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Dendritic cells are important mediators in the early presentation of antigen and regulation of the differentiation of T cells. Peanut oral immunotherapy (POIT) results in desensitization in most peanut allergic individuals (responders), but not in others due to allergic reactions (non-responders). Delineation of early immunologic changes contributing to desensitization would help clarify the POIT mechanism of action. We analyzed dendritic cells in 15 pediatric subjects (5-12 years) undergoing a phase 1 single-center POIT study. We examined dendritic cells at baseline, 6-, 12-, 18- and 24-weeks after initiation of POIT and responders of therapy were compared to non-responders and healthy controls. The distribution frequency of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) from peripheral blood samples were measured in vitro. A general linear mixed model was used, and included fixed effects for cohort (responder, non-responder, or healthy control), time (0-, 6-, 12-, 18-, and 24-weeks), and the cohort-time interaction term. P-values were adjusted for multiple hypothesis testing using Tukey's method. We observed that POIT responders had reduced TNFa producing myeloid dendritic cells (mDCs) compared to non-responders. Additionally, non-responders had increased OX40L expressing mDCs at 18-weeks compared to responders. In conclusion, our findings suggest that a reduced pro-inflammatory phenotype in DCs could potentially serve as a predictor of early outcome and success of POIT desensitization.

Quorum sensing (QS) and nucleotide-based second messengers are vital signaling systems that regulate bacterial physiology in response to changing environments. Disrupting bacterial signal transduction is a promising direction to combat infectious diseases, and QS and the second messengers are undoubtedly potential targets. In Vibrio cholerae, both QS and the second messenger 3', 5'-cyclic diguanylate (c-di-GMP) play a central role in controlling motility, motile-to-sessile life transition, and virulence. In this study, we found that water-soluble extract from the North American cranberry could significantly inhibit V. cholerae biofilm formation during the development/maturation stage by reducing the biofilm matrix production and secretion. The anti-biofilm effect by water-soluble cranberry extract was possibly through modulating the intracellular c-di-GMP level and was independent of QS and the QS master regulator HapR. Our results suggest an opportunity to explore more functional foods to fight stubborn infections through interference with the bacterial signaling systems.

We demonstrate ensemble three-dimensional cell cultures and quantitative analysis of angiogenic growth from uniform endothelial monolayers. Our approach combines two key elements: a micro-fluidic assay that enables parallelized angiogenic growth instances subject to common extracellular conditions, and an automated image acquisition and processing scheme enabling high-throughput, unbiased quantification of angiogenic growth. Because of the increased throughput of the assay in comparison to existing three-dimensional morphogenic assays, statistical properties of angiogenic growth can be reliably estimated. We used the assay to evaluate the combined effects of vascular endothelial growth factor (VEGF) and the signaling lipid sphingoshine-1-phosphate (S1P). Our results show the importance of S1P in amplifying the angiogenic response in the presence of VEGF gradients. Furthermore, the application of S1P with VEGF gradients resulted in angiogenic sprouts with higher aspect ratio than S1P with background levels of VEGF, despite reduced total migratory activity. This implies a synergistic effect between the growth factors in promoting angiogenic activity. Finally, the variance in the computed angiogenic metrics (as measured by ensemble standard deviation) was found to increase linearly with the ensemble mean. This finding is consistent with stochastic agent-based mathematical models of angiogenesis that represent angiogenic growth as a series of independent stochastic cell-level decisions.

In recent years, real-world studies (RWS) are gaining increasing interests, because they can generate more realistic and generalizable results than randomized controlled clinical trials (RCT). In 2017, we published a RCT in 741 early childhood care and education providers (CCPs). It is the Phase I of our iLookOut for Child Abuse project (iLookOut), an online, interactive learning module about reporting suspected child maltreatment. That study demonstrated that in a RCT setting, the iLookOut is efficient at improving CCPs' knowledge of and attitudes towards child maltreatment reporting. However, the generalizability of that RCT's results in a RWS setting remains unknown. To address this question, we design and conduct this large RWS in 11,065 CCPs, which is the Phase II of the iLookOut. We hypothesize replication of the earlier RCT findings, i.e., the iLookOut can improve CCPs' knowledge of and attitudes toward child maltreatment reporting in a real world setting. In addition, this RWS also explores whether demographic factors affect CCPs' performance. Results of this RWS confirmed the generalizability of the previous RCT's results in a real world setting. It yielded similar effect sizes for knowledge and attitudes as were found in the earlier RCT. Cohen's d for knowledge improvement was 0.95 in that RCT, 0.96 in this RWS; Cohen's d for attitude improvement was 0.98 in that RCT, 0.80 in this RWS. Also, we found several significant differences in knowledge and attitude improvement with regard to age, race, education, and employment status. In conclusion, iLookOut improves knowledge and attitudes of CCPs about child maltreatment prevention and reporting in a real-world setting. The generalizability of the initial RCT findings to this RWS provides strong evidence that the iLookout will be effective in other real world settings. It can be a useful model for other interventions aimed at preventing child maltreatment. Clinical trial registration for the original RCT: NCT02225301 (ClinicalTrials.gov Identifier)., Competing Interests: The authors have declared that no competing interests exist.

Heterotopic ossification (HO) is the pathologic formation of bone separate from the normal skeleton. Although several models exist for studying HO, an understanding of the common in vitro properties of cells isolated from these models is lacking. We studied three separate animal models of HO including two models of trauma-induced HO and one model of genetic HO, and human HO specimens, to characterize the properties of cells derived from tissue containing pre-and mature ectopic bone in relation to analogous mesenchymal cell populations or osteoblasts obtained from normal muscle tissue. We found that when cultured in vitro, cells isolated from the trauma sites in two distinct models exhibited increased osteogenic differentiation when compared to cells isolated from uninjured controls. Furthermore, osteoblasts isolated from heterotopic bone in a genetic model of HO also exhibited increased osteogenic differentiation when compared with normal osteoblasts. Finally, osteoblasts derived from mature heterotopic bone obtained from human patients exhibited increased osteogenic differentiation when compared with normal bone from the same patients. These findings demonstrate that across models, cells derived from tissues forming heterotopic ossification exhibit increased osteogenic differentiation when compared with either normal tissues or osteoblasts. These cell types can be used in the future for in vitro investigations for drug screening purposes.

Heterotopic ossification (HO) is the growth of extra-skeletal bone which occurs following trauma, burns, and in patients with genetic bone morphogenetic protein (BMP) receptor mutations. The clinical and laboratory evaluation of HO is dependent on radiographic imaging to identify and characterize these lesions. Here we show that despite its inadequacies, plain film radiography and single modality microCT continue to serve as a primary method of HO imaging in nearly 30% of published in vivo literature. Furthermore, we demonstrate that detailed microCT analysis is superior to plain film and single modality microCT radiography specifically in the evaluation of HO formed through three representative models due to its ability to 1) define structural relationships between growing extra-skeletal bone and normal, anatomic bone, 2) provide accurate quantification and growth rate based on volume of the space-occupying lesion, thereby facilitating assessments of therapeutic intervention, 3) identify HO at earlier times allowing for evaluation of early intervention, and 4) characterization of metrics of bone physiology including porosity, tissue mineral density, and cortical and trabecular volume. Examination of our trauma model using microCT demonstrated two separate areas of HO based on anatomic location and relationship with surrounding, normal bone structures. Additionally, microCT allows HO growth rate to be evaluated to characterize HO progression. Taken together, these data demonstrate the need for a paradigm shift in the evaluation of HO towards microCT as a standard tool for imaging.

Introduction: The function of Glycogen Synthase Kinases 3β (GSK-3β) has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification., Methods: Palates were harvested from GSK-3β, embryonic days 15.0-18.5 (e15.0-e18.5), and e15.5 Indian Hedgehog (Ihh) null embryos, and their wild-type littermates. The phenotype of GSK-3β null embryos was analyzed with skeletal whole mount and pentachrome stains. Spatiotemporal regulation of osteogenic gene expression, in addition to Wnt and Hedgehog signaling activity, were examined in vivo on GSK-3β and Ihh +/+ and -/- e15.5 embryos using in situ hybridization and immunohistochemistry. To corroborate these results, expression of the same molecular targets were assessed by qRT-PCR of e15.5 palates, or e13.5 palate cultures treated with both Wnt and Hedgehog agonists and anatagonists., Results: GSK-3β null embryos displayed a 48 percent decrease (*p<0.05) in palatine bone formation compared to wild-type littermates. GSK-3β null embryos also exhibited decreased osteogenic gene expression that was associated with increased Wnt and decreased Hedgehog signaling. e13.5 palate culture studies demonstrated that Wnt signaling negatively regulates both osteogenic gene expression and Hedgehog signaling activity, while inhibition of Wnt signaling augments both osteogenic gene expression and Hedgehog signaling activity. In addition, no differences in Wnt signaling activity were noted in Ihh null embryos, suggesting that canonical Wnt may be upstream of Hedgehog in secondary palate development. Lastly, we found that GSK-3β -/- palate cultures were "rescued" with the Wnt inhibitor, Dkk-1., Conclusions: Here, we identify a critical role for GSK-3β in palatogenesis through its direct regulation of canonical Wnt signaling. These findings shed light on critical developmental pathways involved in palatogenesis and may lead to novel molecular targets to prevent cleft palate formation.

Background: Human adipose-derived stromal cells (hASCs) represent a multipotent cell stromal cell type with proven capacity to differentiate along an osteogenic lineage. This suggests that they may be used to heal defects of the craniofacial or appendicular skeleton. We sought to substantiate the use of undifferentiated hASCs in the regeneration of a non-healing mouse skeletal defect., Methodology/principal Findings: Human ASCs were harvested from female lipoaspirate. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an apatite coated PLGA scaffold alone, or a scaffold with human ASCs. MicroCT scans were obtained at stratified time points post-injury. Histology, in situ hybridization, and histomorphometry were performed. Near complete healing was observed among hASC engrafted calvarial defects. This was in comparison to control groups that showed little healing (*P<0.01). Human ASCs once engrafted differentiate down an osteogenic lineage, determined by qRT-PCR and histological co-expression assays using GFP labeled cells. ASCs were shown to persist within a defect site for two weeks (shown by sex chromosome analysis and quantified using Luciferase+ ASCs). Finally, rBMP-2 was observed to increase hASC osteogenesis in vitro and osseous healing in vivo., Conclusions/significance: Human ASCs ossify critical sized mouse calvarial defects without the need for pre-differentiation. Recombinant differentiation factors such as BMP-2 may be used to supplement hASC mediated repair. Interestingly, ASC presence gradually dissipates from the calvarial defect site. This study supports the potential translation for ASC use in the treatment of human skeletal defects.