Effects of Emulsifiers on an In vitro Model of Intestinal Epithelial Tight Junctions and the Transport of Food Allergens.

SCOPE: Certain food emulsifiers may interfere with gut barrier function in ways correlating to increased exposure to allergens. Understanding the consequences of interactions between these food ingredients and the intestinal epithelium is important for evaluating allergen dose exposure characteristics. METHODS AND RESULTS: This study challenged Caco-2 cell monolayers, an in vitro model of human intestinal epithelial tight junctions with synthetic polysorbate-80 or natural lecithin alone, or in combination with known allergens (egg proteins: ovalbumin, ovomucoid, and ovotransferrin; and a synthetic form of galactose-alpha-1,3-galactose [alpha-gal], an allergen of increasing concern). For most doses of individual emulsifiers and allergens, >90% cell viability and <15% cytotoxicity are observed; however, toxicity increased at a 0.5% concentration of emulsifiers. At low cytotoxic concentration (0.2%), only polysorbate-80 treatment reduced monolayer integrity (≈20%) with increased lucifer yellow passage. Dose-related differences in expression of tight junction-associated genes and occludin protein are observed with emulsifier treatments. The transport of all tested allergens across the cell monolayers, excluding ovotransferrin, nearly doubled in the presence of 0.2% polysorbate-80 compared to lecithin and untreated control. CONCLUSION: By modulating paracellular permeability, polysorbate-80 may enhance absorption of allergens in a size-dependent manner.

A pre-registered, multi-lab non-replication of the action-sentence compatibility effect (ACE).

The Action-sentence Compatibility Effect (ACE) is a well-known demonstration of the role of motor activity in the comprehension of language. Participants are asked to make sensibility judgments on sentences by producing movements toward the body or away from the body. The ACE is the finding that movements are faster when the direction of the movement (e.g., toward) matches the direction of the action in the to-be-judged sentence (e.g., Art gave you the pen describes action toward you). We report on a pre-registered, multi-lab replication of one version of the ACE. The results show that none of the 18 labs involved in the study observed a reliable ACE, and that the meta-analytic estimate of the size of the ACE was essentially zero.

Presence of Native and Non-native Ants Linked to Lower Emergence Success of Loggerhead Sea Turtle Nests: Implications for Management.

Ants have been suggested as one of many population pressures sea turtles face potentially affecting nesting-beach survival of eggs and hatchlings. However, little is known about the extent to which ants act as incidental or primary mortality factors. Most research has focused on New World fire ants (genus Solenopsis), with confirmed records of other ant species interactions with sea turtle nests in situ being rare. Our study documented the ant species associated with loggerhead sea turtle Caretta caretta (Linnaeus) (Testudines: Cheloniidae) nests in Georgia and determined if ant presence was linked to lower hatching or emergence success. Samples (n = 116) collected from sea turtle nests on eight islands contained 14 ant species including Solenopsis invicta Buren (Hymenoptera: Formicidae), the red imported fire ant, which was the most common ant species encountered. Ant presence was not correlated with lower hatching success, but when other known disturbances were removed, correlated with significantly lower nest emergence success (P < 0.0001). Logistic modeling suggests that proximity of sea turtle nests to the primary dune significantly increases risk of ant predation on hatchling sea turtles. Population managers can reduce this risk by maintaining a 1-m buffer shoreward between dune vegetation and relocated sea turtle nests. Our results suggest that ants may exert a density-dependent pressure on nesting sea turtle populations and call for additional investigations to determine if managing native and invasive ants augments other efforts to improve hatchling survival.

Real-time clinical note monitoring to detect conditions for rapid follow-up: A case study of clinical trial enrollment in drug-induced torsades de pointes and Stevens-Johnson syndrome.

Identifying acute events as they occur is challenging in large hospital systems. Here, we describe an automated method to detect 2 rare adverse drug events (ADEs), drug-induced torsades de pointes and Stevens-Johnson syndrome and toxic epidermal necrolysis, in near real time for participant recruitment into prospective clinical studies. A text processing system searched clinical notes from the electronic health record (EHR) for relevant keywords and alerted study personnel via email of potential patients for chart review or in-person evaluation. Between 2016 and 2018, the automated recruitment system resulted in capture of 138 true cases of drug-induced rare events, improving recall from 43% to 93%. Our focused electronic alert system maintained 2-year enrollment, including across an EHR migration from a bespoke system to Epic. Real-time monitoring of EHR notes may accelerate research for certain conditions less amenable to conventional study recruitment paradigms.

Long-lived zebrafish Rohon-Beard cells.

During normal development of the nervous system, extensive neuronal proliferation as well as death occurs. The extent of development death varies considerably between neuronal populations from little to almost 100%. Early born somatosensory neurons, known as Rohon-Beard cells, have served as an example of neurons that disappear during early developmental stages, presumably as their function is taken over by later developing dorsal root ganglion neurons. However, recent studies have raised questions about the extent to which zebrafish Rohon-Beard cells die during embryogenesis. While Rohon-Beard cells have distinguishing morphological features during embryonic stages development, they subsequently undergo substantial changes in their shape, size and position that hinder their unambiguous identification at later stages. To overcome this obstacle, we identify Rohon-Beard cells at one day, and using a combination of mosaic and stable transgenic labeling and repeated observation, follow them for 13-16 days post fertilization. We find that about 40% survive to late larval stages. Our studies also reveal that Rohon-Beard cells display an unusual repertoire of cell death properties. At one day, about 25% Rohon-Beard cells expose phosphatidyl serine at the surface membrane, but less than one Rohon-Beard cell/embryo expresses activated-caspase-3. Further, the temporal delay between detection of cell death markers and loss of the soma ranges from

Identifying patterns in foraging-area origins in breeding aggregations of migratory species: Loggerhead turtles in the Northwest Atlantic.

Population assessments conducted at reproductive sites of migratory species necessitate understanding the foraging-area origins of breeding individuals. Without this information, efforts to contextualize changes in breeding populations and develop effective management strategies are compromised. We used stable isotope analysis of tissue samples collected from loggerhead sea turtles (Caretta caretta) nesting at seven sites in the Northern Recovery Unit (NRU) of the eastern United States (North Carolina, South Carolina and Georgia) to assign females to three separate foraging areas in the Northwest Atlantic Ocean (NWA). We found that the majority of the females at NRU nesting sites (84.4%) use more northern foraging areas in the Mid-Atlantic Bight, while fewer females use more proximate foraging areas in the South Atlantic Bight (13.4%) and more southerly foraging areas in the Subtropical Northwest Atlantic (2.2%). We did not find significant latitudinal or temporal trends in the proportions of NRU females originating from different foraging areas. Combining these findings with previous data from stable isotope and satellite tracking studies across NWA nesting sites showed that variation in the proportion of adult loggerheads originating from different foraging areas is primarily related differences between recovery units: individuals in the NRU primarily use the Mid-Atlantic Bight foraging area, while individuals from the three Florida recovery units primarily use the Subtropical Northwest Atlantic and Eastern Gulf of Mexico foraging areas. Because each foraging area is associated with its own distinct ecological characteristics, environmental fluctuations and anthropogenic threats that affect the abundance and productivity of individuals at nesting sites, this information is critical for accurately evaluating population trends and developing effective region-specific management strategies.

Susceptibility of aging mice to listeriosis: Role of anti-inflammatory responses with enhanced Treg-cell expression of CD39/CD73 and Th-17 cells.

Foodborne Listeria monocytogenes (Lm) causes serious illness and death in immunosuppressed hosts, including the elderly population. We investigated Lm susceptibility and inflammatory cytokines in geriatric mice. Young-adult and old mice were gavaged with a Lm strain Lmo-InlAm. Tissues were assayed for Lm burden and splenocytes were analyzed for Th1/Th2/Th17/Treg responses and expression of CD39 and CD73. Old Lm-infected mice lost body-weight dose-dependently, had higher Lm colonization, and showed higher inflammatory responses than Lm-infected young-adult mice. After infection, IL-17 levels increased significantly in old mice whereas IFN-γ levels were unchanged. Levels of IL-10 and Treg cells were increased in infected old mice as compared to infected young-adult mice. Age-dependent enhanced expression of CD39/CD73 was observed in purified Treg prior to infection, suggesting increased baseline adenosine production in old mice. Lm lysate-treated splenocytes from older mice produced significantly higher levels of IL-10, IL17, and IL-1ß, produced less IFN-γ and IL-2, and proliferated less than splenocytes from young-adult mice. Data suggests that older mice maybe more susceptible to Lm infection due to an imbalance of Th cell responses with disproportionate and persistent anti-inflammatory responses. Lm infection enhanced differentiation of proinflammatory Th17 cells, which may also exacerbate pathological responses during listeriosis.

Enhanced quantitation of egg allergen in foods using incurred standards and antibodies against processed egg in a model ELISA.

Underestimation of egg allergen from processed foods prompted the evaluation of critical Enzyme-Linked Immunosorbent Assay (ELISA) parameters: (1) extraction of egg proteins from a processed matrix; (2) use of anti-heat processed egg antibodies (Abs) on detectability of modified proteins, and (3) utilization of incurred material as standards. The relative affinity of two combinations of raw (R), boiled (B) and fried (F) Abs to unprocessed/processed egg proteins with or without matrix was determined from antibody (Ab) binding curves. In ELISAs using RBF-Abs and BF-Abs, denaturing buffer, and incurred standards, the Limit of Detection (LOD) and Limit of Quantitation (LOQ) were 0.47 and 0.25; and 1.58 and 0.85, respectively, and the linear range was 0-24 µg g-1 egg protein. The recoveries of egg protein from cookies, cereal bar, and muffin (incurred levels 4.8-48 µg g-1) with the developed ELISAs were in an acceptable range (50-130%). These ELISAs consistently detected more declared/undeclared egg proteins in market samples compared to assays using PBS for extraction. Overall, better assay performance was observed using BF-Abs. An ELISA combining anti-processed egg Abs, denaturing buffer, and incurred standards promises improved quantitation of egg proteins in processed foods.

Extraction Conditions Affect the Immunoreactivity of Peanut Allergens.

Peanut allergic consumers rely on food package labels to avoid foods containing peanut. The inadvertent presence of peanut in foods due to cross-contact can be fatal if ingestion of such food leads to an allergic reaction. Analytical methods are available to detect undeclared peanut in foods. However, depending on the type of food matrix and food processing parameters, method performance can be adversely affected due to reduction in the extraction efficiency of peanut proteins. Temperature and probe sonication were used as a preincubation treatment for peanut flour slurries to assess their effect on the total peanut protein solubility from raw, light-roasted, and dark-roasted peanut flours. The effect of these treatments on the immunoreactivity of peanut allergens (Ara h 1, Ara h 2, Ara h 3, and Ara h 6) was determined by an indirect enzyme-linked immunosorbent assay using antibodies raised against these individual peanut proteins. Preincubation at 50 °C did not significantly improve the peanut protein solubility, whereas an increase in protein solubility was observed when light- and dark-roasted peanut flour slurries were preincubated at 90 °C or sonicated. The immunoreactivity of peanut allergens varied depending on the degree of peanut flour roasting and type of preincubation treatment. Overall, the immunoreactivity of peanut allergens from most peanut flour slurries was unaffected when preincubated at 50 °C for up to 60 min or sonicated with a probe for up to 5 min, whereas preincubation at 90 °C resulted in a time-dependent reduction in immunoreactivity of peanut allergens. Sonication treatment may improve peanut protein extraction without markedly affecting their immunoreactivity. PRACTICAL APPLICATION: Extraction of peanut proteins is vital for developed analytical methods to estimate peanut allergens in foods. The manuscript describes the effect of two different temperatures (50 and 90 °C) and probe-type sonication on peanut protein solubility. The findings suggest sonication can improve peanut protein solubility without markedly affecting their immunoreactivity.

HLA-A*32:01 is strongly associated with vancomycin-induced drug reaction with eosinophilia and systemic symptoms.

BACKGROUND: Vancomycin is a prevalent cause of the severe hypersensitivity syndrome drug reaction with eosinophilia and systemic symptoms (DRESS), which leads to significant morbidity and mortality and commonly occurs in the setting of combination antibiotic therapy, affecting future treatment choices. Variations in HLA class I in particular have been associated with serious T cell-mediated adverse drug reactions, which has led to preventive screening strategies for some drugs. OBJECTIVE: We sought to determine whether variation in the HLA region is associated with vancomycin-induced DRESS. METHODS: Probable vancomycin-induced DRESS cases were matched 1:2 with tolerant control subjects based on sex, race, and age by using BioVU, Vanderbilt's deidentified electronic health record database. Associations between DRESS and carriage of HLA class I and II alleles were assessed by means of conditional logistic regression. An extended sample set from BioVU was used to conduct a time-to-event analysis of those exposed to vancomycin with and without the identified HLA risk allele. RESULTS: Twenty-three subjects met the inclusion criteria for vancomycin-associated DRESS. Nineteen (82.6%) of 23 cases carried HLA-A*32:01 compared with 0 (0%) of 46 of the matched vancomycin-tolerant control subjects (P = 1 × 10-8) and 6.3% of the BioVU population (n = 54,249, P = 2 × 10-16). Time-to-event analysis of DRESS development during vancomycin treatment among the HLA-A*32:01-positive group indicated that 19.2% had DRESS and did so within 4 weeks. CONCLUSIONS: HLA-A*32:01 is strongly associated with vancomycin-induced DRESS in a population of predominantly European ancestry. HLA-A*32:01 testing could improve antibiotic safety, help implicate vancomycin as the causal drug, and preserve future treatment options with coadministered antibiotics.

The CDK9-cyclin T1 complex mediates saturated fatty acid-induced vascular calcification by inducing expression of the transcription factor CHOP.

Vascular calcification (or mineralization) is a common complication of chronic kidney disease (CKD) and is closely associated with increased mortality and morbidity rates. We recently reported that activation of the activating transcription factor 4 (ATF4) pathway through the saturated fatty acid (SFA)-induced endoplasmic reticulum (ER) stress response plays a causative role in CKD-associated vascular calcification. Here, using mouse models of CKD, we 1) studied the contribution of the proapoptotic transcription factor CCAAT enhancer-binding protein homologous protein (CHOP) to CKD-dependent medial calcification, and 2) we identified an additional regulator of ER stress-mediated CHOP expression. Transgenic mice having smooth muscle cell (SMC)-specific CHOP expression developed severe vascular apoptosis and medial calcification under CKD. Screening of a protein kinase inhibitor library identified 16 compounds, including seven cyclin-dependent kinase (CDK) inhibitors, that significantly suppressed CHOP induction during ER stress. Moreover, selective CDK9 inhibitors and CRISPR/Cas9-mediated CDK9 reduction blocked SFA-mediated induction of CHOP expression, whereas inhibitors of other CDK isoforms did not. Cyclin T1 knockout inhibited SFA-mediated induction of CHOP and mineralization, whereas deletion of cyclin T2 and cyclin K promoted CHOP expression levels and mineralization. Of note, the CDK9-cyclin T1 complex directly phosphorylated and activated ATF4. These results demonstrate that the CDK9-cyclin T1 and CDK9-cyclin T2/K complexes have opposing roles in CHOP expression and CKD-induced vascular calcification. They further reveal that the CDK9-cyclin T1 complex mediates vascular calcification through CHOP induction and phosphorylation-mediated ATF4 activation.

Diet-induced obesity precipitates kidney dysfunction and alters inflammatory mediators in mice treated with Shiga Toxin 2.

Shiga Toxin (Stx)-producing E. coli (STEC) continue to be a prominent cause of foodborne outbreaks of hemorrhagic colitis worldwide, and can result in life-threatening diseases, including hemolytic uremic syndrome (HUS), in susceptible individuals. Obesity-associated immune dysfunction has been shown to be a risk factor for infectious diseases, although few studies have addressed the role of obesity in foodborne diseases. We hypothesized that obesity may affect the development of HUS through an alteration of immune responses and kidney function. We combined diet-induced obese (DIO) and HUS mouse models to look for differences in disease outcome between DIO and wild-type (WT) male and female C57 B l/6 mice. Following multiple intraperitoneal injections with endotoxin-free saline or sublethal doses of purified Stx2, we examined DIO and WT mice for signs of HUS development. DIO mice receiving Stx2 injections lost more body weight, and had significantly higher (p < 0.001) BUN, serum creatinine, and neutrophil counts compared to WT mice or DIO mice receiving saline injections. Lymphocyte counts were significantly (p < 0.05) lower in Stx2-treated obese mice compared to WT mice or saline-treated DIO mice. In addition to increased Stx2-induced kidney dysfunction, DIO mouse kidneys also had significantly increased expression of IL-1α, IL-1ß, IL-6, TNF-α, MCP-1, and KC RNA compared to saline controls (p < 0.05). Serum cytokine levels of IL-6 and KC were also significantly higher in Stx2-treated mice compared to saline controls, but there were no significant differences between the WT and DIO mice. WT and DIO mice treated with Stx2 exhibited significantly higher degrees of kidney tubular dilation and necrosis as well as some signs of tissue repair/regeneration, but did not appear to progress to the full pathology typically associated with human HUS. Although the combined obesity/HUS mouse model did not manifest into HUS symptoms and pathogenesis, these data demonstrate that obesity alters kidney function, inflammatory cells and cytokine production in response to Stx2, and may play a role in HUS severity in a susceptible model of infection.

Investigation of Islet2a function in zebrafish embryos: Mutants and morphants differ in morphologic phenotypes and gene expression.

Zebrafish primary motor neurons differ from each other with respect to morphology, muscle targets and electrophysiological properties. For example, CaP has 2-3-fold larger densities of both inward and outward currents than do other motor neurons. We tested whether the transcription factor Islet2a, uniquely expressed in CaP, but not other primary motor neurons, plays a role in specifying its stereotypic electrophysiological properties. We used both TALEN-based gene editing and antisense morpholino approaches to disrupt Islet2a function. Our electrophysiology results do not support a specific role for Islet2a in determining CaP's unique electrical properties. However, we also found that the morphological phenotypes of CaP and a later-born motor neuron differed between islet2a mutants and morphants. Using microarrays, we tested whether the gene expression profiles of whole embryo morphants, mutants and controls also differed. Morphants had 174 and 201 genes that were differentially expressed compared to mutants and controls, respectively. Further, islet2a was identified as a differentially expressed gene. To examine how mutation of islet2a affected islet gene expression specifically in CaPs, we performed RNA in situ hybridization. We detected no obvious differences in expression of islet1, islet2a, or islet2b in CaPs of mutant versus sibling control embryos. However, immunolabeling studies revealed that an Islet protein persisted in CaPs of mutants, albeit at a reduced level compared to controls. While we cannot exclude requirement for some Islet protein, we conclude that differentiation of the CaP's stereotypic large inward and outward currents does not have a specific requirement for Islet2a.

SJS/TEN 2017: Building Multidisciplinary Networks to Drive Science and Translation.

Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) is a life-threatening, immunologically mediated, and usually drug-induced disease with a high burden to individuals, their families, and society with an annual incidence of 1 to 5 per 1,000,000. To effect significant reduction in short- and long-term morbidity and mortality, and advance clinical care and research, coordination of multiple medical, surgical, behavioral, and basic scientific disciplines is required. On March 2, 2017, an investigator-driven meeting was held immediately before the American Academy of Dermatology Annual meeting for the central purpose of assembling, for the first time in the United States, clinicians and scientists from multiple disciplines involved in SJS/TEN clinical care and basic science research. As a product of this meeting, this article summarizes the current state of knowledge and expert opinion related to SJS/TEN covering a broad spectrum of topics including epidemiology and pharmacogenomic networks; clinical management and complications; special populations such as pediatrics, the elderly, and pregnant women; regulatory issues and the electronic health record; new agents that cause SJS/TEN; pharmacogenomics and immunopathogenesis; and the patient perspective. Goals include the maintenance of a durable and productive multidisciplinary network that will significantly further scientific progress and translation into prevention, early diagnosis, and management of SJS/TEN.

Immunogenicity of Influenza Vaccination in Patients With Cancer.

BACKGROUND: Influenza leads to significant morbidity and mortality in patients with cancer. Patients with cancer receiving chemotherapy may not mount an adequate immune response to the vaccine. We performed this pilot study to evaluate the immunogenicity of influenza vaccination in patients with cancer receiving chemotherapy. MATERIALS AND METHODS: During the 2011 to 2012 influenza season, patients undergoing chemotherapy for solid tumors were given trivalent inactivated influenza vaccine either on the day of chemotherapy (schedule A) or a week before chemotherapy (schedule B) by a single 0.5 mL injection in the deltoid muscle region. This was not a randomized trial. Hemagglutination inhibition assays were performed on blood samples from these patients taken at baseline, and 4 weeks postvaccination. Seroconversion rate (>4-fold increase in titers) and seroprotection rates (postvaccination titers of >1:40) were calculated for each vaccine component: influenza A (H1N1), A (H3N2) and B. RESULTS: A total of 18 patients received influenza vaccination as part of this pilot study. Of these, 8 patients received the vaccine on schedule A and 10 patients received the vaccine on schedule B. Geometric mean titers against each strain significantly improved after vaccination for both groups, as measured by signed rank test. Seroconversion to at least 1 strain was observed in 75% of patients on schedule A, and 70% of patients vaccinated on schedule B. Seroprotection to at least 1 strain was observed in 100% of patients in the schedule A group, and 60% of patients vaccinated on schedule B. Seroconversion and seroprotection rates against the 3 influenza strains were not significantly different between the 2 groups. CONCLUSIONS: Patients with nonhematological malignancies who are receiving chemotherapy mount an immune response to influenza vaccination. Timing of influenza vaccination in relation to chemotherapy does not seem to matter.

Extracellular adenosine produced by ecto-5'-nucleotidase (CD73) regulates macrophage pro-inflammatory responses, nitric oxide production, and favors Salmonella persistence.

Surface enzymes CD39 (nucleoside triphosphate dephosphorylase) and CD73 (ecto-5'-nucleotidase) mediate the synthesis of extracellular adenosine that can regulate immune responses. Adenosine produced by CD39/CD73 acts via adenosine receptors (ARs). CD73 is expressed by a variety of cell types and mediates anti-inflammatory responses. Because efficient innate immune responses are required for clearance of Salmonella infection, we investigated the role of CD73 in macrophage function, including phagocytosis, intracellular killing of Salmonella, and anti-bacterial pro-inflammatory responses to Salmonella-whole cell lysate (ST-WCL) or Salmonella infection. Additionally, RAW 264.7 macrophage mRNA expression of CD39, CD73, and all ARs were measured by qPCR after ST-WCL treatment. Pro-inflammatory cytokine mRNA and nitric oxide (NO) production were quantitated in the ST-WCL treated macrophage with and without CD73-inhibitor (APCP) treatment. Phagocytosis and intracellular killing by peritoneal macrophages from CD73-deficent mice were also evaluated using E. coli BioParticles® and GFP-Salmonella infection, respectively. CD73, CD39, and A2BAR mRNA were predominantly expressed in RAW cells. ST-WCL treatment significantly reduced CD73 expression, suggesting endogenous down-regulation of CD73, and an enhanced pro-inflammatory response. ST-WCL treated and CD73-inhibited macrophages produced more NO and a higher level of pro-inflammatory cytokines than CD73-competent macrophages (e.g. IL-1ß, TNF-α). Phagocytosis of E. coli BioParticles® was significantly higher in the macrophages treated with APCP and in the peritoneal macrophages from CD73-deficent mice as compared to APCP-untreated, and CD73-competent macrophages. Internalized bacteria were more efficiently cleared from macrophages in the absence of CD73, as observed by fluorescence-microscopy and Salmonella-DNA measurement by qPCR from the infected cells. CD73 down-regulation or CD73-inhibition of macrophages during Salmonella infection can enhance the production of pro-inflammatory cytokines and NO production, improving intracellular killing and host survivability. Extracellular adenosine synthesized by CD73 suppresses antibacterial responses of macrophages, which may weaken macrophage function and impair innate immune responses to Salmonella infection.

Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells.

Emerging evidence indicates that upregulation of the ER stress-induced pro-osteogenic transcription factor ATF4 plays an important role in vascular calcification, a common complication in patients with aging, diabetes, and chronic kidney disease (CKD). In this study, we demonstrated the pathophysiological role of ATF4 in vascular calcification using global Atf4 KO, smooth muscle cell-specific (SMC-specific) Atf4 KO, and transgenic (TG) mouse models. Reduced expression of ATF4 in global ATF4-haplodeficient and SMC-specific Atf4 KO mice reduced medial and atherosclerotic calcification under normal kidney and CKD conditions. In contrast, increased expression of ATF4 in SMC-specific Atf4 TG mice caused severe medial and atherosclerotic calcification. We further demonstrated that ATF4 transcriptionally upregulates the expression of type III sodium-dependent phosphate cotransporters (PiT1 and PiT2) by interacting with C/EBPß. These results demonstrate that the ER stress effector ATF4 plays a critical role in the pathogenesis of vascular calcification through increased phosphate uptake in vascular SMCs.

Oral exposure to Listeria monocytogenes in aged IL-17RKO mice: A possible murine model to study listeriosis in susceptible populations.

Foodborne Listeria monocytogenes (LM) is a cause of serious illness and death in the US. The case-fatality rate of invasive LM infection in the elderly population is >50%. The goal of this study is to establish a murine model of oral LM infection that can be used as a surrogate for human foodborne listeriosis in the geriatric population. Adult C57BL/6 (wild-type, WT) and adult or old IL17R-KO (knock-out) mice were gavaged with a murinized LM strain (Lmo-InlAm) and monitored for body-weight loss and survivability. Tissues were collected and assayed for bacterial burden, histology, and cytokine responses. When compared to WT mice, adult IL17R-KO mice are more susceptible to LM infection and showed increased LM burden and tissue pathology and a higher mortality rate. Older LM-infected KO-mice lost significantly (p < 0.02, ANOVA) more body-weight and had a higher bacterial burden in the liver (p = 0.03) and spleen as compared to adult mice. Uninfected, aged KO-mice showed a higher baseline pro-inflammatory response when compared to uninfected adult-KO mice. After infection, the pro-inflammatory cytokine, IFN-γ, mRNA in the liver was higher in the adult mice as compared to the old mice. The anti-inflammatory cytokine, IL-10, mRNA and regulatory T-cells (CD4+CD25+h or CD4+Foxp3+) cells in the aged mice increased significantly after infection as compared to adult mice. Expression of the T-cell activation marker, CD25 (IL-2Rα) in the aged mice did not increase significantly over baseline. These data suggest that aged IL17R-KO mice can be used as an in vivo model to study oral listeriosis and that aged mice are more susceptible to LM infection due to dysregulation of pro- and anti-inflammatory responses compared to adult mice, resulting in a protracted clearance of the infection.

Survey of undeclared soy allergen levels in the most frequently recalled food categories with or without precautionary labelling.

A comprehensive study was designed to determine the frequency and levels of soy allergen in packaged bakery and snack food products. A representative sample of products with no soy allergen disclosed on the label was analysed using two widely used enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no soy identified on the product label or which had a soy precautionary statement. Among 558 bakery and snack products, soy protein was detected in 17% of the products using the Neogen (NE) kit and 11% of the products using the Elisa Systems (ES) kit. The disagreement rates between kits were 8.8% for bakery products and 3.3% for snack products. Overall soy protein was detected at higher frequency in bakery products than in snack foods. Among 284 bakery samples, soy protein was detected in 25% of the samples with no precautionary statement and 19% of the samples which had a precautionary statement. Among 274 snack samples, soy protein was detected in 11% of the samples with no precautionary statement and 9% of the samples which had a precautionary statement. The sample repeatability was at an acceptable level (< 9%) for each method and food commodity. The reproducibility between kits was 23% for bakery foods and 36% for snack foods. None of the bakery (21) and snack (6) products without precautionary labelling (measured level > 5 ppm) had a higher level of soy protein per serving compared with the eliciting dose10 (ED10) of 10.6 mg for soy allergic patients. But the level of soy protein per serving may be clinically relevant to a subpopulation of soy allergic patients if a more stringent eliciting dose is applied. These findings emphasise that suitable detection methodologies and references doses are crucial for labelling accuracy and the safety of soy allergic consumers.