A Holocene record of environmental change from Río Zeballos, Central Patagonia

There are few continuous palaeoenvironmental records spanning the Holocene in Andean Southern Patagonia near the Northern Patagonian Ice Field (~47°S). Insights into the environmental context for human-landscape interactions have relied mostly on data extrapolated from distant extra-Andean locations that suggest limited environmental change during the Holocene. La Frontera (46°52’S), a high altitude site on the southern beech forest-steppe ecotone boundary in the Río Zeballos valley provides lithostratigraphical and palaeoecological evidence, constrained by 14C dating and tephrochronology, for dynamic environmental change during the last ~8000 years. An initial amelioration in environmental conditions after c. 8210 cal. BP was followed by a reversal to colder conditions between c. 7420 and 6480 cal. BP, coincident with initial human occupation within the Paso Roballos and Lago Pueyrredón basin. Between c. 6480 and 3700 cal. BP the woodland / steppe composition continued to fluctuate in response to climatic change. After c. 3700 cal. BP a gradual shift to more stable and temperate conditions, punctuated by increased fire activity, is contemporary with the later phases of human occupation extending up into the Paso Roballos-Río Zeballos corridor

Vitamin D3-Induced Tolerogenic Dendritic Cells Modulate the Transcriptomic Profile of T CD4 + Cells Towards a Functional Hyporesponsiveness

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most robust approaches due to their immune regulatory properties, which are currently being tested in clinical trials. However, the mechanisms that vitD3-tolDC trigger for the induction of tolerance remain elusive. For this reason, we performed a full phenotypical, functional, and transcriptomic characterization of T cells upon their interaction with autologous, antigen-specific vitD3-tolDC. We observed a strong antigen-specific reduction of T cell proliferation, combined with a decrease in the relative prevalence of T1 subpopulations and IFN- γ production. The analysis of the transcriptomic profile of T CD4 + cells evidenced a significant down-modulation of genes involved in cell cycle and cell response to mainly pro-inflammatory immune-related stimuli, highlighting the role of JUNB gene as a potential biomarker of these processes. Consequently, our results show the induction of a strong antigen-specific hyporesponsiveness combined with a reduction on the T1 immune profile of T cells upon their interaction with vitD3-tolDC, which manifests the regulatory properties of these cells and, therefore, their therapeutic potential in the clinic. https://doi.org/10.13039/5011000033295https://doi.org/10.13039/5011000033293https://doi.org/10.13039/5011000033296https://doi.org/10.13039/501100003329 https://doi.org/10.13039/501100003329_https://doi.org/10.13039/501100003329_https://doi.org/10.13039/501100003329 https://doi.org/10.13039/50110000332

Ethyl Pyruvate Induces Tolerogenic Dendritic Cells

Altres ajuts: Cost Action BM1305Dendritic cells (DC) are professional antigen presenting cells that have a key role in shaping the immune response. Tolerogenic DC (tolDC) have immuno-regulatory properties and they are a promising prospective therapy for multiple sclerosis and other autoimmune diseases. Ethyl pyruvate (EP) is a redox analog of dimethyl fumarate (Tecfidera), a drug for multiple sclerosis treatment. We have recently shown that EP ameliorates experimental autoimmune encephalomyelitis, a multiple sclerosis murine model. Here, we expanded our study to its tolerogenic effects on DC. Phenotypic analysis has shown that DC obtained from mice or humans reduce expression of molecules required for T cell activation such as CD86, CD83, and HLA-DR under the influence of EP, while CD11c expression and viability of DC are not affected. Furthermore, EP-treated DC restrain proliferation and modulate cytokine production of allogeneic lymphocytes. These results demonstrate that EP has the ability to direct DC toward tolDC

In-vivo activity of IFN-λ and IFN-α against bovine-viral-diarrhea virus in a mouse model

Bovine-viral-diarrhea virus (BVDV) can cause significant economic losses in livestock. The disease is controlled with vaccination and bovines are susceptible until vaccine immunity develops and may remain vulnerable if a persistently infected animal is left on the farm; therefore, an antiviral agent that reduces virus infectivity can be a useful tool in control programs. Although many compounds with promising in-vitro efficacy have been identified, the lack of laboratory-animal models limited their potential for further clinical development. Recently, we described the activity of type I and III interferons, IFN-α and IFN-λ respectively, against several BVDV strains in-vitro. In this study, we analyzed the in-vivo efficacy of both IFNs using a BALB/c-mouse model. Mice infected with two type-2 BVDV field strains developed a viremia with different kinetics, depending on the infecting strain's virulence, that persisted for 56 days post-infection (dpi). Mice infected with the low-virulence strain elicited high systemic TNF-α levels at 2 dpi. IFNs were first applied subcutaneously 1 day before or after infection. The two IFNs reduced viremia with different kinetics, depending on whether either one was applied before or after infection. In a second experiment, we increased the number of applications of both IFNs. All the treatments reduced viremia compared to untreated mice. The application of IFN-λ pre- and post-infection reduced viremia over time. This study is the first proof of the concept of the antiviral potency of IFN-λ against BVDV in-vivo, thus encouraging further trails for a potential use of this cytokine in cattle.Instituto de VirologíaFil: Quintana, María Eugenia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina.Fil: Barone, Lucas Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina.Fil: Trotta, Myrian Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Turco, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Mansilla, Florencia Celeste. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Capozzo, Alejandra Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina.Fil: Cardoso, Nancy Patricia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina.Fil: Quintana, María Eugenia. Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET); ArgentinaFil: Barone, Lucas Jose. Consejo Nacional de Investigaciones Científicas y Tecnológicas; ArgentinaFil: Capozzo, Alejandra Victoria.Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET); ArgentinaFil: Cardoso, Nancy Patricia. Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET); Argentin

Transfection of Vitamin D3-Induced Tolerogenic Dendritic Cells for the Silencing of Potential Tolerogenic Genes. Identification of CSF1R-CSF1 Signaling as a Glycolytic Regulator

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising strategy to re-establish immune tolerance in autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (VitD3-tolDC) has been widely tested because of their immune regulatory properties. To identify molecules and pathways involved in the generation of VitD3-tolDC, we established an easy and fast gene silencing method based on the use of Viromer blue to introduce siRNA into monocytes on day 1 of culture differentiation. The analysis of the effect of CD209 (DC-SIGN) and CD115 (CSF1R) down-modulation on the phenotype and functionality of transfected VitD3-tolDC revealed a partial role of CD115 in their tolerogenicity. Further investigations showed that CSF1R-CSF1 signaling is involved in the induction of cell metabolic reprogramming, triggering glycolysis to produce high amounts of lactate, a novel suppressive mechanism of T cell proliferation, recently found in autologous tolerogenic dendritic cells (ATDCs)

Potent Induction of Envelope-Specific Antibody Responses by Virus-Like Particle Immunogens Based on HIV-1 Envelopes from Patients with Early Broadly Neutralizing Responses

Longitudinal studies in HIV-1 infected individuals have indicated that 2-3 years of infection are required to develop broadly neutralizing antibodies. However, we have previously identified individuals with broadly neutralizing activity (bNA) in early HIV-1 infection, indicating that a vaccine may be capable of bNA induction after short periods of antigen exposure. Here, we describe 5 HIV-1 envelope sequences from individuals who have developed bNA within the first 100 days of infection (early neutralizers) and selected two of them to design immunogens based on HIV-1-Gag virus like particles (VLPs). These VLPs were homogeneous and incorporated the corresponding envelopes (7 to 9μg of gp120 in 1010 VLPs). Both envelopes bound to well-characterized bNAbs, including trimer-specific antibodies (PGT145, VRC01 and 35022). For immunogenicity testing, we immunized rabbits with the Env-VLPs or with the corresponding stabilized soluble Envelope trimers. A short immunization protocol (105 days) was used to recapitulate the early nAb induction observed after HIV-1 infection in these two individuals. All VLP and trimeric Envelope immunogens induced a comparably strong anti-gp120 response, despite having immunized rabbits with 30 times less gp120 in the case of the Env-VLPs. In addition, animals immunized with VLP-formulated Envs induced antibodies that cross-recognized the corresponding soluble stabilized trimer and vice versa, even though no neutralizing activity was observed. Nevertheless, our data may provide a new platform of immunogens, based on HIV-1 envelopes from patients with early broadly neutralizing responses, with the potential to generate protective immune responses using vaccination protocols similar to those used in classical preventive vaccines. Importance: It is generally accepted that an effective HIV-1 vaccine should be able to induce broad-spectrum neutralizing antibodies. Since most of these antibodies require long periods of somatic maturation in vivo, several groups are developing immunogens, based on the HIV envelope protein, that require complex and lengthy immunization protocols that would be difficult to implement to the general population. Here, we show that rabbits immunized with new envelopes (VLP-formulated) from two individuals who demonstrated broadly neutralizing activity very early after infection, induced specific HIV-1 antibodies after a short immunization protocol. This evidence provides the basis for generating protective immune responses with classic vaccination protocols with vaccine prototypes based on HIV envelope sequences from individuals who have developed early broadly neutralizing responses.This project received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 681137 to I.B., N.G., A.O., C.B., J.A., R.W.S., and E.Y. It was also partially supported by the Spanish AIDS Research Network (RIS), funded by the Instituto de Salud Carlos III and co-funded by the European Regional Development Fund (ERDF) “A way to build Europe” (projects RD16CIII/0002/0001, RD16CIII/0002/0005, and RD16CIII/0025/0041), Plan Estatal de I1D1I 2013-2016 to N.G., A.M.M., J.A., V.S.M., E.Y., M.P., A.O., and C.B.; by IDIBAPS to J.M.M. (80:20 Research grant); by the Fondation Dormeur, Vaduz to C.B.; by the Ministerio de Economía, Industria y Competitividad to N.G., V.S.M., and E.Y. (PI17CIII/00049); by the Ministerio de Ciencia e Innovación to N.G., V.S.M., and E.Y. (PI20CIII/00039); by the Consejo Nacional de Innovación, Ciencia y Tecnología to C.B.P.; and by the HHS/ National Institutes of Health (NIH) to C.B. (P01-AI131568).S

MAP7 and MUCL1 are biomarkers of Vitamin D3-induced tolerogenic dendritic cells in multiple sclerosis patients

The administration of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases, such as multiple sclerosis (MS). Specifically, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most widely studied approaches, as it has evidenced significant immune regulatory properties, both in vitro and in vivo. In this article, we generated human vitD3-tolDC from monocytes from healthy donors and MS patients, characterized in both cases by a semi-mature phenotype, secretion of IL-10 and inhibition of allogeneic lymphocyte proliferation. Additionally, we studied their transcriptomic profile and selected a number of differentially expressed genes compared to control mature and immature dendritic cells for their analysis. Among them, qPCR results validated CYP24A1, MAP7 and MUCL1 genes as biomarkers of vitD3-tolDC in both healthy donors and MS patients. Furthermore, we constructed a network of protein interactions based on the literature, which manifested that MAP7 and MUCL1 genes are both closely connected between them and involved in immune-related functions. In conclusion, this study evidences that MAP7 and MUCL1 constitute robust and potentially functional biomarkers of the generation of vitD3-tolDC, opening the window for their use as quality controls in clinical trials for MS

Occurrence of antibiotics in Lettuce (Lactuca sativa L.) and Radish (Raphanus sativus L.) following organic soil fertilisation under plot-scale conditions: Crop and human health implications

Recent studies have demonstrated the crop uptake of antibiotics (ABs) from soils treated with AB-carrying fertilisers. However, there is a lack of plot-scale studies linking their effects at the agronomic and metabolomic/transcriptomic level to their impact on human health. This paper assesses the plant uptake of 23 ABs following two productive cycles of lettuce and radish cropped with sewage sludge, pig slurry, the organic fraction of municipal solid waste, or chemical fertilisation under plot-scale conditions (32 plots spanning 3-10 m2 each). AB uptake by plants depended on both the vegetable and the AB class and was higher in radish than in lettuce edible parts. Levels ranged from undetectable to up to 76 ng/g (fresh weight). Repetitive organic fertilisation resulted in an increase in the concentration of ABs in lettuce leaves, but not in radish roots. Significant metabolomic and transcriptomic changes were observed following soil fertilisation. Nevertheless, a human health risk assessment indicates that the occurrence of ABs in lettuce or radish edible parts does not pose any risk. To our knowledge, this is the first holistic plot-scale study demonstrating that the use of organic fertilisers containing ABs is safe for crop security and human health.The authors gratefully acknowledge the financial support of the Spanish Ministry of Science and Innovation through projects AGL2017- 89518-R and RTI2018-096175-B-I00. IDAEA-CSIC is a Severo Ochoa Centre of Excellence (Spanish Ministry of Science and Innovation, Project CEX2018-000794-S). Mònica Escolà Casas wishes to thank the Beatriu de Pinós 2018 grant programme (MSCA grant agreement number 801370) for the funding.Peer ReviewedPostprint (published version

AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress

Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL). The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. Herein, we developed a miniaturized screening assay to identify inhibitors of PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. After screening a library of 627 kinase inhibitors, we found that targeting the pro-survival kinase AKT leads to strong impairment of PCNA ubiquitylation. Mechanistically, we found that AKT-mediated modulation of Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation after UV requires the upstream activity of DNA PKcs, without affecting PCNA ubiquitylation levels in unperturbed cells. Moreover, we confirmed that persistent AKT inhibition blocks the recruitment of TLS polymerases to sites of DNA damage and impairs DNA replication forks processivity after UV irradiation, leading to increased DNA replication stress and cell death. Remarkably, when we compared the differential survival of HR-proficient vs HR-deficient cells, we found that the combination of UV irradiation and AKT inhibition leads to robust SL induction in HR-deficient cells. We link this phenotype to AKT ability to inhibit PCNA ubiquitylation, since the targeted knockdown of PCNA E3-ligase (RAD18) and a non-ubiquitylable (PCNA K164R) knock-in model recapitulate the observed SL induction. Collectively, this work identifies AKT as a novel regulator of PCNA ubiquitylation and provides the proof-of-concept of inhibiting TLS as a therapeutic approach to selectively kill HR-deficient cells submitted to replication stress.Fil: Villafañez, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: García, Iris Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Carbajosa González, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Pansa, Maria Florencia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mansilla, Sabrina Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Llorens de Los Ríos, María Candelaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Angiolini, Virginia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; ArgentinaFil: Guantay, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Jacobs, Heinz. Tumor Biology And Immunology-netherlands Cancer Institu; Países BajosFil: Madauss, Kevin P.. Glaxosmithkline; Reino UnidoFil: Gloger, Israel. Glaxosmithkline; Reino UnidoFil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Soria, Ramiro Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Assessing Blood-Based Biomarkers to Define a Therapeutic Window for Natalizumab

Natalizumab is a monoclonal antibody that binds CD49d. Although it is one of the most effective treatments for Relapsing-Remitting Multiple Sclerosis (RRMS), a dosing regimen has not been optimized for safety and efficacy in individual patients. We aimed to identify biomarkers to monitor Natalizumab treatment and to establish a personalized dose utilizing an ongoing longitudinal study in 29 RRMS patients under Natalizumab with standard interval dose (SD) of 300 mg/4 wks or extended interval dose (EID) of 300 mg/6 wks. Blood samples were analyzed by flow cytometry to determine CD49d saturation and expression in several T and B lymphocytes subpopulations. Each patient was analyzed at two different timepoints separated by 3 Natalizumab administrations. Natalizumab and sVCAM-1 levels in serum were also analyzed using ELISA. To determine the reproducibility of various markers, two different timepoints were compared and no significant differences were observed for CD49d expression nor for saturation; SD patients had higher saturation levels (~80%) than EID patients (~60%). A positive correlation exists between CD49d saturation and Natalizumab serum levels. CD49d expression and saturation are stable parameters that could be used as biomarkers in the immunomonitoring of Natalizumab treatment. Moreover, Natalizumab and sVCAM-1 serum levels could be used to optimize an individual's dosing schedule