Fecal glucocorticoid metabolites as biomarkers in equids : assay choice matters

Free ranging animals are exposed to environmental, demographic, and ecological challenges over time, which can affect their health and fitness. Non‐invasive biomarkers can provide insight into how animals cope with these challenges and assess the effectiveness of conservation management strategies. We evaluated how free ranging ponies (Equus ferus caballus) on the Carneddau Mountain range, North Wales respond to 2 stimuli: an acute stressor of an annual roundup event in November 2014, and spatial and temporal variation in ecological factors in 2018. We evaluated fecal glucocorticoid metabolites using 2 enzyme immunoassays (EIAs): an 11‐oxoetiocholanolone EIA (measuring 11,17‐dioxoandrostanes [11,17‐DOAs]) and a corticosterone EIA. The former assay has been validated in equids, whereas there is limited evidence for the suitability of the latter. We used an additional parent testosterone EIA to measure fecal androgen metabolites in response to the ecological challenges. Following the roundup, the metabolite concentrations measured by the 2 glucocorticoid EIAs were not correlated. The 11,17‐DOAs were elevated from the second day following the roundup and then slowly returned to pre‐round levels over the next 2 weeks. In contrast, the metabolites measured by the corticosterone assay showed no response to the roundup. For the ecological data, all 3 assays detected a positive correlation between metabolites and social group size in males but not in females. The metabolite concentrations measured by the testosterone and corticosterone assays were highly correlated and were temporally associated with the onset of the breeding season, whereas the 11,17‐DOAs were not. The co‐variance of metabolites measured by the corticosterone and testosterone assays, and the lack of an acute response in the corticosterone assay to the roundup, suggests that metabolites detected by the corticosterone assay were not primarily associated with increased glucocorticoid production. We recommend using well‐validated fecal biomarker assays of hypothalamus‐pituitary‐adrenal axis activity to evaluate and compare the effect of different management interventions and environmental change. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society

Fecal Glucocorticoid Metabolites as Biomarkers in Equids: Assay Choice Matters

Free ranging animals are exposed to environmental, demographic, and ecological challenges over time, which can affect their health and fitness. Non-invasive biomarkers can provide insight into how animals cope with these challenges and assess the effectiveness of conservation management strategies. We evaluated how free ranging ponies (Equus ferus caballus) on the Carneddau Mountain range, North Wales respond to 2 stimuli: an acute stressor of an annual roundup event in November 2014, and spatial and temporal variation in ecological factors in 2018. We evaluated fecal glucocorticoid metabolites using 2 enzyme immunoassays (EIAs): an 11-oxoetiocholanolone EIA (measuring 11,17-dioxoandrostanes [11,17-DOAs]) and a corticosterone EIA. The former assay has been validated in equids, whereas there is limited evidence for the suitability of the latter. We used an additional parent testosterone EIA to measure fecal androgen metabolites in response to the ecological challenges. Following the roundup, the metabolite concentrations measured by the 2 glucocorticoid EIAs were not correlated. The 11,17-DOAs were elevated from the second day following the roundup and then slowly returned to pre-round levels over the next 2 weeks. In contrast, the metabolites measured by the corticosterone assay showed no response to the roundup. For the ecological data, all 3 assays detected a positive correlation between metabolites and social group size in males but not in females. The metabolite concentrations measured by the testosterone and corticosterone assays were highly correlated and were temporally associated with the onset of the breeding season, whereas the 11,17-DOAs were not. The co-variance of metabolites measured by the corticosterone and testosterone assays, and the lack of an acute response in the corticosterone assay to the roundup, suggests that metabolites detected by the corticosterone assay were not primarily associated with increased glucocorticoid production. We recommend using well-validated fecal biomarker assays of hypothalamus-pituitary-adrenal axis activity to evaluate and compare the effect of different management interventions and environmental change. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society

Reconstitution of huPBL-NSG Mice with Donor-Matched Dendritic Cells Enables Antigen-Specific T-cell Activation

Humanized mouse models provide a unique opportunity to study human immune cells in vivo, but traditional models have been limited to the evaluation of non-specific T-cell interactions due to the absence of antigen-presenting cells. In this study, immunodeficient NOD/SCID/IL2r-γnull (NSG) mice were engrafted with human peripheral blood lymphocytes alone or in combination with donor-matched monocyte-derived dendritic cells (DC) to determine whether antigen-specific T-cell activation could be reconstituted. Over a period of 3 weeks, transferred peripheral blood lymphocytes reconstituted the spleen and peripheral blood of recipient mice with predominantly human CD45-positive lymphocytes. Animals exhibited a relatively normal CD4/CD8 ratio (average 1.63:1) as well as reconstitution of CD3/CD56 (averaging 17.8%) and CD20 subsets (averaging 4.0%). Animals reconstituted with donor-matched CD11c+ DC also demonstrated a CD11c+ population within their spleen, representing 0.27% to 0.43% of the recovered human cells with concurrent expression of HLA-DR, CD40, and CD86. When immunized with adenovirus, either as free replication-incompetent vector (AdV) or as vector-transduced DC (DC/AdV), there was activation and expansion of AdV-specific T-cells, an increase in Th1 cytokines in serum, and skewing of T-cells toward an effector/memory phenotype. T-cells recovered from animals challenged with AdV in vivo proliferated and secreted a Th1-profile of cytokines in response to DC/AdV challenge in vitro. Our results suggest that engrafting NSG mice with a combination of lymphocytes and donor-matched DC can reconstitute antigen responsiveness and allow the in vivo assessment of human immune response to viruses, vaccines, and other immune challenges

Analysis of clinical uncertainties by health professionals and patients: an example from mental health

<p>Abstract</p> <p>Background</p> <p>The first step in practising Evidence Based Medicine (EBM) has been described as translating clinical uncertainty into a structured and focused clinical question that can be used to search the literature to ascertain or refute that uncertainty. In this study we focus on questions about treatments for schizophrenia posed by mental health professionals and patients to gain a deeper understanding about types of questions asked naturally, and whether they can be reformulated into structured and focused clinical questions.</p> <p>Methods</p> <p>From a survey of uncertainties about the treatment of schizophrenia we describe, categorise and analyse the type of questions asked by mental health professionals and patients about treatment uncertainties for schizophrenia. We explore the value of mapping from an unstructured to a structured framework, test inter-rater reliability for this task, develop a linguistic taxonomy, and cross tabulate that taxonomy with elements of a well structured clinical question.</p> <p>Results</p> <p>Few of the 78 Patients and 161 clinicians spontaneously asked well structured queries about treatment uncertainties for schizophrenia. Uncertainties were most commonly about drug treatments (45.3% of clinicians and 41% of patients), psychological therapies (19.9% of clinicians and 9% of patients) or were unclassifiable.(11.8% of clinicians and 16.7% of patients). Few naturally asked questions could be classified using the well structured and focused clinical question format (i.e. PICO format). A simple linguistic taxonomy better described the types of questions people naturally ask.</p> <p>Conclusion</p> <p>People do not spontaneously ask well structured clinical questions. Other taxonomies may better capture the nature of questions. However, access to EBM resources is greatly facilitated by framing enquiries in the language of EBM, such as posing queries in PICO format. People do not naturally do this. It may be preferable to identify a way of searching the literature that more closely matches the way people naturally ask questions if access to information about treatments are to be made more broadly available.</p

Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice

BACKGROUND:The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research. METHODOLOGY/PRINCIPAL FINDINGS:We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor gamma-chain knockout (NOD-scid IL2rgamma(null)) mice engrafted with human hematopoietic stem cells. Human CD45(+) cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rgamma(null) mice with HLA-A2(+) human cord blood hematopoietic stem cells, were able to secrete IFN-gamma, IL-2 and TNF-alpha in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353((111-119)), NS4b 2423((181-189)), and NS4a 2148((56-64)). CONCLUSIONS/SIGNIFICANCE:This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections

Selection of a core set of RILs from Forrest × Williams 82 to develop a framework map in soybean

Soybean BAC-based physical maps provide a useful platform for gene and QTL map-based cloning, EST mapping, marker development, genome sequencing, and comparative genomic research. Soybean physical maps for “Forrest” and “Williams 82” representing the southern and northern US soybean germplasm base, respectively, have been constructed with different fingerprinting methods. These physical maps are complementary for coverage of gaps on the 20 soybean linkage groups. More than 5,000 genetic markers have been anchored onto the Williams 82 physical map, but only a limited number of markers have been anchored to the Forrest physical map. A mapping population of Forrest × Williams 82 made up of 1,025 F8 recombinant inbred lines (RILs) was used to construct a reference genetic map. A framework map with almost 1,000 genetic markers was constructed using a core set of these RILs. The core set of the population was evaluated with the theoretical population using equality, symmetry and representativeness tests. A high-resolution genetic map will allow integration and utilization of the physical maps to target QTL regions of interest, and to place a larger number of markers into a map in a more efficient way using a core set of RILs

Human Solid Tumor Xenografts in Immunodeficient Mice Are Vulnerable to Lymphomagenesis Associated with Epstein-Barr Virus

Xenografting primary human solid tumor tissue into immunodeficient mice is a widely used tool in studies of human cancer biology; however, care must be taken to prove that the tumors obtained recapitulate parent tissue. We xenografted primary human hepatocellular carcinoma (HCC) tumor fragments or bulk tumor cell suspensions into immunodeficient mice. We unexpectedly observed that 11 of 21 xenografts generated from 16 independent patient samples resembled lymphoid neoplasms rather than HCC. Immunohistochemistry and flow cytometry analyses revealed that the lymphoid neoplasms were comprised of cells expressing human CD45 and CD19/20, consistent with human B lymphocytes. In situ hybridization was strongly positive for Epstein-Barr virus (EBV) encoded RNA. Genomic analysis revealed unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements in each B-cell neoplasm. These data demonstrate that the lymphoid neoplasms were EBV-associated human B-cell lymphomas. Analogous to EBV-associated lymphoproliferative disorders in immunocompromised humans, the human lymphomas in these HCC xenografts likely developed from reactivation of latent EBV in intratumoral passenger B lymphocytes following their xenotransplantation into immunodeficient recipient mice. Given the high prevalence of latent EBV infection in humans and the universal presence of B lymphocytes in solid tumors, this potentially confounding process represents an important pitfall of human solid tumor xenografting. This phenomenon can be recognized and avoided by routine phenotyping of primary tumors and xenografts with human leukocyte markers, and provides a compelling biological rationale for exclusion of these cells from human solid tumor xenotransplantation assays