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Salik Hussain1,*, Faris Al-Nsour1,*, Annette B Rice1, Jamie Marshburn1, Zhaoxia Ji2, Jeffery I Zink2, Brenda Yingling1, Nigel J Walker3, Stavros Garantziotis11Clinical Research Unit, National Institute of Environmental Health Sciences/National Institute of Health, Research Triangle Park, NC, 2UC Center for Environmental Implications of Nanotechnology University of California, Los Angeles, CA, 3Division of National Toxicology Program, National Institute of Environmental Health Sciences/National Institute of Health, Research Triangle Park, NC, USA*Both are principal authorsBackground: Cerium dioxide (CeO2) nanoparticles have potential therapeutic applications and are widely used for industrial purposes. However, the effects of these nanoparticles on primary human cells are largely unknown. The ability of nanoparticles to exacerbate pre-existing inflammatory disorders is not well documented for engineered nanoparticles, and is certainly lacking for CeO2 nanoparticles. We investigated the inflammation-modulating effects of CeO2 nanoparticles at noncytotoxic concentrations in human peripheral blood monocytes.Methods: CD14+ cells were isolated from peripheral blood samples of human volunteers. Cells were exposed to either 0.5 or 1 µg/mL of CeO2 nanoparticles over a period of 24 or 48 hours with or without lipopolysaccharide (10 ng/mL) prestimulation. Modulation of the inflammatory response was studied by measuring secreted tumor necrosis factor-alpha, interleukin-1beta, macrophage chemotactic protein-1, interferon-gamma, and interferon gamma-induced protein 10.Results: CeO2 nanoparticle suspensions were thoroughly characterized using dynamic light scattering analysis (194 nm hydrodynamic diameter), zeta potential analysis (-14 mV), and transmission electron microscopy (irregular-shaped particles). Transmission electron microscopy of CD14+ cells exposed to CeO2 nanoparticles revealed that these nanoparticles were efficiently internalized by monocytes and were found either in vesicles or free in the cytoplasm. However, no significant differences in secreted cytokine profiles were observed between CeO2 nanoparticle-treated cells and control cells at noncytotoxic doses. No significant effects of CeO2 nanoparticle exposure subsequent to lipopolysaccharide priming was observed on cytokine secretion. Moreover, no significant difference in lipopolysaccharide-induced cytokine production was observed after exposure to CeO2 nanoparticles followed by lipopolysaccharide exposure.Conclusion: CeO2 nanoparticles at noncytotoxic concentrations neither modulate pre-existing inflammation nor prime for subsequent exposure to lipopolysaccharides in human monocytes from healthy subjects.Keywords: cerium dioxide, nanoparticle, nanomedicine, inflammation, human monocyte, lipopolysaccharides

Detrimental Th17 driven inflammatory and autoimmune disease such as Crohn's disease, graft versus host disease and multiple sclerosis remain a significant cause of morbidity and mortality worldwide. Multipotent stromal/stem cell (MSC) inhibit Th17 polarization and activation in vitro and in rodent models. As such, MSC based therapeutic approaches are being investigated as novel therapeutic approaches to treat Th17 driven diseases in humans. The significance of naturally occurring diseases in dogs is increasingly recognized as a realistic platform to conduct pre-clinical testing of novel therapeutics. Full characterization of Th17 cells in dogs has not been completed. We have developed and validated a flow-cytometric method to detect Th17 cells in canine blood. We further demonstrate that Th17 and other IL17 producing cells are present in tissues of dogs with naturally occurring chronic inflammatory diseases. Finally, we have determined the kinetics of a canine specific Th17 polarization in vitro and demonstrate that canine MSC inhibit Th17 polarization in vitro, in a PGE2 independent mechanism. Our findings provide fundamental research tools and suggest that naturally occurring diseases in dogs, such as inflammatory bowel disease, may be harnessed to translate novel MSC based therapeutic strategies that target the Th17 pathway.

To address gaps in the quality of care for osteo-arthritis, the authors developed a Web-based computer program to provide patients with personalized feedback designed to improve the quality of their osteoarthritis care. The current study was designed to examine satisfaction as well as the potential effects of the feedback on patients' perceptions of their osteoarthritis care by randomizing patients to use the site before or after they answered questions about the quality of their osteoarthritis care. On average, participants received 8.7 recommendations to change their osteoarthritis care. Satisfaction with osteo-arthritis care was similar between subjects in both groups. Most subjects believed that the Web site would help them get better care from their doctor (77.7%), and most would recommend it to others (94.3%). Overall, the Web site is well accepted and has no negative effect on patients' satisfaction with their osteo-arthritis care. [ABSTRACT FROM AUTHOR]

Benzo[α]pyrene (B[α]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[α]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[α]P, with and without the addition of exogenous epoxide hydrolase, and B[α]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 μM. HPLC analysis detected eight major metabolites of B[α]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[α]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[α]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[α]P metabolism, P450 1B1 may significantly contribute to B[α]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans. [ABSTRACT FROM PUBLISHER]

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent tumor promoter in two-stage initiation-promotion models and induces cell proliferation and development of enzyme-altered hepatic foci. It is believed that increased cell proliferation is a necessary step in carcinogenesis. Therefore, the analysis of the effect of TCDD on cell proliferation in rat liver may aid in the understanding of the mechanism of hepatocarcinogenesis induced by TCDD. The aim of this study was to investigate the time course and reversibility of cell proliferation in non-initiated and diethylnitrosamine-initiated female rats exposed biweekly to a daily averaged dose of 125 ng TCDD/kg/day for up to 60 weeks. In addition we evaluated the suitability of different dose metrics for the evaluation of TCDD-induced changes in cell proliferation and CYP1A1 enzyme induction. Cell proliferation was measured as the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into hepatocytes undergoing replicative DNA synthesis. Mean BrdU labeling indices in TCDD-treated animals were not increased over controls after 14 weeks exposure, but were increased 8- and 2-fold after 30 and 60 weeks' treatment respectively, despite similar liver levels of TCDD at all these times (23-30 p.p.b.). In comparison, CYP1A1 activity, as measured by ethoxyresorufin deethylase activity, was significantly induced at all times points analyzed. Sixteen weeks following cessation of TCDD treatment, labeling indices were still significantly elevated over controls, but after 30 weeks of withdrawal, labeling indices were no different from controls, indicating that TCDD-induced changes in cell proliferation were reversible. Dosimetric analysis indicated that rat liver tissue burden was suitable for prediction of CYP1A1 expression but not cell proliferation and that the area under the curve was unsuitable for prediction of both TCDD-induced changes in CYP1A1 expression and cell proliferation. [ABSTRACT FROM PUBLISHER]

Knowledge of the response of cytochrome P450 1B1 (CYP1B1) to exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in both humans and rodents is limited. To improve the analysis of CYP1 proteins, specific CYP1B1 and CYP1A1 polypeptides were expressed as hexahistidine-tagged fusion proteins in Escherichia coli, purified to homogeneity and used to produce polyclonal antibodies in rabbits. Immunoblot analyses showed that these antibodies were specific and sensitive, detecting both the human and rat forms of the respective isozymes and exhibiting negligible cross-reactivity between the two known CYP1 subfamilies. We show that CYP1B1, CYP1A1 and CYP1A2 protein levels were induced in the livers of female Sprague-Dawley rats following either acute (single dose of 25 μg TCDD/kg or chronic (125 ng TCDD/kg/day for 30 weeks) exposure to TCDD. CYP1B1 protein exhibited a dose-response to TCDD that was different from those of CYP1A1 and CYP1A2. CYP1B1 induction appeared to be less sensitive to TCDD exposure, with induction occurring at higher doses of TCDD than that required for induction of CYP1A1 or CYP1A2. Immunohistochemical analysis showed that in animals chronically exposed to TCDD (35 ng/kg/day for 30 weeks), CYP1B1 was induced only in centrilobular hepatocytes, a pattern of expression similar to that of CYP1A1 and CYP1A2. These observations of cellular co-localization of the CYP1 cytochrome in livers of TCDD-treated rats and apparent differences in both protein amounts and dose-response are indicative of both common and unique regulation of CYP1 induction. [ABSTRACT FROM PUBLISHER]

Tularemia is a zoonotic disease caused by the facultative intracellular gram-negative bacterium Francisella tularensis. F. tularensis has a very low infection dose by the aerosol route which can result in an acute, and potentially lethal, infection in humans. Consequently, it is classified as a Category A bioterrorism agent by the US Centers for Disease Control (CDC) and is a pathogen of concern for the International Biodefence community. There are currently no licenced tularemia vaccines. In this study we report on the continued assessment of a tularemia subunit vaccine utilising β-glucan particles (GPs) as a vaccine delivery platform for immunogenic F. tularensis antigens. Using a Fischer 344 rat infection model, we demonstrate that a GP based vaccine comprising the F. tularensis lipopolysaccharide antigen together with the protein antigen FTT0814 provided partial protection of F344 rats against an aerosol challenge with a high virulence strain of F. tularensis, SCHU S4. Inclusion of imiquimod as an adjuvant failed to enhance protective efficacy. Moreover, the level of protection afforded was dependant on the challenge dose. Immunological characterisation of this vaccine demonstrated that it induced strong antibody immunoglobulin responses to both polysaccharide and protein antigens. Furthermore, we demonstrate that the FTT0814 component of the GP vaccine primed CD4+ and CD8+ T-cells from immunised F344 rats to express interferon-γ, and CD4+ cells to express interleukin-17, in an antigen specific manner. These data demonstrate the development potential of this tularemia subunit vaccine and builds on a body of work highlighting GPs as a promising vaccine platform for difficult to treat pathogens including those of concern to the bio-defence community., Competing Interests: The authors have declared that no competing interest exist., (Copyright: © 2024 Crown copyright, Dstl. This material is licensed under the terms of the Open Government Licence except where otherwise stated, which permits unrestricted use, distribution, and reproduction in any medium, provided you acknowledge the source of the Information or link to any attribution statement specified in the license. To view this licence, visit http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3 or write to the Information Policy Team, The National Archives, Kew, London TW9 4DU, or email: psi@nationalarchives.gov.uk.)

Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

Background: Transcription factors bind DNA in specific sequence contexts. In addition to distinguishing one nucleobase from another, some transcription factors can distinguish between unmodified and modified bases. Current models of transcription factor binding tend not to take DNA modifications into account, while the recent few that do often have limitations. This makes a comprehensive and accurate profiling of transcription factor affinities difficult., Results: Here, we develop methods to identify transcription factor binding sites in modified DNA. Our models expand the standard A/C/G/T DNA alphabet to include cytosine modifications. We develop Cytomod to create modified genomic sequences and we also enhance the MEME Suite, adding the capacity to handle custom alphabets. We adapt the well-established position weight matrix (PWM) model of transcription factor binding affinity to this expanded DNA alphabet. Using these methods, we identify modification-sensitive transcription factor binding motifs. We confirm established binding preferences, such as the preference of ZFP57 and C/EBPβ for methylated motifs and the preference of c-Myc for unmethylated E-box motifs., Conclusions: Using known binding preferences to tune model parameters, we discover novel modified motifs for a wide array of transcription factors. Finally, we validate our binding preference predictions for OCT4 using cleavage under targets and release using nuclease (CUT&RUN) experiments across conventional, methylation-, and hydroxymethylation-enriched sequences. Our approach readily extends to other DNA modifications. As more genome-wide single-base resolution modification data becomes available, we expect that our method will yield insights into altered transcription factor binding affinities across many different modifications., (© 2023. The Author(s).)

DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine., (© 2023. The Author(s).)

Background: Pseudothrombocytopenia may lead to the erroneous diagnosis of thrombocytopenia, resulting in unnecessary testing and treatment. The addition of exogenous substances to blood samples prior to collection has been shown to mitigate platelet (PLT) clumps in blood samples. Postcollection additives aiming to disaggregate PLT clumps have been largely unexplored., Objectives: We aimed to determine if the addition of amikacin to blood samples postcollection aids in the disaggregation of PLT clumps in cats and dogs., Methods: For this prospective study, EDTA-collected blood samples from 28 cats and 17 dogs were obtained from a hospital population at UC Davis Veterinary Medical Teaching Hospital. Samples had PLT clumps detected on blood smears and thrombocytopenia per analyzer count. Amikacin was added to samples postcollection, and an additional CBC was performed. Flow cytometry was performed to assess PLT-fibrinogen binding in amikacin-treated aliquots., Results: PLT-clumped samples treated with amikacin significantly increased PLT numbers by 134% and decreased mean platelet volume (MPV) values by 14% (P ≤ 0.0001) in cats, and increased PLT numbers by 32% (P = 0.04) and increased MPV values by 9% (P = 0.02) in dogs. Mean cell volume (MCV) slightly increased (<4%) for both species. No other CBC parameters were substantially affected by the addition of amikacin. Flow cytometry showed decreased PLT-fibrinogen binding in the majority of cats but was not significant (P > 0.05)., Conclusions: Adding amikacin to PLT-clumped blood samples postcollection may be a convenient solution for pseudothrombocytopenia in cats and dogs. Future studies are needed to elucidate the mechanism of amikacin and its effectiveness under different storage conditions. This is the first reported use of amikacin postcollection to disaggregate PLT clumps in blood samples from animals., (© 2023 The Authors. Veterinary Clinical Pathology published by Wiley Periodicals LLC on behalf of American Society for Veterinary Clinical Pathology.)

Early detection of cancer will improve survival rates. The blood biomarker 5-hydroxymethylcytosine has been shown to discriminate cancer. In a large covariate-controlled study of over two thousand individual blood samples, we created, tested and explored the properties of a 5-hydroxymethylcytosine-based classifier to detect colorectal cancer (CRC). In an independent validation sample set, the classifier discriminated CRC samples from controls with an area under the receiver operating characteristic curve (AUC) of 90% (95% CI [87, 93]). Sensitivity was 55% at 95% specificity. Performance was similar for early stage 1 (AUC 89%; 95% CI [83, 94]) and late stage 4 CRC (AUC 94%; 95% CI [89, 98]). The classifier could detect CRC even when the proportion of tumor DNA in blood was undetectable by other methods. Expanding the classifier to include information about cell-free DNA fragment size and abundance across the genome led to gains in sensitivity (63% at 95% specificity), with similar overall performance (AUC 91%; 95% CI [89, 94]). We confirm that 5-hydroxymethylcytosine can be used to detect CRC, even in early-stage disease. Therefore, the inclusion of 5-hydroxymethylcytosine in multianalyte testing could improve sensitivity for the detection of early-stage cancer., (© 2022. The Author(s).)

Background: The need for an updated plague vaccine is highlighted by outbreaks in endemic regions together with the pandemic potential of this disease. There is no easily available, approved vaccine. Methods: Here we have used a murine model of pneumonic plague to examine the factors that maximise immunogenicity and contribute to survival following vaccination. We varied vaccine type, as either a genetic fusion of the F1 and V protein antigens or a mixture of these two recombinant antigens, as well as antigen dose-level and formulation in order to correlate immune response to survival. Results: Whilst there was interaction between each of the variables of vaccine type, dose level and formulation and these all contributed to survival, vaccine formulation in protein-coated microcrystals (PCMCs) was the key contributor in inducing antibody titres. From these data, we propose a cut-off in total serum antibody titre to the F1 and V proteins of 100 µg/mL and 200 µg/mL, respectively. At these thresholds, survival is predicted in this murine pneumonic model to be >90%. Within the total titre of antibody to the V antigen, the neutralising antibody component correlated with dose level and was enhanced when the V antigen in free form was formulated in PCMCs. Antibody titre to F1 was limited by fusion to V, but this was compensated for by PCMC formulation. Conclusions: These data will enable clinical assessment of this and other candidate plague vaccines that utilise the same vaccine antigens by identifying a target antibody titre from murine models, which will guide the evaluation of clinical titres as serological surrogate markers of efficacy.

Background: Multiple surgical techniques are described for basal joint osteoarthritis. This study compares clinical outcomes and patient satisfaction with trapeziectomy and abductor pollicis longus (APL) suspensionplasty compared to trapeziectomy with ligament reconstruction and tendon interposition (LRTI) when performed by 2 fellowship-trained hand surgeons. Methods: A retrospective review of 51 consecutive patients undergoing APL suspensionplasty (53 hands) was performed. With this technique, a distally based APL slip is brought through and sewn to the flexor carpi radialis (FCR). The remaining APL is placed in the trapeziectomy void. This technique was compared to 151 patients (166 hands) who underwent LRTI using the FCR tendon. Outcomes assessed included postoperative pain relief, grip and pinch strength, complications, and need for reoperation. Two-tailed, Fisher's exact test was used for data analysis. Results: APL suspensionplasty resulted in postoperative pain relief in 92.5% (n = 49) compared to 94.0% (n = 156) with LRTI ( P = .758). Mean postoperative grip and pinch strengths with APL suspensionplasty were 41.2 and 10.4 kg, respectively. With LRTI, average grip strength was 42.0 kg, and pinch was 10.1 kg. Both techniques were well tolerated with minimal complications. In the APL group, 1 patient had a postoperative infection requiring drainage. Among the LRTI cases, 1 wound dehiscence required closure, and 2 minor postoperative wound infections resolved with oral antibiotics. Mean follow-up time among APL suspensionplasty patients was 3.3 months compared to 8.4 months following LRTI. Conclusions: APL suspensionplasty is a safe, effective procedure which provides similar pain relief and functional outcomes compared to LRTI.

Two monoclonal antibodies directed to the V antigen of Yersinia pestis have been tested for protective efficacy in a murine model of bubonic plague. Mice were infected with a current clinical isolate from Madagascar, designated Y. pestis 10-21/S. Mab7.3, delivered to mice intra-periteoneally at either 24 h prior to, or 24 h post-infection, was fully protective, building on many studies which have demonstrated the protective efficacy of this Mab against a number of different clinical isolates of Y. pestis . Mab 29.3, delivered intra-peritoneally at either -24 h or +24 h, protected 4/5 mice in either condition; this has demonstrated the protective efficacy of this Mab in vivo for the first time. These results add to the cumulative data about Mab7.3, which is currently being humanized and highlight its potential as a human immunotherapeutic for plague, which is an enduring endemic disease in Madagascar and other regions of Africa, Asia, and South America., (© Crown copyright 2021.)

Background: Feline adipose-derived mesenchymal stem cells (ASCs) engage with a variety of immune cells and have been used in several clinical trials for the treatment of inflammatory and immune-dysregulated diseases in cats, but the impact they exert on the functional characteristics on T cells, particularly CD8+ T cells, remains to be elucidated., Methods: Modified mixed leukocyte reaction was performed between feline ASCs and PBMCs. Changes of cell cycle stages, phenotype and cellular senescence were determined through flow cytometry and gene expression analysis. Cytotoxicity assay was performed to evaluate CD8+ T cell effector function., Results: Feline ASCs induce cell cycle arrest on CD8+ T cells in a contact-dependent manner, downregulate CD8 surface expression, and shift their phenotype toward terminally differentiated effector cells (CD57+, CD45R+, CD62L-). CD8 T cells interacted with feline ASCs also upregulated granzyme B, IL-2 and KLRG-1 expression and have enhanced cytotoxic potential, evident by the increased percentage of lysis on target cells., Conclusions: Our findings suggest that feline ASCs (1) alter CD8+ T cells toward terminally differentiated, proinflammatory effector phenotype with limited proliferative capacity, and (2) enhance their cytotoxic potential through granzyme B upregulation. These cytotoxic CD8+ T cells could aid in disease cure in cases caused by an underlying, unresolved viral infection., (© 2021. The Author(s).)

Background: The effect of body mass index (BMI) on complication rates in prepectoral implant-based breast reconstruction is not well established. The purpose of this study was to compare complication rates between different BMI groups in subpectoral and prepectoral reconstruction., Methods: A single-surgeon, 4-year, retrospective review was performed of consecutive prosthetic breast reconstructions. During this time, the senior author's practice shifted from a subpectoral to prepectoral technique. Patients were stratified into BMI subgroups (<25, 25-35, and >35 kg/m2) and complication rates were analyzed. A survey was administered to blinded medical personnel and patients comparing esthetic results., Results: Implant-based reconstructions were performed in 195 patients (103 subpectoral and 92 prepectoral). No significant difference in major complication rate was observed between techniques. Among patients with BMI greater than 35 kg/m2, implant exposure occurred at a significantly higher rate in the prepectoral group (P = 0.04). In patients with BMI greater than 25 kg/m2, minor asymmetry was more prevalent with prepectoral reconstruction (12.3% vs 0%; P = 0.02). Regardless of technique, the odds of reoperation increased by 7% per point increase in BMI, although this did not reach statistical significance (P = 0.07; odds ratio, 1.07; 95% confidence interval, 0.99-1.15).A total of 66 survey responses were received. Physicians rated esthetic results more positively than patients did. Patients with a BMI of less than 25 kg/m2 were rated better than other BMI groups in nearly all categories. The position of submuscular reconstruction was rated significantly better than prepectoral., Conclusions: There is a trend toward higher complication rates in prepectoral versus subpectoral breast reconstruction with increasing BMI. Nonetheless, the technique appears to be safe, with comparable clinical and cosmetic results., Competing Interests: Conflicts of interest and sources of funding: none declared., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Reconstruction of soft tissue defects after skin cancer excision remains a challenge. Options for reconstruction are numerous, including primary repair, local tissue rearrangement, and skin grafts, among others. In this series, the authors present a novel technique: The triangular dart flap. This is a single-stage tissue rearrangement that uses the redundant tissue of the dog-ear to aid in the closure of these wounds., Methods: A retrospective review was conducted of all patients undergoing local tissue rearrangements by the senior author from 2009 to 2018. Factors were collected and analyzed, including age, size and cause of defect, comorbidities, smoking history, and postoperative complications., Results: Twenty-four patients underwent reconstruction with a triangular dart flap for repair of malignant defects. Mean defect size was 7.3 cm2 (0.8-20 cm2), and mean repair size was 29.7 cm2 (6-80 cm2). Initial pathology included basal cell carcinoma (45.8%), melanoma in situ (29.2%), and squamous cell carcinoma (16.7%), among others. Location varied widely among face and extremities. Anesthesia was predominantly local only (79.1%). There were no major complications, and 5 (20.8%) minor complications., Conclusions: The triangular dart flap is a novel single-stage procedure, generally performed under local anesthesia only, for correction of Mohs defects. By using the redundant tissue of dog-ears to better approximate the wound edges, a tension-free primary closure can be achieved in sensitive areas, such as the nasal tip., Competing Interests: Conflicts of interest and sources of funding: none declared., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objectives: The aim of this pilot study was to determine the safety, efficacy and immunomodulatory function of systemically administered adipose-derived mesenchymal stem cells (ASCs) in cats affected by feline chronic gingivostomatitis (FCGS) prior to full-mouth tooth extractions., Methods: Five client-owned cats affected with FCGS that did not undergo full-mouth tooth extractions for FCGS treatment received two intravenous injections of 20 million fresh, allogeneic or autologous ASCs. An oral examination with photographs, a complete blood count, blood immune cell phenotyping and a biochemical profile were completed at 0 and 6 months after treatment., Results: Four cats completed the study and one cat exited the study 3 months after treatment. While the treatment was determined to be clinically safe, no positive clinical response was observed in three cats and a mild response was noted in two cats. Furthermore, none of the cats exhibited immune modulation, as evidenced by no alteration in circulating CD8 + T cells, normalization of the CD4:CD8 ratio or neutrophil counts., Conclusions and Relevance: Unlike the reported efficacy of ASCs in treating cats with non-responsive FCGS after full-mouth tooth extraction, the systemic administration of ASCs prior to full-mouth tooth extraction lacks substantial clinical efficacy and is not recommended at this time.

Background: Canine inflammatory brain disease (IBD) is a severe inflammatory disorder characterized by infiltration of activated immune cell subsets into the brain and spinal cord. Multipotent mesenchymal stromal cells (MSCs) are a promising therapy for IBD, based on their potent pro-angiogenic, neuroprotective, and immunomodulatory properties. The aims of this study were to compare the immunomodulatory attributes of canine adipose-derived MSCs (ASCs) and placenta-derived MSCs (PMSCs) in vitro. These data will serve as potency information to help inform the optimal MSC cell source to treat naturally occurring canine IBD., Methods: Indoleamine 2,3 dioxygenase (IDO) activity and prostaglandin E 2 (PGE 2 ) concentration at baseline and after stimulation with interferon gamma (IFNγ) and/or tumor necrosis factor alpha (TNFα) were measured from canine ASC and PMSC cultures. Leukocyte suppression assays (LSAs) were performed to compare the ability of ASCs and PMSCs to inhibit activated peripheral blood mononuclear cell (PBMC) proliferation. IDO activity and PGE 2 ; interleukin (IL)-2, IL-6, and IL-8; TNFα; and vascular endothelial growth factor (VEGF) concentrations were also measured from co-culture supernatants. Cell cycle analysis was performed to determine how ASCs and PMSCs altered lymphocyte proliferation., Results: Activated canine MSCs from both tissue sources secreted high concentrations of IDO and PGE 2 , after direct stimulation with IFNγ and TNFα, or indirect stimulation by activated PBMCs. Both ASCs and PMSCs inhibited activated PBMC proliferation in LSA assays; however, PMSCs inhibited PBMC proliferation significantly more than ASCs. Blocking PGE 2 and IDO in LSA assays determined that PGE 2 is important only for ASC inhibition of PBMC proliferation. Activated ASCs increased IL-6 and VEGF secretion and decreased TNFα secretion, while activated PMSCs increased IL-6, IL-8, and VEGF secretion. ASCs inhibited lymphocyte proliferation via cell cycle arrest in the G0/G1 and PMSCs inhibited lymphocyte proliferation via induction of lymphocyte apoptosis., Conclusion: Our results demonstrate that ASCs and PMSCs have substantial in vitro potential as a cell-based therapy for IBD; however, PMSCs more potently inhibited lymphocyte proliferation by inducing apoptosis of activated lymphocytes. These data suggest that the mechanism by which ASCs and PMSCs downregulate PBMC proliferation differs. Additional studies may elucidate additional mechanisms by which canine MSCs modulate neuroinflammatory responses.

Feline chronic gingivostomatitis (FCGS) is an immune-mediated inflammatory condition affecting the oral mucosa that results in substantial pain and suffering. The goal of this study was to complete an in-depth immunohistochemistry analysis of affected FCGS mucosa, to perform and compare immune cell phenotypes in the blood of FCGS and healthy controls cats, and to determine a transcriptomic profile of the affected and normal oral mucosa of FCGS cats. We hypothesized that cats with FCGS would have circulating activated CD8+ T cells and that tissues would be infiltrated with activated B and T cells with a highly proinflammatory transcriptome. We found that oral mucosal tissues from cats with FCGS have high tissue infiltration of B cells and that T cells include both CD4+ and CD8+ lymphocytes. Cells positive for CD25 (IL2 receptor, indicative of lymphocyte activation) and FOXP3 (indicative of regulatory T cells) were scattered throughout the mucosa. Compared to healthy individuals, cats with FCGS had high circulating CD8+ effector memory cells with a concurrent decrease in central memory cells and evidence of circulating activated CD8+ T cells (CD25+, CD62L-). Gene expression in the affected tissues was enriched for genes associated with T-cell signaling, cell adhesion molecules, leukocyte migration, inflammatory signaling pathways, extracellular matrix-receptor interactions, cytokine-cytokine receptor interactions, and natural killer cell-mediated cytotoxicity, among others. These data are essential to understand disease pathogenesis, to inform mechanism of action studies for future and current therapies, and to help select prognostic biomarkers and potency assays for stem cell treatment of FCGS., (Copyright © 2020 Vapniarsky, Simpson, Arzi, Taechangam, Walker, Garrity, Bulkeley and Borjesson.)

Background: The ability of mesenchymal stem cells (MSCs) to modulate immune responses inspired a series of clinical trials addressing oral mucosal inflammation. We previously reported on the safety and efficacy of fresh, allogeneic and autologous, adipose-derived mesenchymal stem cells (ASCs) to treat feline gingivostomatitis (FCGS), an oral mucosal inflammatory disease that shares similarities with human oral lichen planus., Methods: To meet clinical demand and goals for future commercialization, we determined the feasibility of shipping fresh ASCs to distant clinics and extended our pilot studies to expand safety and efficacy data for shipped and non-shipped ASCs in a cohort of 18 FCGS cats enrolled locally and at a few different locations within the USA., Results: We found that ASCs retained their viability, phenotype, and function after shipment. ASCs administered systemically resulted in a 72% positive response rate, identical to that noted in our previous studies. Cats that responded to ASC therapy had a significant decrease in circulating globulin concentration and histological evidence of decreased CD3+ T cells and CD20+ B cells in the oral mucosa. Responder cats also had significantly decreased percentages of CD8 lo cells in blood prior to and at 3 months post-ASC therapy. CD8 lo cells may serve as a potential "predictor" for response to systemic ASC therapy., Conclusion: Fresh feline ASCs can be successfully shipped and administered to cats with FCGS. ASCs modulate the immune response and demonstrate efficacy for chronic oral mucosal inflammatory lesions that are characterized by CD8+ T cell inflammation and T cell activation. FCGS is a potentially useful naturally occurring large animal model of human oral inflammatory diseases.

Equine recurrent uveitis (ERU) is an immune-mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T-cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T-cell phenotype. Adipose-derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4 + T-cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T-cells and B-cells in horses with ERU were similar to normal horses. However, CD4 + T-cells from horses with ERU expressed higher amounts of IFNγ indicating a pro-inflammatory Th1 phenotype. When co-incubated with MSCs, activated CD4 + T-cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell-cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4 + T-cell activation phenotype through a combination of cell-cell contact and prostaglandin signaling., (© 2019 The Authors. Veterinary Ophthalmology published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Ophthalmologists.)

Botanical safety science continues to evolve as new tools for risk assessment become available alongside continual desire by consumers for "natural" botanical ingredients in consumer products. Focusing on botanical food/dietary supplements a recent international roundtable meeting brought together scientists to discuss the needs, available tools, and ongoing data gaps in the botanical safety risk assessment process. Participants discussed the key elements of botanical safety evaluations. They provided perspective on the use of a decision tree methodology to conduct a robust risk assessment and concluded with alignment on a series of consensus statements. This discussion highlighted the strengths and vulnerabilities in common assumptions, and the participants shared additional perspective to ensure that this end-to-end safety approach is sufficient, actionable and timely. Critical areas and data gaps were identified as opportunities for future focus. These include, better context on history of use, systematic assessment of weight of evidence, use of in silico approaches, inclusion of threshold of toxicological concern considerations, individual substances/matrix interactions of plant constituents, assessing botanical-drug interactions and adaptations needed to apply to in vitro and in vivo pharmacokinetic modelling of botanical constituents., (Copyright © 2019. Published by Elsevier B.V.)

Fullerene C60 is used in a variety of industrial and consumer capacities. As part of a comprehensive evaluation of the toxicity of fullerene C60 by the National Toxicology Program, the disposition following intratracheal (IT) instillation and intravenous (IV) administration of 1 or 5 mg/kg b.wt. fullerene C60 was investigated in male Fischer 344 rats. Following IT instillation, fullerene C60 was detected in the lung as early as 0.5 h post-exposure with minimal clearance over the 168 h period; the concentration increased ≥20-fold with a 5-fold increase in the dose. Fullerene C60 was not detected in extrapulmonary tissues. Following IV administration, fullerene C60 was rapidly eliminated from the blood and was undetectable after 0.5 h post-administration. The highest tissue concentrations of fullerene C60 occurred in the liver, followed by the spleen, lung and kidney. Fullerene C60 was cleared slowly from the kidney and the lung with estimated half-lives of 24 and 139 h, respectively. The liver concentration of fullerene C60 did not change much with time; over 90% of the fullerene C60 remained there over the study duration up to 168 h. Fullerene C60 was also not detected in urine or feces. These data support the hypothesis that fullerene C60 accumulates in the body and therefore has the potential to induce detrimental health effects following exposure.

Background: Feline adipose-derived mesenchymal stem cells (ASCs) have been successfully used in clinical trials for the treatment of immune-mediated diseases with T cell dysregulation. However, the immunomodulatory pathways utilized by feline ASCs to suppress T cell activation have not been fully determined. We investigated the mechanisms used by feline ASCs to inhibit T cell proliferation, including the soluble factors and the cell-cell contact ligands responsible for ASC-T cell interaction., Methods: The immunomodulatory activity of feline ASCs was evaluated via cell cycle analysis and in vitro mixed leukocyte reaction using specific immunomodulatory inhibitors. Cell-cell interactions were assessed with static adhesion assays, also with inhibitors., Results: Feline ASCs decrease T cell proliferation by causing cell cycle arrest in G0-G1. Blocking prostaglandin (PGE 2 ), but not IDO, partially restored lymphocyte proliferation. Although PDL-1 and CD137L are both expressed on activated feline ASCs, only the interaction of intercellular adhesion molecule 1 (ICAM-1, CD54) with its ligand, lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), was responsible for ASC-T cell adhesion. Blocking this interaction reduced cell-cell adhesion and mediator (IFN-γ) secretion and signaling., Conclusions: Feline ASCs utilize PGE 2 and ICAM-1/LFA-1 ligand interaction to inhibit T cell proliferation with a resultant cell cycle arrest in G0-G1. These data further elucidate the mechanisms by which feline ASCs interact with T cells, help define appropriate T cell-mediated disease targets in cats that may be amenable to ASC therapy, and may also inform potential translational models for human diseases.

Background: The opioid crisis in America has sparked a shift toward a multimodality perioperative pain regimen. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) in the perioperative period decreases opioid consumption and increases efficacy. However, many plastic surgeons avoid their use because of antiplatelet effects. The purpose of this article is to systematically review the plastic surgery literature to assess the risk of intraoperative or postoperative bleeding and hematoma formation., Methods: A systematic review of articles published in PubMed was performed in September 2018 to investigate the incidence of increased bleeding and hematoma formation with use of NSAIDs in the perioperative period in plastic surgery. All articles were reviewed for primary outcome measures, and a selective literature review was performed to examine perioperative NSAID use in other surgical subspecialties. Random-effect meta-analysis was performed., Results: Our search yielded 806 total articles, with 15 meeting inclusion criteria, and this included 3064 patients (1679 with perioperative NSAIDs, 1385 with no NSAIDs). There was no significant difference in overall incidence of bleeding/hematoma in the treatment group versus control (no NSAIDs). The overall pooled odds ratio (OR) and corresponding 95% confidence interval were 1.20 and 0.73 to 1.97 (P = 0.48). When separated by drug administered across all plastic surgery procedures, there were no statistically significant differences in incidences of hematoma or increased bleeding with use of ketorolac (OR, 1.48 [0.86-2.56]; P = 0.57), ibuprofen (OR, 0.55 [0.14-2.14]; P = 0.87), or celecoxib (OR, 0.22 [0.02-2.52]; P = 0.39). When examining NSAID use in breast surgery, there was no statistically significant difference in incidence of hematoma or increased bleeding when combining all 3 drug types (OR, 1.39 [0.82-2.37]; P = 0.60). Some individual studies demonstrated trends toward increased bleeding/hematoma in reduction mammoplasties., Conclusions: Nonsteroidal anti-inflammatory drugs significantly improve pain control and decrease opioid use when used in plastic surgery. The majority of evidence in plastic surgery does not support an increased incidence of bleeding/hematoma with the use of perioperative NSAIDs.

Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection., (© 2019 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

Background: Forehead flap reconstruction of large nasal defects can be challenging. The senior author has used a paramedian forehead flap modification using the supratrochlear artery on the contralateral side of the defect., Methods: A 9-year retrospective review (2008-2016) was performed for patients undergoing nasal reconstruction with the cross-paramedian forehead flap. Outcomes were analyzed by comparing our previous reviews, which allows us to analyze patient outcomes for over 19 years., Results: Fifty-three patients were identified. The aasal defect was most frequently due to basal cell carcinoma (n = 37, 69.8%). Twenty-three (43%) patients were smokers, and nine (17%) had diabetes. The mean defect size was 12.9 cm 2 , involving an average of 2.6 nasal subunits. One-third of the patients had cartilage defects (n = 18) and mucosal lining defects (n = 19). Periorbital involvement was present in five patients. Complications included partial flap loss (n = 6), donor site dehiscence (n = 4), flap dehiscence (n = 2), and postoperative infection (n = 1). Only two of the partial flap losses were considered significant, as they required additional reconstructive procedures for soft tissue coverage. Complications were 12 times as likely as those in diabetes (OR = 11.97, p = 0.007, 95% CI 1.94-72.44), six times as likely as those in cartilage defects (OR = 6.4, p = 0.007, 95% CI 1.64-24.92), and nearly five times as likely as those in mucosal lining defects (OR = 4.8, p = 1.27-18.09, 95% CI 1.27-18.09). Thirty-one patients required revisions most commonly for flap debulking (n = 16)., Conclusion: The cross-paramedian forehead flap is a reliable option in the armamentarium of the reconstructive surgeon for large and complex defects in addition to those with periorbital extension., Summary: Coverage of distal nasal defects after tumor extirpation remains a challenge to the reconstructive surgeon. Our institution uses the cross-paramedian forehead flap for these defects. This flap is based on the supratrochlear artery on the contralateral side of the defect and is oriented obliquely across the forehead for additional length and an improved donor site scar at the level of the eyebrow. The technique and outcomes were published in 2009, and this manuscript serves as an update on outcomes and applications during the past 9 years. By including all our data, we can analyze outcomes for over 19 years. During the past 9 years, 53 patients underwent the cross-paramedian forehead flap technique between 2008 and 2016. These patients were found to have an average defect size of 12.9 cm 2 and an average loss of 2.6 nasal subunits. Cartilage defects were present in 34.6% (n = 18) and mucosal defects were present in 36.5% (n = 19) of patients. Five patients had periorbital reconstruction with the forehead flap, of which three patients underwent a single-stage islandized forehead flap reconstruction. Given the large defect size, additional local flaps were frequently used, including nasolabial flaps (n = 16) and cheek rearrangement (n = 11). Complications included partial flap loss (n = 6), donor site dehiscence (n = 4), and postoperative infection (n = 1). Only two of these partial flap losses were considered significant, as they required additional reconstructive procedures to address areas of soft tissue loss. Increased rates of complications were associated with the presence of diabetes and defect characteristics, which reflects increased complexity including mucosal and cartilage loss. When comparing with our prior review of this technique, the more recent population have had increasing complexity of the nasal defects with a large surface area involvement. Overall, the cross-paramedian forehead flap is a reliable option in the armamentarium of the reconstructive surgeon for large and distal nasal defects., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Increasing evidence from rodent studies indicates that inhaled multi-walled carbon nanotubes (MWCNTs) have harmful effects on the lungs. In this study, we examined the effects of inhalation exposure to MWCNTs on allergen-induced airway inflammation and fibrosis. We hypothesized that inhalation pre-exposure to MWCNTs would render mice susceptible to developing allergic lung disease induced by house dust mite (HDM) allergen. Methods: Male B6C3F1/N mice were exposed by whole-body inhalation for 6 h a day, 5 d a week, for 30 d to air control or 0.06, 0.2, and 0.6 mg/m 3 of MWCNTs. The exposure atmospheres were agglomerates (1.4-1.8 µm) composed of MWCNTs (average diameter 16 nm; average length 2.4 µm; 0.52% Ni). Mice then received 25 µg of HDM extract by intranasal instillation 6 times over 3 weeks. Necropsy was performed at 3 and 30 d after the final HDM dose to collect serum, bronchoalveolar lavage fluid (BALF), and lung tissue for histopathology. Results: MWCNT exposure at the highest dose inhibited HDM-induced serum IgE levels, IL-13 protein levels in BALF, and airway mucus production. However, perivascular and peribronchiolar inflammatory lesions were observed in the lungs of mice at 3 d with MWCNT and HDM, but not MWCNT or HDM alone. Moreover, combined HDM and MWCNT exposure increased airway fibrosis in the lungs of mice. Conclusions: Inhalation pre-exposure to MWCNTs inhibited HDM-induced TH2 immune responses, yet this combined exposure resulted in vascular inflammation and airway fibrosis, indicating that MWCNT pre-exposure alters the immune response to allergens.

Background: The National Health System in Wales has developed a novel national electronic dashboard which reports a daily "escalation score," reflecting management's opinion of the pressure each hospital is facing, primarily due to unscheduled care. The aim of this study was to examine the possibility of replacing human scores with a quantitative model, based on the relationship between reported escalation scores and selected hospital metrics., Methods: Generalized linear mixed models were used to model the association between hospital metrics and escalation scores between October one year and October the next year utilizing hospital bed occupancy rate, ambulance hours lost waiting outside emergency departments, number of "boarded out" patients in the hospital, and the daily ratio of admissions to discharges in the hospital. These models were tested against a subsequent period (December unto May the following year), using three models: "general," "hospital-specific," and "group-specific." The model generated by the initial time frame was tested against data from the subsequent time frame using weighted κ., Results: Across 16 hospitals, using 3343 escalation scores, the rates of agreement and weighted κ were: general model (48.8%; 0.16), hospital-specific model (45.0%; 0.25), and group-specific model (43.1%; 0.25). A 17th small hospital was excluded due to missing data., Conclusions: This is novel research as no similar studies were identified, although the topic is important as it addresses a major current health-care challenge. Automated scores can be derived which have the advantage of being derived objectively, avoiding human inter- and intraindividual variation. Prospective testing is recommended to assess potential service planning benefit., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Introduction: Recent nationwide surveys found that natural products, including botanical dietary supplements, are used by ∼18% of adults. In many cases, there is a paucity of toxicological data available for these substances to allow for confident evaluations of product safety. The National Toxicology Program (NTP) has received numerous nominations from the public and federal agencies to study the toxicological effects of botanical dietary supplements. The NTP sought to evaluate the utility of in vitro quantitative high-throughput screening (qHTS) assays for toxicological assessment of botanical and dietary supplements. Materials and Methods: In brief, concentration-response assessments of 90 test substances, including 13 distinct botanical species, and individual purported active constituents were evaluated using a subset of the Tox21 qHTS testing panel. The screen included 20 different endpoints that covered a broad range of biologically relevant signaling pathways to detect test article effects upon endocrine activity, nuclear receptor signaling, stress response signaling, genotoxicity, and cell death signaling. Results and Discussion: Botanical dietary supplement extracts induced measurable and diverse activity. Elevated biological activity profiles were observed following treatments with individual chemical constituents relative to their associated botanical extract. The overall distribution of activity was comparable to activities exhibited by compounds present in the Tox21 10K chemical library. Conclusion: Botanical supplements did not exhibit minimal or idiosyncratic activities that would preclude the use of qHTS platforms as a feasible method to screen this class of compounds. However, there are still many considerations and further development required when attempting to use in vitro qHTS methods to characterize the safety profile of botanical/dietary supplements., Competing Interests: The authors declare no apparent conflicts of interest.

There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure media (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.

The National Toxicology Program (NTP) receives requests to evaluate chemicals with potential to cause adverse health effects, including developmental neurotoxicity (DNT). Some recent requests have included classes of chemicals such as flame retardants, polycyclic aromatic compounds, perfluoroalkyl substances, and bisphenol A analogs with approximately 20-50 compounds per class, many of which include commercial mixtures. However, all the compounds within a class cannot be tested using traditional DNT animal testing guideline studies due to resource and time limitations. Hence, a rapid and biologically relevant screening approach is required to prioritize compounds for further in vivo testing. Because neurodevelopment is a complex process involving multiple distinct cellular processes, one assay will unlikely address the complexity. Hence, the NTP sought to characterize a battery of in vitro and alternative animal assays to quantify chemical effects on a variety of neurodevelopmental processes. A culmination of this effort resulted in a NTP-hosted collaborative project with approximately 40 participants spanning across domains of academia, industry, government, and regulatory agencies; collaborators presented data on cell-based assays and alternative animal models that was generated using a targeted set of compounds provided by the NTP. The NTP analyzed the assay results using benchmark concentration (BMC) modeling to be able to compare results across the divergent assays. The results were shared with the contributing researchers on a private web application during the workshop, and are now publicly available. This article highlights the overview and goals of the project, and describes the NTP's approach in creating the chemical library, development of NTPs data analysis strategy, and the structure of the web application. Finally, we discuss key issues with emphasis on the utility of this approach, and knowledge gaps that need to be addressed for its use in regulatory decision making.

Imprinting is the preferential expression of one parental allele over the other. It is controlled primarily through differential methylation of cytosine at CpG dinucleotides. Here we combine 285 methylomes and 11,617 transcriptomes from peripheral blood samples with parent-of-origin phased haplotypes, to produce a new map of imprinted methylation and gene expression patterns across the human genome. We demonstrate how imprinted methylation is a continuous rather than a binary characteristic. We describe at high resolution the parent-of-origin methylation pattern at the 15q11.2 Prader-Willi/Angelman syndrome locus, with nearly confluent stochastic paternal methylation punctuated by 'spikes' of maternal methylation. We find examples of polymorphic imprinted methylation unrelated (at VTRNA2-1 and PARD6G) or related (at CHRNE) to nearby SNP genotypes. We observe RNA isoform-specific imprinted expression patterns suggestive of a methylation-sensitive transcriptional elongation block. Finally, we gain new insights into parent-of-origin-specific effects on phenotypes at the DLK1/MEG3 and GNAS loci.

Francisella tularensis is an intracellular pathogen causing the disease tularemia, and an organism of concern to biodefence. There is no licensed vaccine available. Subunit approaches have failed to induce protection, which requires both humoral and cellular immune memory responses, and have been hampered by a lack of understanding as to which antigens are immunoprotective. We undertook a preliminary in silico analysis to identify candidate protein antigens. These antigens were then recombinantly expressed and encapsulated into glucan particles (GPs), purified Saccharomyces cerevisiae cell walls composed primarily of β-1,3-glucans. Immunological profiling in the mouse was used to down-selection to seven lead antigens: FTT1043 (Mip), IglC, FTT0814, FTT0438, FTT0071 (GltA), FTT0289, FTT0890 (PilA) prior to transitioning their evaluation to a Fischer 344 rat model for efficacy evaluation. F344 rats were vaccinated with the GP protein antigens co-delivered with GP-loaded with Francisella LPS. Measurement of cell mediated immune responses and computational epitope analysis allowed down-selection to three promising candidates: FTT0438, FTT1043 and FTT0814. Of these, a GP vaccine delivering Francisella LPS and the FTT0814 protein was able to induce protection in rats against an aerosol challenge of F. tularensis SchuS4, and reduced organ colonisation and clinical signs below that which immunisation with a GP-LPS alone vaccine provided. This is the first report of a protein supplementing protection induced by LPS in a Francisella vaccine. This paves the way for developing an effective, safe subunit vaccine for the prevention of inhalational tularemia, and validates the GP platform for vaccine delivery where complex immune responses are required for prevention of infections by intracellular pathogens., Competing Interests: We have the following interests. Jane Homan is employed by the commercial company: ioGenetics. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

The National Toxicology Program's (NTP) mission is "to evaluate agents of public health concern, by developing and applying the tools of modern toxicology and molecular biology." Botanical dietary supplements (BDS) represent agents of public health concern due to widespread exposure to high doses, a lack of safety data for most products, variable quality, and reports of adverse events. This commentary will address lessons learned in NTP testing activities with BDS and recommendations for moving forward., (© 2018 American Society for Clinical Pharmacology and Therapeutics.)

Due to the extensive use of botanical dietary supplements by consumers in the United States, there is a need for appropriate research and data to support safety assessments. Complexity and variability, both natural and introduced, of botanical dietary supplements make research on these products difficult. Botanical dietary supplements are regulated by the Food and Drug Administration (FDA) under the Federal Food, Drug, and Cosmetic Act (FD&C Act), as amended by the 1994 Dietary Supplement Health and Education Act (DSHEA). They are regulated as a category of food, which differs from the regulation of pharmaceutical products. Both manufacturers and the FDA are faced with the challenge of determining the best approaches for evaluating and monitoring the safety of botanical products. High quality botanicals research requires accurate identification and characterization of the material being studied. Inconsistent results in efficacy studies of botanical dietary supplements have led to efforts to improve the rigor and reproducibility of research in the field. Addressing the challenges associated with botanical dietary supplement safety is a global effort requiring coordination between numerous stakeholders, including researchers, suppliers, manufacturers, and regulators, all of whom play a role in ensuring that high quality products are available on the market.

Joint injury is a common cause of premature retirement for the human and equine athlete alike. Implantation of engineered cartilage offers the potential to increase the success rate of surgical intervention and hasten recovery times. Mesenchymal stem cells (MSCs) are a particularly attractive cell source for cartilage engineering. While bone marrow-derived MSCs (BM-MSCs) have been most extensively characterized for musculoskeletal tissue engineering, studies suggest that cord blood MSCs (CB-MSCs) may elicit a more robust chondrogenic phenotype. The objective of this study was to determine a superior equine MSC source for cartilage engineering. MSCs derived from bone marrow or cord blood were stimulated to undergo chondrogenesis through aggregate redifferentiation and used to generate cartilage through the self-assembling process. The resulting neocartilage produced from either BM-MSCs or CB-MSCs was compared by measuring mechanical, biochemical, and histological properties. We found that while BM constructs possessed higher tensile properties and collagen content, CB constructs had superior compressive properties comparable to that of native tissue and higher GAG content. Moreover, CB constructs had alkaline phosphatase activity, collagen type X, and collagen type II on par with native tissue suggesting a more hyaline cartilage-like phenotype. In conclusion, while both BM-MSCs and CB-MSCs were able to form neocartilage, CB-MSCs resulted in tissue more closely resembling native equine articular cartilage as determined by a quantitative functionality index. Therefore, CB-MSCs are deemed a superior source for the purpose of articular cartilage self-assembly.

Summary: The National Institute of Environmental Health Sciences (NIEHS) introduces a new translational research framework that builds upon previous biomedical models to create a more comprehensive and integrated environmental health paradigm. The framework was developed as a graphical construct that illustrates the complexity of designing, implementing, and tracking translational research in environmental health. We conceptualize translational research as a series of concentric rings and nodes, defining "translation" as movement either from one ring to another or between nodes on a ring. A "Fundamental Questions" ring expands upon the research described in other frameworks as "basic" to include three interrelated concepts critical to basic science research: research questions, experimental settings, and organisms. This feature enables us to capture more granularity and thus facilitates an approach for categorizing translational research and its growth over time. We anticipate that the framework will help researchers develop compelling long-term translational research stories and accelerate public health impacts by clearly mapping out opportunities for collaborations. By using this paradigm, researchers everywhere will be better positioned to design research programs, identify research partners based on cross-disciplinary research needs, identify stakeholders who are likely to use the research for environmental decision-making and intervention, and track progress toward common goals. https://doi.org/10.1289/EHP3657.