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Microarray patches (MAPs) have the potential to be a safer, more acceptable, easier to use and more cost-effective method for administration of vaccines when compared to the needle and syringe. Since MAPs deliver vaccine to the dermis and epidermis, a degree of local immune response at the site of application is expected. In a phase 1 clinical trial (ACTRN 12618000112268), the Vaxxas high-density MAP (HD-MAP) was used to deliver a monovalent, split inactivated influenza virus vaccine into the skin. HD-MAP immunisation led to significantly enhanced humoral responses on day 8, 22 and 61 compared with IM injection of a quadrivalent commercial seasonal influenza vaccine (Afluria Quadrivalent®). Here, the aim was to analyse cellular responses to HD-MAPs in the skin of trial subjects, using flow cytometry and immunohistochemistry. HD-MAPs were coated with a split inactivated influenza virus vaccine (A/Singapore/GP1908/2015 [H1N1]), to deliver 5 μg haemagglutinin (HA) per HD-MAP. Three HD-MAPs were applied to the volar forearm (FA) of five healthy volunteers (to achieve the required 15 μg HA dose), whilst five control subjects received three uncoated HD-MAPs (placebo). Local skin response was recorded for over 61 days and haemagglutination inhibition antibody titres (HAI) were assessed on days 1, 4, 8, 22, and 61. Skin biopsies were taken before (day 1), and three days after HD-MAP application (day 4) and analysed by flow-cytometry and immunohistochemistry to compare local immune subset infiltration. HD-MAP vaccination with 15 μg HA resulted in significant HAI antibody titres compared to the placebo group. Application of uncoated placebo HD-MAPs resulted in mild erythema and oedema in most subjects, that resolved by day 4 in 80% of subjects. Active, HA-coated HD-MAP application resulted in stronger erythema responses on day 4, which resolved between days 22–61. Overall, these erythema responses were accompanied by an influx of immune cells in all subjects. Increased cell infiltration of CD3+, CD4+, CD8+ T cells as well as myeloid CD11b+ CD11c+ and non-myeloid CD11b- dendritic cells were observed in all subjects, but more pronounced in active HD-MAP groups. In contrast, CD19+/CD20+ B cell counts remained unchanged. Key limitations include the use of an influenza vaccine, to which the subjects may have had previous exposure. Different results might have been obtained with HD-MAPs inducing a primary immune response. In conclusion, influenza vaccine administered to the forearm (FA) using the HD-MAP was well-tolerated and induced a mild to moderate skin response with lymphocytic infiltrate at the site of application.

Our group has developed two transplantation models for the engraftment of Human Intestinal Organoids (HIOs): the renal subcapsular space (RSS) and the mesentery each with specific benefits for study. While engraftment at both sites generates laminated intestinal structures, a direct comparison between models has not yet been performed. Embryonic stem cells were differentiated into HIOs, as previously described. HIOs from the same batch were transplanted on the same day into either the RSS or mesentery. 10 weeks were allowed for engraftment and differentiation, at which time they were harvested and assessed. Metrics for comparison included: mortality, engraftment rate, gross size, number and grade of lumens, and expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism. Mortality was significantly increased when undergoing mesentery transplantation, however engraftment was significantly higher. Graft sizes were similar between groups. Morphometric parameters were similar between groups, however m-tHIOs presented with significantly fewer lumens than k-tHIO. Transcript and protein level expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism were similar between groups. Transplantation into both sites yields viable tissue of similar quality based on our assessments with enhanced engraftment and a dominant lumen for uniform study benefiting the mesenteric site and survival benefiting RSS.

Background In service to its core mission of improving the health and well-being of veterans, Veterans Affairs (VA) leadership is committed to supporting research best practices in the VA. Recognizing that the behavior of researchers is influenced by the organizational climates in which they work, efforts to assess the integrity of research climates and share such information with research leadership in VA may be one way to support research best practices. The Survey of Organizational Research Climate (SOuRCe) is the first validated survey instrument specifically designed to assess the organizational climate of research integrity in academic research organizations. The current study reports on an initiative to use the SOuRCe in VA facilities to characterize the organizational research climates and pilot test the effectiveness of using SOuRCe data as a reporting and feedback intervention tool. Methods We administered the SOuRCe using a cross-sectional, online survey, with mailed follow-up to non-responders, of research-engaged employees in the research services of a random selection of 42 VA facilities (e.g., Hospitals/Stations) believed to employ 20 or more research staff. We attained a 51% participation rate, yielding more than 5,200 usable surveys. Results We found a general consistency in organizational research climates across a variety of sub-groups in this random sample of research services in the VA. We also observed similar SOuRCe scale score means, relative rankings of these scales and their internal reliability, in this VA-based sample as we have previously documented in more traditional academic research settings. Results also showed more substantial variability in research climate scores within than between facilities in the VA research service as reflected in meaningful subgroup differences. These findings suggest that the SOuRCe is suitable as an instrument for assessing the research integrity climates in VA and that the tool has similar patterns of results that have been observed in more traditional academic research settings. Conclusions The local and specific nature of organizational climates in VA research services, as reflected in variability across sub-groups within individual facilities, has important policy implications. Global, “one-size-fits-all” type initiatives are not likely to yield as much benefit as efforts targeted to specific organizational units or sub-groups and tailored to the specific strengths and weaknesses documented in those locations.

The cattle rumen has a diverse microbial ecosystem that is essential for the host to digest plant material. Extremes in body weight (BW) gain in mice and humans have been associated with different intestinal microbial populations. The objective of this study was to characterize the microbiome of the cattle rumen among steers differing in feed efficiency. Two contemporary groups of steers (n=148 and n=197) were fed a ration (dry matter basis) of 57.35% dry-rolled corn, 30% wet distillers grain with solubles, 8% alfalfa hay, 4.25% supplement, and 0.4% urea for 63 days. Individual feed intake (FI) and BW gain were determined. Within contemporary group, the four steers within each Cartesian quadrant were sampled (n=16/group) from the bivariate distribution of average daily BW gain and average daily FI. Bacterial 16S rRNA gene amplicons were sequenced from the harvested bovine rumen fluid samples using next-generation sequencing technology. No significant changes in diversity or richness were indicated, and UniFrac principal coordinate analysis did not show any separation of microbial communities within the rumen. However, the abundances of relative microbial populations and operational taxonomic units did reveal significant differences with reference to feed efficiency groups. Bacteroidetes and Firmicutes were the dominant phyla in all ruminal groups, with significant population shifts in relevant ruminal taxa, including phyla Firmicutes and Lentisphaerae, as well as genera Succiniclasticum, Lactobacillus, Ruminococcus, and Prevotella. This study suggests the involvement of the rumen microbiome as a component influencing the efficiency of weight gain at the 16S level, which can be utilized to better understand variations in microbial ecology as well as host factors that will improve feed efficiency.

SOX17 is a key transcriptional regulator that can act by regulating other transcription factors including HNF1β and FOXA2, which are known to regulate postnatal β cell function. Given this, we investigated the role of SOX17 in the developing and postnatal pancreas and found a novel role for SOX17 in regulating insulin secretion. Deletion of the Sox17 gene in the pancreas (Sox17-paLOF) had no observable impact on pancreas development. However, Sox17-paLOF mice had higher islet proinsulin protein content, abnormal trafficking of proinsulin, and dilated secretory organelles suggesting that Sox17-paLOF adult mice are prediabetic. Consistant with this, Sox17-paLOF mice were more susceptible to aged-related and high fat diet-induced hyperglycemia and diabetes. Overexpression of Sox17 in mature β cells using Ins2-rtTA driver mice resulted in precocious secretion of proinsulin. Transcriptionally, SOX17 appears to broadly regulate secretory networks since a 24-hour pulse of SOX17 expression resulted in global transcriptional changes in factors that regulate hormone transport and secretion. Lastly, transient SOX17 overexpression was able to reverse the insulin secretory defects observed in MODY4 animals and restored euglycemia. Together, these data demonstrate a critical new role for SOX17 in regulating insulin trafficking and secretion and that modulation of Sox17-regulated pathways might be used therapeutically to improve cell function in the context of diabetes.

International audience; Neurogenin3(+) (Ngn3(+)) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach, where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors, but the lineage and regulators of pancreatic gastrin(+) cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin(+) cells in the developing pancreas co-express glucagon, ghrelin or pancreatic polypeptide, but many gastrin(+) cells do not express any other islet hormone. Pancreatic gastrin(+) cells express the transcription factors Nkx6.1, Nkx2.2 and low levels of Pdx1, and derive from Ngn3(+) endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3, Nkx2.2, NeuroD1 and Arx, but not Pax4 or Pax6. Finally, gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus, gastrin(+) cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells.

The Sry-related high mobility group box transcription factor Sox17 is required for diverse developmental processes including endoderm formation, vascular development, and fetal hematopoietic stem cell maintenance. Expression of Sox17 in mature respiratory epithelial cells causes proliferation and lineage respecification, suggesting that Sox17 can alter adult lung progenitor cell fate. In this paper, we identify mechanisms by which Sox17 influences lung epithelial progenitor cell behavior and reprograms cell fate in the mature respiratory epithelium. Conditional expression of Sox17 in epithelial cells of the adult mouse lung demonstrated that cell cluster formation and respecification of alveolar progenitor cells toward proximal airway lineages were rapidly reversible processes. Prolonged expression of Sox17 caused the ectopic formation of bronchiolar-like structures with diverse respiratory epithelial cell characteristics in alveolar regions of lung. During initiation of progenitor cell behavior, Sox17 induced proliferation and increased the expression of the progenitor cell marker Sca-1 and genes involved in cell cycle progression. Notably, Sox17 enhanced cyclin D1 expression in vivo and activated cyclin D1 promoter activity in vitro. Sox17 decreased the expression of transforming growth factor-beta (TGF-beta)-responsive cell cycle inhibitors in the adult mouse lung, including p15, p21, and p57, and inhibited TGF-beta1-mediated transcriptional responses in vitro. Further, Sox17 interacted with Smad3 and blocked Smad3 DNA binding and transcriptional activity. Together, these data show that a subset of mature respiratory epithelial cells retains remarkable phenotypic plasticity and that Sox17, a gene required for early endoderm formation, activates the cell cycle and reinitiates multipotent progenitor cell behavior in mature lung cells.

Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6-8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs.