Immunophenotyping of sheep paraffin-embedded peripheral lymph nodes

Sheep are not only a major livestock species globally, they are also an important large animal model for biomedical research and have contributed to our understanding of the ontogeny and architecture of the mammalian immune system. In this study, we applied immunohistochemistry and multicolor immunofluorescence in fixed and paraffin-embedded lymph nodes to phenotype the key populations of antigen presenting cells, lymphocytes and stromal cells that orchestrate the host adaptive immune response. We used an extensive panel of antibodies directed against markers associated with dendritic cells (MHC class II, CD83 and CD208), macrophages (CD11b, CD163 and CD169), stromal cells (CNA.42, S100 and CD83), and lymphocytes (CD3, Pax5, CD4, CD8). Using different methods of tissue fixation and antigen retrieval, we provide a detailed immunophenotyping of sheep lymph nodes including the identification of potential subpopulations of antigen presenting cells and stromal cells. By characterizing cells expressing combinations of these markers in the context of their morphology and location within the lymph node architecture, we provide valuable new tools to investigate the structure, activation and regulation of the sheep immune system in health and disease

Demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies

<p>Abstract</p> <p>Background</p> <p>Bovine neonatal pancytopenia (BNP) is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD) vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows (“BNP dams”). Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and β-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC) cultured <it>in vitro</it> from sternal biopsies taken at 24 hours and 6 days post-challenge.</p> <p>Results</p> <p>Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell) colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid) colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage) was markedly reduced.</p> <p>Conclusion</p> <p>This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types.</p

autonomic cardiovascular control in hyperthyroid women during sleep

Hyperthyroidism is characterized by hyperadrenergic symptoms (i.e., tachycardia, anxiety, and increased metabolic state). Although hyperthyroid patients often complain about an impairment of sleep, no data are available on sleep characteristics and autonomic cardiovascular control during sleep in these patients. We aimed to assess sleep qualitative indices and autonomic cardiovascular regulation during sleep in hyperthyroidism (Hyperthyr) and after treatment. Six subjects with a first diagnosis of Graves' disease or hyperfunctioning nodule underwent a complete polysomnographic study (PSG) at the time of diagnosis and after the treatment, when they became euthyroid (Euthyr). ECG and respiratory signals were extracted and samples of consecutive 250–300 beats were analyzed using linear spectral and nonlinear entropy analysis of heart rate variability (HRV), during the different sleep stages. Heart rate was decreased and total power increased in Euthyr compared to Hyperthyr, both during wake and sleep; no changes of the sympathovagal balance were observed. Entropy analysis showed that regularity index was reduced in Euthyr compared to Hyperthyr, suggesting changes in the complexity of the cardiovascular control. Periodic leg movements (PLM) were reduced in Euthyr compared to Hyperthyr. In conclusion, hyperthyroidism seems to be associated with an increased sleep fragmentation, due to PLM and an altered cardiac autonomic control

Congo River sand and the equatorial quartz factory

A never solved problem in sedimentary petrology is the origin of sandstone consisting exclusively of quartz and most durable heavy minerals. The Congo River offers an excellent test case to investigate under which tectonic, geomorphological, climatic, and geochemical conditions pure quartzose sand is generated today. In both upper and lowermost parts of the catchment, tributaries contain significant amounts of feldspars, rock fragments, or moderately stable heavy minerals pointing at the central basin as the main location of the "quartz factory". In Congo sand, quartz is enriched relatively to all other minerals including zircon, as indicated by Si/Zr ratios much higher than in the upper continental crust. Selective elimination of old zircons that accumulated radiation damage through time is suggested by low percentages of grains yielding Archean U-Pb ages despite the basin being surrounded by Archean cratonic blocks. Intense weathering is documented by the lack of carbonate grains in sand and by dominant kaolinite and geochemical signatures in mud. In sand, composed almost entirely of SiO2, the weathering effect is masked by massive addition of quartz grains recycled during multiple events of basin inversion since the Proterozoic. Changes in mineralogical, geochemical, and geochronological signatures across Bas-Congo concur to suggest that approximately 10% of the sand supplied to the Atlantic Ocean is generated by rapid fluvial incision into the recently uplifted Atlantic Rise. The Congo River connects with a huge canyon similar to 30 km upstream of the mouth, and pure quartzose sand is thus funnelled directly toward the deep-sea to feed a huge turbidite fan. Offshore sediments on both sides of the canyon are not derived from the Congo River. They reflect mixed provenance, including illite-rich dust wind-blown from the arid Sahel and augite, hypersthene, and smectite ejected from volcanic centres probably situated along the Cameroon Line in the north. Because mixing of detritus from diverse sources and supply of polycyclic grains almost invariably occurs in the terminal lowland tract of a sediment-routing-system, no ancient sandstone can be safely considered as entirely first-cycle. Moreover, the abundance of pure quartzarenite in the rock record can hardly be explained by chemical weathering or physical recycling alone. The final cleansing of minerals other than quartz, zircon, tourmaline, and rutile requires one or more cycles of chemical dissolution during diagenesis, which operates at higher temperatures and over longer periods than weathering at the Earth's surface

Cellular residual disease (CRD) in early breast cancer –Expanding the concept of minimal residual disease monitoring?

Despite a significant evolution in treatment strategies for early breast cancer (EBC) patients, up to 30% of them experience recurrence due to occult micrometastasis. The minimal residual disease (minimal RD) in EBC patients after the treatment with curative intent cannot be easily detected by clinical examination and radiological imaging, as they are both burdened by limited sensitivity. A new frontier and promising approach to address this unmet need is the study of liquid biopsy (LB). The most studied tumor-derived analytes in the peripheral blood for minimal RD monitoring are currently: i) the circulating tumor DNA (ctDNA), for the detection of somatic DNA alterations, so referred to as molecular residual disease (MRD); ii) circulating tumor cells (CTCs), for the detection of cellular residual disease (CRD). MRD detection, while reaching a high specificity, is still presenting a number of limitations. On the other hand, CRD allows a real-time disease monitoring, detecting live cells, and possess the potential to provide an enormous amount of biological information. Indeed, CTCs can provide a multi-level portrait (i.e., DNA, RNA and proteins) of the tumor, longitudinally depicting its evolving landscape, and can be used for functional (in vitro/in vivo) characterization. Moreover, CRD goes beyond the association with the risk of recurrence: predictive biomarkers for treatment response can also be evaluated. Nevertheless, CTCs are less studied in this context, because of their need to be immediately processed and their limited detection in a small fraction of patients in the early and post-surgery setting. These limitations could however be overcome by the use of newly developed technologies that enable an increased CTC detection rate and retrospective studies. Here, we review the strengths and limitations of using MRD and CRD for minimal RD detection, focusing on the methodologies available for LB analysis in this setting, and on the main clinical studies investigating MRD and CRD in EBC. Considering the limits and the advantages of both MRD and CRD, we propose the integration of ctDNA and CTCs as complementary tools for minimal RD assessment to achieve a synergistic and novel approach for minimal RD analysis

Archaeometrical characterization of pigments and paintings on Pre-Hispanic pottery from the regions of Fiambalá and Chaschuil (Catamarca, Argentina)

The results of an analysis of pigments used in slips and designs deployed in ceramic assemblages recovered from archaeological sites located in different environments, expressing the socio-historical process developed in the regions of Fiambalá and Chaschuil (Catamarca, Argentina) during the last 1500 years are presented. The sample, formed by fragments of different ceramic styles and natural pigments, was analysed via Raman spectroscopy and X-ray diffraction. Results indicate the continuity in use of certain pigments (mainly hematite for red hues and Mn oxides in combination with magnetite for black paints) within the different socio-political organizations that inhabited the region from the first to the 14th centuries ce, in spite of the diverse shades of colour, which suggests an intentional search linked with their cultural conventions. For the Inca Period, this scenery of continuities in minerals employed for reds and blacks is complemented by the use of new compounds (Ti oxide, apatite and gypsum) in order to generate the ‘cream’ tones applied as slips, all of which have not been identified for previous moments.Fil: Ratto, Norma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Saavedra 15. Instituto de las Culturas. Universidad de Buenos Aires. Instituto de las Culturas; ArgentinaFil: Reinoso, Maria Elba. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica; ArgentinaFil: Basile, Mara Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Saavedra 15. Instituto de las Culturas. Universidad de Buenos Aires. Instituto de las Culturas; ArgentinaFil: Freire Espeleta, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica; ArgentinaFil: Halac, Emilia Beatriz. Universidad Nacional de San Martín; Argentina. Comisión Nacional de Energía Atómica; Argentin

Road tunnel risk-based safety design methodology by GU@LARP Quantum risk model

The ALARP concept is used in different countries for different sectors of activity where a risk assessment or measure is requested. In this paper a model is developed based upon ALARP principle for tunnel risk-based design in case of fire accident scenarios. In Italy, ALARP risk acceptability and tolerability criteria have been adopted then the compliance with them has to be verified in order to guarantee a minimum-sufficient level of safety. The quantum of risk coupled with any design scenario is defined and modelled and the consequent individual quantum of risk coupled with the single exposed unit in the scenario is defined too. The methodologies for the identification of the requested design scenario, in number and type, are outlined. The scenarios are described in a shape suitable as INPUTS in the thermo-dynamical numerical simulations for fire generation and exposed units evacuation. The expected OUTPUTS of the numerical simulation are the estimations of the number of the fatalities (N) coupled with the single specific scenarios. In parallel with the above physical deterministic scenario simulations, a conceptual and operational procedure has been also established for the scenarios probabilities assessment. Merging the resulting data of both the above separate models, the risk quanta Gu@larp model is finally established. A case study is developed considering scenarios related to a virtual limit tunnel to support the description of the model itself, properties, advantages and perspectives

Investigating Employees’ Concerns and Wishes Regarding Digital Stress Management Interventions With Value Sensitive Design: Mixed Methods Study

Background: Work stress places a heavy economic and disease burden on society. Recent technological advances include digital health interventions for helping employees prevent and manage their stress at work effectively. Although such digital solutions come with an array of ethical risks, especially if they involve biomedical big data, the incorporation of employees' values in their design and deployment has been widely overlooked. Objective: To bridge this gap, we used the value sensitive design (VSD) framework to identify relevant values concerning a digital stress management intervention (dSMI) at the workplace, assess how users comprehend these values, and derive specific requirements for an ethics-informed design of dSMIs. VSD is a theoretically grounded framework that front-loads ethics by accounting for values throughout the design process of a technology. Methods: We conducted a literature search to identify relevant values of dSMIs at the workplace. To understand how potential users comprehend these values and derive design requirements, we conducted a web-based study that contained closed and open questions with employees of a Swiss company, allowing both quantitative and qualitative analyses. Results: The values health and well-being, privacy, autonomy, accountability, and identity were identified through our literature search. Statistical analysis of 170 responses from the web-based study revealed that the intention to use and perceived usefulness of a dSMI were moderate to high. Employees' moderate to high health and well-being concerns included worries that a dSMI would not be effective or would even amplify their stress levels. Privacy concerns were also rated on the higher end of the score range, whereas concerns regarding autonomy, accountability, and identity were rated lower. Moreover, a personalized dSMI with a monitoring system involving a machine learning-based analysis of data led to significantly higher privacy (P=.009) and accountability concerns (P=.04) than a dSMI without a monitoring system. In addition, integrability, user-friendliness, and digital independence emerged as novel values from the qualitative analysis of 85 text responses. Conclusions: Although most surveyed employees were willing to use a dSMI at the workplace, there were considerable health and well-being concerns with regard to effectiveness and problem perpetuation. For a minority of employees who value digital independence, a nondigital offer might be more suitable. In terms of the type of dSMI, privacy and accountability concerns must be particularly well addressed if a machine learning-based monitoring component is included. To help mitigate these concerns, we propose specific requirements to support the VSD of a dSMI at the workplace. The results of this work and our research protocol will inform future research on VSD-based interventions and further advance the integration of ethics in digital health

Adhesion of human gingival fibroblasts/Streptococcus mitis co-culture on the nanocomposite system Chitlac-nAg

10noComposite materials are increasingly used as dental restoration. In the field of biomaterials, infections remain the main reason of dental devices failure. Silver, in the form of nanoparticles (AgNPs), ions and salt, well known for its antimicrobial properties, is used in several medical applications in order to avoid bacterial infection. To reduce both bacterial adhesion to dental devices and cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilized AgNPs with a polyelectrolyte solution containing Chitlac. Here we analyzed the proliferative and adhesive ability of human gingival fibroblasts (HGFs) on BisGMA/TEGDMA thermosets uncoated and coated with AgNPs in a coculture model system with Streptococcus mitis. After 48 h, HGFs well adhered onto both surfaces, while S. mitis cytotoxic response was higher in the presence of AgNPs coated thermosets. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release raised up to 20 %. Moreover the presence of S. mitis reduced this release mainly when HGFs adhered to Chitlac-nAg coated thermosets. The reduced secretion of collagen type I was significant in the presence of both surfaces with the co-culture system even more when saliva is added. Integrin β1 localized closely to cell membranes onto Chitlac-nAg thermosets and PKCα translocated into nuclei. These data confirm that Chitlac-nAg have a promising utilization in the field of restorative dentistry exerting their antimicrobial activity due to AgNPs without cytotoxicity for eukaryotic cells.openopenCataldi, Amelia; Gallorini, Marialucia; Di Giulio, Mara; Guarnieri, Simone; Mariggiò, Maria Addolorata; Traini, Tonino; Di Pietro, Roberta; Cellini, Luigina; Marsich, Eleonora; Sancilio, SilviaCataldi, Amelia; Gallorini, Marialucia; Di Giulio, Mara; Guarnieri, Simone; Mariggiò, Maria Addolorata; Traini, Tonino; Di Pietro, Roberta; Cellini, Luigina; Marsich, Eleonora; Sancilio, Silvi

Real-time assessment of HER2 status in circulating tumor cells of breast cancer patients: Methods of detection and clinical implications

The human epidermal growth factor receptor 2 (HER2) plays a central role in breast cancer (BC). Therefore, it is critical to develop a method that can capture its spatial and temporal heterogeneity. Nowadays, therapeutic decisions for BC patients relies on evaluation of HER2 status from tissue biopsies using immunohistochemistry and in situ hybridization. Nevertheless, considering the technical and logistical challenges associated with tissue biopsies, there is an unmet need for a non-invasive and accurate approach to obtain real-time assessment of HER2 status. In this context, circulating biomarkers, particularly circulating tumor cells (CTCs), emerged as promising candidates. HER2 assessment on CTCs can be performed at genomic, transcriptomic, and protein levels on both bulk CTCs and at the single-cell resolution. However, the main limitation of the literature to date is the lack of a consistent definition of HER2-positive CTCs, which poses a major challenge for both, future research and clinical applications. Several studies revealed discordance in HER2 status between the primary tumor and corresponding CTCs. For instance, HER2-positive CTCs have been detected among patients with HER2-negative BC and vice versa. As a result, researchers have evaluated the prognostic and predictive value of HER2 status in CTCs, both in the early and metastatic settings, to increase the possibility of using anti-HER2 therapy also for these patients and to dissect mechanisms of treatment resistance. This review aims to provide an overview of the methods to determine HER2 status in CTCs and to summarize the evidence and future perspective on how CTCs-HER2 assessment can be integrated into the clinical management of BC patients