nuestro objetivo fue evaluar si la infección con PPRV podía alterar la funcionalidad de las DC, obstaculizando el desarrollo de una respuesta inmune adaptativa eficaz frente al virus. Las DC pueden encontrarse principalmente en dos estados funcionales generales, inmaduro y maduro, por lo que se estudió el efecto de la infección por PPRV en ambas etapas. Las células CD14+ (monocitos) seleccionadas positivamente a partir de células mononucleares de sangre periférica (PBMC) se diferenciaron a DC derivadas de monocito inmaduras (iMoDC) con GM-CSF e IL-4. Las iMoDC se maduraron a DC derivadas de monocito maduras (mMoDC) mediante la transfección de Poly I:C. La infección con PPRV modificó la expresión de marcadores clave en las DC, tales como MHC-I, MHC-II, CD80,CD86, CD14, CD209, CD11b y CD11c, tanto en iMoDC como en mMoDC. La funcionalidad de las MoDC también se vio alterada por PPRV: la capacidad fagocítica de las iMoDC infectadas estaba reducida, mientras que las mMoDC infectadas eran incapaces de estimular linfocitos T..., Peste des Petits Ruminants virus (PPRV) is a Morbillivirus, Paramyxoviridae family, closely related to measles and rinderpest virus. It is the causative agent of Peste des Petits Ruminants (PPR), an economically important disease, which severely impacts developing countries with heavy dependence on sheep and goat livestock. Dendritic cells (DC), which are professional antigen presenting cells that act as a bridge between the innate and the adaptive immunity to trigger a correct immune response to pathogens, are among the immune cells targeted by the virus. Our goal was to assess whether PPRV infection could alter DC functionality, hindering the development of an efficient adaptive immune response against the virus. Since DC primarily exist in two basic functional states, immature and mature, we studied the effects of PPRV infection in both cell phases. CD14+ cells (monocytes) positively selected from Peripheral Blood Mononuclear Cells (PBMC) were differentiated into immature monocyte-derived DC (iMoDC) with GM-CSF and IL-4. iMoDC were matured into mature monocyte-derived DC (mMoDC) by Poly I:C transfection. Expression of key DC markers MHCI, MHC-II, CD80, CD86, CD14, CD209, CD11b and CD11c was affected by PPRV infection in iMoDC and mMoDC. PPRV infection also altered MoDC functionality: phagocytic activity of infected iMoDC was reduced, while infected mMoDC were unable to stimulate T cells...

SARS-CoV-2 vaccines currently in use have contributed to controlling the COVID-19 pandemic. Notwithstanding, the high mutation rate, fundamentally in the spike glycoprotein (S), is causing the emergence of new variants. Solely utilizing this antigen is a drawback that may reduce the efficacy of these vaccines. Herein we present a DNA vaccine candidate that contains the genes encoding the S and the nucleocapsid (N) proteins implemented into the non-replicative mammalian expression plasmid vector, pPAL. This plasmid lacks antibiotic resistance genes and contains an alternative selectable marker for production. The S gene sequence was modified to avoid furin cleavage (Sfs). Potent humoral and cellular immune responses were observed in C57BL/6J mice vaccinated with pPAL-Sfs + pPAL-N following a prime/boost regimen by the intramuscular route applying in vivo electroporation. The immunogen fully protected K18-hACE2 mice against a lethal dose (105 PFU) of SARS-CoV-2. Viral replication was completely controlled in the lungs, brain, and heart of vaccinated mice. Therefore, pPAL-Sfs + pPAL-N is a promising DNA vaccine candidate for protection from COVID-19.

Viruses have evolved numerous strategies to impair immunity so that they can replicate more efficiently. Among those, the immunosuppressive effects of morbillivirus infection can be particularly problematic, as they allow secondary infections to take hold in the host, worsening disease prognosis. In the present work, we hypothesized that the highly contagious morbillivirus peste des petits ruminants virus (PPRV) could target monocytes and dendritic cells (DC) to contribute to the immunosuppressive effects produced by the infection. Monocytes isolated from healthy sheep, a natural host of the disease, were able be infected by PPRV and this impaired the differentiation and phagocytic ability of immature monocyte-derived DC (MoDC). We also assessed PPRV capacity to infect differentiated MoDC. Ovine MoDC could be productively infected by PPRV, and this drastically reduced MoDC capacity to activate allogeneic T cell responses. Transcriptomic analysis of infected MoDC indicated that several tolerogenic DC signature genes were upregulated upon PPRV infection. Furthermore, PPRV-infected MoDC could impair the proliferative response of autologous CD4+ and CD8+ T cell to the mitogen concanavalin A (ConA), which indicated that DC targeting by the virus could promote immunosuppression. These results shed new light on the mechanisms employed by morbillivirus to suppress the host immune responses. IMPORTANCE Morbilliviruses pose a threat to global health given their high infectivity. The morbillivirus peste des petits ruminants virus (PPRV) severely affects small-ruminant-productivity and leads to important economic losses in communities that rely on these animals for subsistence. PPRV produces in the infected host a period of severe immunosuppression that opportunistic pathogens exploit, which worsens the course of the infection. The mechanisms of PPRV immunosuppression are not fully understood. In the present work, we demonstrate that PPRV can infect professional antigen-present

In the past decade, the use of wearable medical devices has been a great breakthrough in clinical practice, trials, and research. In the Parkinson's disease field, clinical evaluation is time limited, and healthcare professionals need to rely on retrospective data collected through patients' self-filled diaries and administered questionnaires. As this often leads to inaccurate evaluations, a more objective system for symptom monitoring in a patient's daily life is claimed. In this regard, the use of wearable medical devices is crucial. This study aims at presenting a review on STAT-ONTM, a wearable medical device Class IIa, which provides objective information on the distribution and severity of PD motor symptoms in home environments. The sensor analyzes inertial signals, with a set of validated machine learning algorithms running in real time. The device was developed for 12 years, and this review aims at gathering all the results achieved within this time frame. First, a compendium of the complete journey of STAT-ONTM since 2009 is presented, encompassing different studies and developments in funded European and Spanish national projects. Subsequently, the methodology of database construction and machine learning algorithms design and development is described. Finally, clinical validation and external studies of STAT-ONTM are presented., Peer Reviewed, Postprint (published version)

Members of the tumour necrosis factor (TNF) superfamily OX40L and CD70 and their receptors are costimulating signalling axes critical for adequate T cell activation in humans and mice but characterisation of these molecules in other species including ruminants is lacking. Here we cloned and expressed the predicted ovine orthologues of the receptors OX40 and CD27, as well as soluble recombinant forms of their potential ovine ligands, OaOX40L and OaCD70. Using biochemical and immunofluorescence analyses, we show that both signalling axes are functional in sheep. We show that oligomeric recombinant ligand constructs are able to induce signalling through their receptors on transfected cells. Recombinant defective human adenoviruses were constructed to express the soluble forms of OaOX40L and OaCD70. Both proteins were detected in the supernatant of adenovirus-infected cells and shown to activate NF-B signalling pathway through their cognate receptor. These adenovirus-secreted OaOX40L and OaCD70 forms could also activate ovine T cell proliferation and enhance IFN- production in CD4+ and CD8+ T cells. Altogether, this study provides the first characterisation of the ovine costimulatory OX40L-OX40 and CD70-CD27 signalling axes, and indicates that their activation in vivo may be useful to enhance vaccination-induced immune responses in sheep and other ruminants.