Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Evaluation of training, patient and practitioner perspectives on community-based monitoring of patients with stable age-related macular degeneration compared to hospital-based care: The FENETRE study report no. 1.

PURPOSE: Describe the development, delivery, acceptability and evaluation of a modular training programme for community-based, non-medical practitioners monitoring patients with quiescent neovascular age related macular degeneration (QnAMD). Also, report on a qualitative process evaluation conducted during the pilot phase of a randomised control trial (the FENETRE Study) exploring patient and practitioner acceptability of community-based QnAMD care relative to hospital-based care. METHODS: Learning outcomes from The College of Optometrists' Medical Retina higher qualifications and the Royal College of Ophthalmologists' Common Clinical Competency Framework were used to develop a competency framework for QnAMD care. Training was delivered online, comprising six asynchronous lectures followed by two synchronous case-based discussion webinars, with an accredited assessment of 24 case vignettes. An anonymous evaluation survey was conducted with the first two FENETRE cohorts (n = 38). Separately, we undertook a qualitative process evaluation, sampling purposively in four hospitals and five community-based practices, interviewing nine patients and eight practitioners. RESULTS: Survey responses (n = 26) showed community optometrists were very satisfied (n = 12; 46%) or satisfied (n = 14; 54%) with the training; feedback reflected by qualitative process evaluation data. Overall, optometrists also felt either confident (n = 15; 58%) or very confident (n = 8; 31%) in conducting AMD monitoring appointments following training, a finding also corroborated by interview data from optometrists participating in the initial pilot phase roll-out. Optometrists identified patient convenience and alleviating pressures in hospital care as the primary reasons for acceptability of community pathways. Data from patients entering community practices suggested they largely found this at least as safe and convenient as hospital care, although some patients randomised to hospital care perceived that as safer. CONCLUSION: This pilot study has shown the development and implementation of a collaborative community monitoring model is feasible, with satisfaction from community optometrists for training and accreditation, and broad acceptance for the pathway by both patients and practitioners.

Magnetic particle characterization-magnetophoretic mobility and particle size.

Quantitative characterization of magnetic particles is useful for analysis and separation of labeled cells and magnetic particles. A particle velocimeter is used to directly measure the magnetophoretic mobility, size, and other parameters of magnetic particle suspensions. The instrument provides quantitative video analysis of particles and their motion. The trajectories of magnetic particles in an isodynamic magnetic field are recorded using a high-definition camera/microscope system for image collection. Image analysis software then converts the image data to the parameters of interest. The distribution of magnetophoretic mobility is determined by combining fast image analysis with velocimetry measurements. Particle size distributions have been characterized to provide a better understanding of sample quality. The results have been used in the development and operation of analyzer protocols for counting particle concentrations accurately and measuring magnetic susceptibility and size for simultaneous display for routine application to particle suspensions and magnetically labeled biological cells. © 2016 International Society for Advancement of Cytometry.

Retrospective evaluation of the impact of functional immunotoxicity testing on pesticide hazard identification and risk assessment.

Conduct of a T-cell-dependent antibody response (TDAR) assay in rodents according to Environmental Protection Agency (EPA) Test Guideline OPPTS 870.7800 is now required for chemical pesticide active ingredients registered in the United States. To assess potential regulatory impact, a retrospective analysis was developed using TDAR tests conducted on 78 pesticide chemicals from 46 separate chemical classes. The objective of the retrospective analysis was to examine the frequency of positive responses and determine the potential for the TDAR to yield lower endpoints than those utilized to calculate reference doses (RfDs). A reduction in the TDAR response was observed at only the high-dose level in five studies, while it was unaltered in the remaining studies. Importantly, for all 78 pesticide chemicals, the TDAR no-observed-adverse-effect levels (TDAR NOAELs) were greater than the NOAELS currently in use as risk assessment endpoints. The TDAR NOAELs were higher than the current EPA-selected endpoints for the chronic RfD, short-term, intermediate and long-term exposure scenarios by 3-27,000, 3-1,688, 3-1,688 and 4.9-1,688 times, respectively. Based on this analysis, conduct of the TDAR assay had minimal impact on hazard identification and did not impact human health risk assessments for the pesticides included in this evaluation. These data strongly support employment of alternative approaches including initial weight-of-evidence analysis for immunotoxic potential prior to conducting functional immunotoxicity testing for pesticide active ingredients.

High-dimensional mapping of human CEACAM1 expression on immune cells and association with melanoma drug resistance.

BACKGROUND: Human carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is an inhibitory cell surface protein that functions through homophilic and heterophilic ligand binding. Its expression on immune cells in human tumors is poorly understood. METHODS: An antibody that distinguishes human CEACAM1 from other highly related CEACAM family members was labeled with 159Tb and inserted into a panel of antibodies that included specificity for programmed cell death protein 1 (PD1) and PD-L1, which are targets of immunotherapy, to gain a data-driven immune cell atlas using cytometry by time-of-flight (CyTOF). A detailed inventory of CEACAM1, PD1, and PD-L1 expression on immune cells in metastatic lesions to lymph node or soft tissues and peripheral blood samples from patients with treatment-naive and -resistant melanoma as well as peripheral blood samples from healthy controls was performed. RESULTS: CEACAM1 is absent or at low levels on healthy circulating immune cells but is increased on immune cells in peripheral blood and tumors of melanoma patients. The majority of circulating PD1-positive NK cells, innate T cells, B cells, monocytic cells, dendritic cells, and CD4+ T cells in the peripheral circulation of treatment-resistant disease co-express CEACAM1 and are demonstrable as discrete populations. CEACAM1 is present on distinct types of cells that are unique to the tumor microenvironment and exhibit expression levels that are highest in treatment resistance; this includes tumor-infiltrating CD8+ T cells. CONCLUSIONS: To the best of our knowledge, this work represents the first comprehensive atlas of CEACAM1 expression on immune cells in a human tumor and reveals an important correlation with treatment-resistant disease. These studies suggest that agents targeting CEACAM1 may represent appropriate partners for PD1-related pathway therapies.

Some proteins, such as programmed cell death protein 1 (PD1), can stop the immune system from attacking cancer cells, allowing cancers to grow. Therapies targeting these proteins can be highly effective, but tumors can become resistant. It is important to identify factors involved in this resistance to develop improved cancer therapies. Human carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a protein that inhibits an immune response and its levels have been associated with poor patient outcomes. We applied a method that allows for the detection of proteins on a single cell to uncover CEACAM1 patterns in melanoma. We found that increased CEACAM1 expression levels on multiple different immune cell types was associated with tumors that were resistant to therapy. These findings may help us to understand the role of CEACAM1 in cancer and to develop better cancer therapies.

Intelectin-1 binds and alters the localization of the mucus barrier-modifying bacterium Akkermansia muciniphila.

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host-microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell-mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.

Application of magnetic particle tracking velocimetry to quadrupole magnetic sorting of porcine pancreatic islets.

Magnetic isolation is a promising method for separating and concentrating pancreatic islets of Langerhans for transplantation in Type 1 diabetes patients. We are developing a continuous magnetic islet sorter to overcome the restrictions of current purification methods that result in limited yield and viability. In Quadrupole Magnetic Sorting (QMS) islets are magnetized by infusing superparamagnetic microbeads into islets' vasculature via arteries that serve the pancreas. The performance of the islet sorter depends on the resulting speed of the islets in an applied magnetic field, a property known as magnetophoretic mobility. Essential to the design and successful operation of the QMS is a method to measure the magnetophoretic mobilities of magnetically infused islets. We have adapted a Magnetic Particle Tracking Velocimeter (MPTV) to measure the magnetophoretic mobility of particles up to 1,000 µm in diameter. Velocity measurements are performed in a well-characterized uniform magnetic energy gradient using video imaging followed by analysis of the video images with a computer algorithm that produces a histogram of absolute mobilities. MPTV was validated using magnetic agarose beads serving as islet surrogates and subjecting them to QMS. Mobility distributions of labeled porcine islets indicated that magnetized islets have sufficient mobility to be captured by the proposed sorting method, with this result confirmed in test isolations of magnetized islets.

Computational Fluid Dynamics Simulation of a Quadrupole Magnetic Sorter Flow Channel: Effect of Splitter Position on Nonspecific Crossover.

In the Quadrupole Magnetic Sorter (QMS) magnetic particles enter a vertical flow annulus and are separated from non-magnetic particles by radial deflection into an outer annulus where the purified magnetic particles are collected via a flow splitter. The purity of magnetically isolated particles in QMS is affected by the migration of nonmagnetic particles across transport lamina in the annular flow channel. Computational Fluid Dynamics (CFD) simulations were used to predict the flow patterns, pressure drop and nonspecific crossover in QMS flow channel for the isolation of pancreatic islets of Langerhans. Simulation results were compared with the experimental results to validate the CFD model. Results of the simulations were used to show that one design gives up to 10% less nonspecific crossover than another and this model can be used to optimise the flow channel design to achieve maximum purity of magnetic particles.

Magnetic characterization of paramagnetic reagents by particle tracking velocimetry.

Magnetic particle characterization determines the quality of magnetic particles and is of great importance in particle technology, drug delivery, cell separation, in vivo diagnostics, and other biomedical applications. The quality of the sample depends on the particle size, intrinsic magnetic properties of the particles, and the uniformity of these properties. A commercial particle tracking velocimeter was used to record and capture dark field images of particle trajectories in an applied isodynamic magnetic field. The calibrated particle size, magnetophoretic mobility, and additional image data were collected for each magnetic bead imaged. Using twenty-one different de-identified calibration beads and transmission electron microscopy to validate the vendor-reported particle size enabled the estimation of intrinsic magnetic properties, namely, apparent magnetic susceptibility and saturation magnetization, of individual paramagnetic particles. The distributions of volume magnetic susceptibility based on the magnetophoretic mobility and size of the particle for different magnetic beads were determined and displayed as two-parameter distributions. The measured apparent susceptibility and saturation magnetization were found to be directly proportional to the percentage of iron oxide in the reagent particles.

Characterization of the effectiveness of a hydrocarbon liquid solidifier.

Solidifiers are dry, granular hydrophobic polymers that form physical bonds with hydrocarbons by molecular interactions (hydrogen bonding, London forces), and are used to immobilize hydrocarbon spill propagation and dispersion. CIAgent© is a non-toxic, proprietary polymer blend listed as an "Oil Solidifier" on the EPA's National Contingency Plan Product Schedule for use on hydrocarbon spills in the navigable waterways of United States. CIAgent solidifies the liquid hydrocarbons through a rapid transformation into a cohesive rubber-like inert mass upon contact and retains the liquid for easier removal and disposal. The objective of this paper is to determine the effectiveness of the solidifier with a variety of hydrocarbon liquids that could be encountered in an oil spill scenario. The effectiveness of the solidifier was characterized in terms of the application rate, temperature change, solubility parameters and solidification time for a variety of hydrocarbon liquids (e. g., gasoline, diesel fuel, crude oil) that could be encountered by measuring the heat of solidification using a solution calorimeter. A thermogram was obtained and the heat of solidification was calculated using the temperature difference upon solidification. The temperature change and the degree of swelling in the solidifier were used to determine the solubility parameter of the solidifier (6.77 Hildebrands). The heat of solidification value was used to determine the ease and speed of the solidification of the hydrocarbon liquids. Solidification times ranged from 40 to 120 s for the liquids tested. The average application ratio in weight of solidifier to weight of hydrocarbon ranged was 3.35.

Epithelial endoplasmic reticulum stress orchestrates a protective IgA response.

Immunoglobulin A (IgA) is the major secretory immunoglobulin isotype found at mucosal surfaces, where it regulates microbial commensalism and excludes luminal factors from contacting intestinal epithelial cells (IECs). IgA is induced by both T cell-dependent and -independent (TI) pathways. However, little is known about TI regulation. We report that IEC endoplasmic reticulum (ER) stress induces a polyreactive IgA response, which is protective against enteric inflammation. IEC ER stress causes TI and microbiota-independent expansion and activation of peritoneal B1b cells, which culminates in increased lamina propria and luminal IgA. Increased numbers of IgA-producing plasma cells were observed in healthy humans with defective autophagy, who are known to exhibit IEC ER stress. Upon ER stress, IECs communicate signals to the peritoneum that induce a barrier-protective TI IgA response.

High-solid enzymatic hydrolysis and fermentation of solka floc into ethanol.

To lower the cost of ethanol distillation of fermentation broths, a high initial glucose concentration is desired. However, an increase in the substrate concentration typically reduces the ethanol yield because of insufficient mass and heat transfer. In addition, different operating temperatures are required to optimize the enzymatic hydrolysis (50 degrees C) and fermentation (30 degrees C). Thus, to overcome these incompatible temperatures, saccharification followed by fermentation (SFF) was employed with relatively high solid concentrations (10% to 20%) using a portion loading method. In this study, glucose and ethanol were produced from Solka Floc, which was first digested by enzymes at 50 degrees for 48 h, followed by fermentation. In this process, commercial enzymes were used in combination with a recombinant strain of Zymomonas mobilis (39679:pZB4L). The effects of the substrate concentration (10% to 20%, w/v) and reactor configuration were also investigated. In the first step, the enzyme reaction was achieved using 20 FPU/g cellulose at 50 degrees C for 96 h. The fermentation was then performed at 30 degrees C for 96 h. The enzymatic digestibility was 50.7%, 38.4%, and 29.4% after 96 h with a baffled Rushton impeller and initial solid concentration of 10%, 15%, and 20% (w/v), respectively, which was significantly higher than that obtained with a baffled marine impeller. The highest ethanol yield of 83.6%, 73.4%, and 21.8%, based on the theoretical amount of glucose, was obtained with a substrate concentration of 10%, 15%, and 20%, respectively, which also corresponded to 80.5%, 68.6%, and 19.1%, based on the theoretical amount of the cell biomass and soluble glucose present after 48 h of SFF.

Midwifery education and technology enhanced learning: Evaluating online story telling in preregistration midwifery education.

BACKGROUND: A major issue regarding the implementation of blended learning for preregistration health programmes is the analysis of students' perceptions and attitudes towards their learning. It is the extent of the embedding of Technology Enhanced Learning (TEL) into the higher education curriculum that makes this analysis so vital. OBJECTIVES: This paper reports on the quantitative results of a UK based study that was set up to respond to the apparent disconnect between technology enhanced education provision and reliable student evaluation of this mode of learning. DESIGN: Employing a mixed methods research design, the research described here was carried to develop a reliable and valid evaluation tool to measure acceptability of and satisfaction with a blended learning approach, specifically designed for a preregistration midwifery module offered at level 4. METHODS: Feasibility testing of 46 completed blended learning evaluation questionnaires - Student Midwife Evaluation of Online Learning Effectiveness (SMEOLE) - using descriptive statistics, reliability and internal consistency tests. RESULTS: Standard deviations and mean scores all followed predicted pattern. Results from the reliability and internal consistency testing confirm the feasibility of SMEOLE as an effective tool for measuring student satisfaction with a blended learning approach to preregistration learning. CONCLUSIONS: The analysis presented in this paper suggests that we have been successful in our aim to produce an evaluation tool capable of assessing the quality of technology enhanced, University level learning in Midwifery. This work can provide future benchmarking against which midwifery, and other health, blended learning curriculum planning could be structured and evaluated.

Innovation in preregistration midwifery education: Web based interactive storytelling learning.

BACKGROUND: through a critical description of the implementation of a web based interactive storytelling learning activity introduced into an undergraduate, preregistration midwifery education programme, this paper will explore how low-cost, low-fidelity online storytelling, designed using Moodle, can be used to enhance students' understanding of compassion and empathy in practice. SAMPLE: cross sectional sample of first year undergraduate Midwifery students (n111) METHOD: drawing from both research and audit data collected in an Higher Education Institution in London England, the paper presents the case for using web based technology to create a sustainable model for midwifery education. FINDINGS: initial results indicate that it is both the low cost and positive student evaluations of web based interactive storytelling, which make this approach to preregistration midwifery education which suggests that this approach has significant potential for learning and teaching in midwifery education in diverse settings around the world. Or how about: global relevance? .

Reintroduced solids increase inhibitor levels in a pretreated corn stover hydrolysate.

Following detoxification of the liquid hydrolysate produced in a corn stover pretreatment process, inhibitor levels are seen to increase with the re-addition of solids for the ensuing hydrolysis and fermentation processes. The solids that were separated from the slurry before detoxification of the liquor contain approx 60% (w/w) moisture, and contamination occurs owing to the diffusion of inhibitors from the moisture entrained in the porous structure of the corn stover solids into the bulk fluid. This evidence suggests the need for additional separation and detoxification steps to purge residual inhibitors entrained in the moisture in the solids. An overliming process to remove furans from the hydrolysate failed to reduce total organic acids concentration, so acids were removed by treatment with an activated carbon powder. Smaller carbon doses proved more efficient in removing organic acids in terms of grams of acid removed per gram of carbon powder. Sugar adsorption by the activated carbon powder was minimal.

Modeling of a continuous pretreatment reactor using computational fluid dynamics.

Computational fluid dynamic simulations are employed to predict flow characteristics in a continuous auger driven reactor designed for the dilute acid pretreatment of biomass. Slurry containing a high concentration of biomass solids exhibits a high viscosity, which poses unique mixing issues within the reactor. The viscosity increases significantly with a small increase in solids concentration and also varies with temperature. A well-mixed slurry is desirable to evenly distribute acid on biomass, prevent buildup on the walls of the reactor, and provides an uniform final product. Simulations provide flow patterns obtained over a wide range of viscosities and pressure distributions, which may affect reaction rates. Results provide a tool for analyzing sources of inconsistencies in product quality and insight into future design and operating parameters.

Continuous Quadrupole Magnetic Separation of Islets during Digestion Improves Purified Porcine Islet Viability.

Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation.

Use of computational fluid dynamics simulations for design of a pretreatment screw conveyor reactor.

Computational fluid dynamics simulations were employed to compare performance of various designs of a pretreatment screw conveyor reactor. The reactor consisted of a vertical screw used to create cross flow between the upward conveying solids and the downward flow of acid. Simulations were performed with the original screw design and a modified design in which the upper flights of the screw were removed. Results of the simulations show visually that the modified design provided favorable plug flow behavior within the reactor. Pressure drop across the length of the reactor without the upper screws in place was predicted by the simulations to be 5 vs 40 kPa for the original design.

Detoxification of actual pretreated corn stover hydrolysate using activated carbon powder.

A technique for the removal of acetic acid from an actual pretreated corn stover hydrolysate was investigated. A powdered form of activated carbon previously shown to be effective in the removal of acetic acid from a synthetic hydrolysate was utilized. The method proved to be effective at lowering acetic acid levels while exhibiting minimal adsorption of the desired sugars from the hydrolysate, although at a lower efficiency in the actual hydrolysate than in the synthetic hydrolysate. Results are obtained for temperatures between 25 and 35 degrees C and agitation rates between 150 and 350 rpm in shake flasks. Adsorption isotherm and kinetic rate data are presented. Temperature differences over this range did not have an effect on adsorption characteristics. Five stages of detoxification were necessary to lower acetic acid concentration to the maximum 2 g/L desired for fermentation.

Enhanced mixing and mass transfer in a recirculation loop results in high cell densities in a roller bottle reactor.

A recirculation loop added to a large-scale roller bottle reactor resulted in high cell densities as compared to standard roller bottles. Four different mammalian cell lines reached an average maximum density equal to 5.4 x 10(6) cells /mL (sigma = 0.263), which was between 2.13 and 2.95 times greater than the densities in roller bottles without recirculation using the same cell lines. The high densities were maintained over long durations (>25 days) while the reactor operated with continuous perfusion. The increased densities are attributed to enhanced liquid mixing and oxygen transfer that occur as a result of the recirculation loop. Models were developed that describe axial liquid flow and oxygen transfer in both the sample loop and the reactor growth chamber. Axial dispersion and oxygen transfer coefficients are presented for a variety of operating conditions. The increased oxygen transfer characteristics of the reactor allow for easy scale-up of roller bottle cultures by operating at larger volumes with greater liquid depths than conventional roller bottles permit. The surface-area-to-volume ratio in the tests performed was 0.206 versus 1.16 cm(-1) in a standard roller bottle.