Effective and Safe Stimulation of Humoral and Cell-Mediated Immunity by Intradermal Immunization with a Cyclic Dinucleotide/Nanoparticle Combination Adjuvant.

Intradermal (ID) immunization is an attractive route of vaccination because it targets tissue rich in dendritic cells, has dose-sparing potential, and allows needle-free delivery. However, few adjuvants are effective, nonreactogenic, and compatible with needle-free delivery devices. In this study, we demonstrate that a combination adjuvant composed of cyclic-di-AMP (cdAMP) and the plant-derived nanoparticle adjuvant Nano-11 significantly enhanced the immune response to ID-injected vaccines in mice and pigs with minimal local reaction at the injection site. The cdAMP/Nano-11 combination adjuvant increased Ag uptake by lymph node-resident and migratory skin dendritic cell subpopulations, including Langerhans cells. ID immunization with cdAMP/Nano-11 expanded the population of germinal center B cells and follicular helper T cells in the draining lymph node and Ag-specific Th1 and Th17 cells in the spleen. It elicited an enhanced immune response with a significant increase of IgG1 and IgG2a responses in mice at a reduced dose compared with i.m. immunization. An increased IgG response was observed following needle-free ID immunization of pigs. Nano-11 and cdAMP demonstrated a strong synergistic interaction, as shown in the activation of mouse, human, and porcine APC, with increased expression of costimulatory molecules and secretion of TNF and IL-1ß. The combination adjuvant induced robust activation of both NF-κB and IFN regulatory factor signaling pathways and the NLRP3 inflammasome. We conclude that the combination of Nano-11 and cdAMP is a promising adjuvant for ID delivery of vaccines that supports a balanced immune response.

Proteomic analysis reveals that aging rabbit vocal folds are more vulnerable to changes caused by systemic dehydration.

BACKGROUND: Older adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol. RESULTS: Young and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of - 5.4% and - 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration. CONCLUSIONS: Water restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

Global landscape of protein complexes in postprandial-state livers from diet-induced obese and lean mice.

Obesity is associated with a spectrum of nonalcoholic fatty liver disease (NAFLD) which is characterized by steatosis. Prolonged fat deposition aggravates liver dysfunctions leading to an advanced form of NAFLD such as steatohepatitis and cirrhosis. As liver function in the postprandial state is critical for macronutrient metabolism and energy homeostasis, we sought to determine the differences in protein complex profiles in lean and fatty livers in the postprandial state. Protein complex profiling is of interest as proteins often do not function alone and the information on the interactions may reveal novel etiology of NAFLD, which is currently limited compared with proteome profiles or RNA-sequencing profiles. To this end, we fractionated liver lysates using size-exclusion chromatography (SEC) and analyzed each fraction using untargeted LC-MS/MS. We identified 1172 proteins that were discovered in lean and fatty livers, and their elution profiles were compared. We found that the majority of liver proteins were present as putative complexes. Also, the fatty liver protein elution profile showed great conservations as lean liver despite the metabolic disease state. Yet, we discovered a few proteins that showed different elution patterns in the fatty liver, including Acadm, Aldh1a7, Aldh1a1, Akr1a1, Eif3l, Fkbp2, G6pdx, Gm20441, Hao1, Pcna, Pkm, Ppif, Prdx4, Stmn1, Tagln, Tubb4b, Ubqln2, and Usp14, which may be involved in high fat diet-induced alterations of protein oligomerization and hepatic functions. Overall, our protein complex profiling could expand our understanding of hepatic abnormalities that cannot be uncovered by simple quantitation of protein expression.

Nutritional management of immediate hypersensitivity to legumes in vegetarians.

Vegetarianism is becoming a common practice among people. Products of vegetable origin are also on the rise, such as vegetable "milk" and legume-based snacks, which may lead to legume sensitivity and allergies in vegetarian diet followers. Furthermore, products derived from legumes, such as lupin flour or fenugreek powder, are often used as food additives. They function as hidden allergens, not always evident on the precautionary labeling, favoring allergic reactions. As dietary allergen restriction is the fundamental pillar in managing patients with food allergies, this review aims to reflect on practical aspects-diagnosis and nutritional management-in managing legume allergies in vegetarians, aiming to reduce the negative nutritional impact of an even more restrictive diet.

Proteomic Analysis of the Effect of Salmonella Challenge on Broiler Chicken.

Salmonella enteritidis is a foodborne pathogen that causes high morbidity in poultry. Proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to study the effects of Salmonella infection on spleen proteome in broiler chickens. Day-old broilers were assigned to control (CON; n = 60) or Salmonella challenge (CON−SE; n = 60), and gavaged with Tryptic soy agar broth or SE. A subset of chicks was euthanized on D3 and D7 (n = 4/group/day) and the spleen was removed, and rapidly frozen, subsequently proteome was measured using label-free LC-MS/MS. Protein spectra were mapped to Gallus gallus Uniprot database. Differentially abundant proteins (DAP; FDR < 0.05) between days and treatments were identified using ANOVA. Cecal content of Salmonella in CON−SE was 3.37 log10 CFU/g and CON were negative. Across the 16 samples, 2625 proteins were identified. Proteins that decreased in abundance between days mediated cell cycle progression, while those that increased in abundance function in cytoskeleton and mRNA processing. SE infection caused an increase in proteins that mediated redox homeostasis, lysosomal activities, and energy production, while proteins decreased in abundance-mediated developmental progression. Proteomic signatures of spleen suggest SE infection was metabolically costly, and energy was diverted from normal developmental processes to potentiate disease resistance mechanisms.

Disruption of pulmonary resolution mediators contribute to exacerbated silver nanoparticle-induced acute inflammation in a metabolic syndrome mouse model.

Pre-existing conditions modulate sensitivity to numerous xenobiotic exposures such as air pollution. Specifically, individuals suffering from metabolic syndrome (MetS) demonstrate enhanced acute inflammatory responses following particulate matter inhalation. The mechanisms associated with these exacerbated inflammatory responses are unknown, impairing interventional strategies and our understanding of susceptible populations. We hypothesize MetS-associated lipid dysregulation influences mediators of inflammatory resolution signaling contributing to increased acute pulmonary toxicity. To evaluate this hypothesis, healthy and MetS mouse models were treated with either 18-hydroxy eicosapentaenoic acid (18-HEPE), 14-hydroxy docosahexaenoic acid (14-HDHA), 17-hydroxy docosahexaenoic acid (17-HDHA), or saline (control) via intraperitoneal injection prior to oropharyngeal aspiration of silver nanoparticles (AgNP). In mice receiving saline treatment, AgNP exposure resulted in an acute pulmonary inflammatory response that was exacerbated in MetS mice. A targeted lipid assessment demonstrated 18-HEPE, 14-HDHA, and 17-HDHA treatments altered lung levels of specialized pro-resolving lipid mediators (SPMs). 14-HDHA and 17-HDHA treatments more efficiently reduced the exacerbated acute inflammatory response in AgNP exposed MetS mice as compared to 18-HEPE. This included decreased neutrophilic influx, diminished induction of inflammatory cytokines/chemokines, and reduced alterations in SPMs. Examination of SPM receptors determined baseline reductions in MetS mice compared to healthy as well as decreases due to AgNP exposure. Overall, these results demonstrate AgNP exposure disrupts inflammatory resolution, specifically 14-HDHA and 17-HDHA derived SPMs, in MetS contributing to exacerbated acute inflammatory responses. Our findings identify a potential mechanism responsible for enhanced susceptibility in MetS that can be targeted for interventional therapeutic approaches.

Laboratory screening test with inhalant and food allergens in atopic Brazilian children and adolescents: a performance.

The Phadiatop Infant® (PhInf) is a panel developed to assess allergic sensitization (immunoglobulin E [IgE]) in children aged <5 years and combines inhalant and food allergens. The test has not been evaluated outside Europe. This is a cross-sectional study conducted at 11 pediatric allergy centers to evaluate PhInf as an allergic disease screening method in Brazilian children. Children as controls and patients (aged 6 months-18 years) were grouped according to their primary disease and age group. PhInf and specific serum IgE (sIgE) screening was performed for Dermatophagoides pteronyssinus (DP), cat and dog epithelia, a mix of grasses and pollens, eggs, cow's milk, peanuts, and shrimp. Values ≥ 0.35 kUA/L (or PAU/L) were considered positive. A total of 470 children and adolescents, which included 385 patients and 85 controls, participated in the study (47.7% boys, average age: 6.3 years). In all, 72.6% of the participants had positive PhInf test (n = 341), with a higher proportion of those having food allergy (92.6%), atopic dermatitis (91.9%), and those aged >13 years having allergy (95%). The PhInf and sIgE agreement between patients (Kappa = 0.94, P < 0.001) and controls (Kappa = 0.84, P < 0.001) was high. PhInf and DP agreement in patients aged >13 years was excellent (Kappa = 0.936, P < 0.001). Compared with sIgE dosage, PhInf had high sensitivity (97%) and specificity (93%). Positivity of PhInf test in this population was high and had an excellent correlation with the allergens comprising the panel. It is a useful method for screening children suspected of having allergic diseases in a non-European country.

Exposure to chronic light-dark phase shifts during the prepartum nonlactating period attenuates circadian rhythms, decreases blood glucose, and increases milk yield in the subsequent lactation.

Maintaining metabolic balance is a key factor in the health of dairy cattle during the transition from pregnancy to lactation. Little is known regarding the role of the circadian timing system in the regulation of physiological changes during the transition period. We hypothesized that disruption of the cow's circadian timing system by exposure to chronic light-dark phase shifts during the prepartum period would negatively affect the regulation of homeostasis and cause metabolic disturbances, leading to reduced milk production in the subsequent lactation. The objective was to determine the effect of exposure to chronic light-dark phase shift during the last 5 wk prepartum of the nonlactating dry period on core body temperature, melatonin, blood glucose, ß-hydroxybutyric acid (BHB) and nonesterified fatty acid (NEFA) concentrations, and milk production. Multiparous cows were moved to tiestalls at 5 wk before expected calving and assigned to control (CTR; n = 16) or phase-shifted (PS; n = 16) treatments. Control cows were exposed to 16 h of light and 8 h of dark. Phase-shifted cows were exposed to the same photoperiod; however, the light-dark cycle was shifted 6 h every 3 d until parturition. Resting behavior and feed intake were recorded daily. Core body temperature was recorded vaginally for 48 h at 23 and 9 d before expected calving using calibrated data loggers. Blood concentrations of melatonin, glucose, BHB, and NEFA were measured during the pre- and postpartum periods. Milk yield and composition were measured through 60 DIM. Treatment did not affect feed intake or body condition. Cosine fit analysis of 24-h core body temperature and circulating melatonin indicated attenuation of circadian rhythms in the PS treatment compared with the CTR treatment. Phase-shifted cows had lower rest consolidation, as indicated by more total resting time, but shorter resting period durations. Phase-shifted cows had lower blood glucose concentration compared with CTR cows (4 mg/mL decrease), but BHB and NEFA concentrations were similar between PS and CTR cows. Milk yield and milk fat yield were greater in PS compared with CTR cows (2.8 kg/d increase). Thus, exposure to chronic light-dark phase shifts during the prepartum period attenuated circadian rhythms of core body temperature, melatonin, and rest-activity behavior and was associated with increased milk fat and milk yield in the postpartum period despite decreased blood glucose pre- and postpartum. Therefore, less variation in central circadian rhythms may create a more constant milieu that supports the onset of lactogenesis.

Phadiatop, Phadiatop Infant and total IgE evaluated in allergic Brazilian children and adolescents.

The clinical history is of importance in the investigation of allergic diseases but does have limitations. Many allergic conditions will be over-diagnosed if anamnesis alone is used for diagnostic criteria. Serum total immunoglobulin E (TIgE) quantification, as well as panels containing allergens prevalent in the studied population, may serve as screening tests and facilitate the diagnosis of allergic disease or its exclusion. We assessed the positivity of two versions of these tests, Phadiatop Europe® (PhEU) and Phadiatop Infant® (PhInf), as well as total IgE (TigE) values in patients with a medical diagnosis of allergic disease and non-allergic individuals. METHODS: A cross-sectional study performed in eleven Brazilian pediatric allergy centers with patients divided into groups according to the primary condition and a group of assessed control subjects. They were submitted to TIgE measurement and screening tests (PhEu and PhInf). RESULTS: TIgE mean serum levels were significantly higher among allergic patients, especially those with asthma/rhinitis or atopic dermatitis. The positivity of the screening tests, considering the total population, was 63.8% for PhEU and 72.6% for PhInf. These increased when we evaluated only the allergic subjects. The concordance index of the two tests was Kappa=0.7 and higher among those of greater age. CONCLUSIONS: In the assessed population, there were significantly higher levels among those with positive screening tests and PhInf showed better performance in the identification of sensitized individuals, regardless of age. This is the first study to evaluate Phadiatop and Phadiatop Infant in the same population.

Bacitracin Methylene Disalicylate (BMD) Treatment Affects Spleen Proteome in Broiler Chicks Infected with Salmonella enteritidis.

Bacitracin Methylene Disalicylate (BMD), as a feed additive to poultry diets, enhances digestion, prevents Salmonella enteritidis (SE) colonization, and treats current infections. The objective of this study was to utilize a quantitative proteomic approach to determine the effect of BMD feed additive on broiler chickens challenged with SE in the spleen proteome. At 1 d of age, chicks were randomly allocated into four groups: control with and without SE challenge (CON, n = 60; CON-SE, n = 60), BMD with and without SE challenge (BMD, n = 60; BMD-SE, n = 60). Birds in the CON-SE and BMD-SE treatment were administered SE inoculum by oral gavage. On day three and day seven post-gavage, the spleen was collected aseptically from birds in each treatment group (CON, n = 4/day; CON-SE, n = 4/day; BMD, n = 4/day; BMD-SE, n = 4/day). Proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed an increased abundance of 115 proteins and decreased of 77 due to the BMD. Proteins that decreased in abundance were enriched for fibrinogen complex and extracellular space, whereas proteins that increased in abundance were enriched for proteasome-mediated ubiquitin-dependent protein catabolic process and mitochondrion. Analysis of the interaction between BMD and the Salmonella challenge found 230 differentially abundant proteins including proteins associated with RNA binding, spliceosome, protein transport, and cell adhesion among the upregulated proteins, and those associated with protein folding, carbon metabolism, biosynthesis of nucleotide sugars, response to oxidative stress, positive regulation of NIK/NF-kappaB signaling, and inflammatory response among the downregulated proteins. The impact of BMD treatment on spleen proteome indicates an anti-apoptotic effect. BMD also modified the response of the spleen to the SE challenge with a marked decrease in proteins that prompt cytokine synthesis and an increase in proteins involved in the selective removal of unfolded proteins.

Proteomic analysis of canine vaccines.

OBJECTIVE: To use proteomic analysis to identify qualitatively and quantitatively mammalian protein components of commercial veterinary vaccines against canine distemper, leptospirosis, borreliosis, and rabies. SAMPLE: 25 licensed veterinary vaccines (from 4 different manufacturers) against canine distemper and leptospirosis, borreliosis, and rabies (3-year and 1-year durations of immunity). PROCEDURES: Duplicate samples from a single-lot vial of each vaccine were prepared by acetone precipitation and proteolysis with trypsin and Lys-C protease mix. Peptides mixtures (1 µg) were analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap Fusion Lumos mass spectrometer. Liquid chromatography-tandem mass spectroscopy data were searched against a Bos taurus protein database using MaxQuant to identify and quantify mammalian proteins in the vaccines. Identified proteins were classified by function and network analysis to visualize interactions. RESULTS: The largest number of mammalian proteins was identified in 3-year rabies vaccines (median, 243 proteins; range, 184 to 339 proteins) and 1-year rabies vaccines (median, 193 proteins; range, 169 to 350 proteins). Borrelia and leptospirosis-distemper (L&D) vaccines had the lowest number of proteins. Rabies vaccines had the highest number of identified proteins in common (n = 316); 33 were unique to 1-year products and 44 were found in 3-year products. Borrelia and L&D vaccines had 16 and 22 uniquely identified proteins, respectively. The protein classifications were primarily modulators of protein-binding activity, enzymes, transfer-carrier proteins, cytoskeletal proteins, defense-immunity proteins, calcium-binding proteins, and extracellular matrix proteins. CLINICAL RELEVANCE: This study demonstrates proteomics application to evaluate quality differences among different vaccines, identifying potential stimulants of desirable and undesirable immune responses.

Development of an Anti-canine PD-L1 Antibody and Caninized PD-L1 Mouse Model as Translational Research Tools for the Study of Immunotherapy in Humans.

Immune checkpoint blockade therapy, one of the most promising cancer immunotherapies, has shown remarkable clinical impact in multiple cancer types. Despite the recent success of immune checkpoint blockade therapy, however, the response rates in patients with cancer are limited (∼20%-40%). To improve the success of immune checkpoint blockade therapy, relevant preclinical animal models are essential for the development and testing of multiple combination approaches and strategies. Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients. Therefore, the canine studies of immuno-oncology drugs can generate knowledge that informs and prioritizes new immuno-oncology therapy in humans. The challenge has been, however, that immunotherapeutic antibodies targeting canine immune checkpoint molecules such as canine PD-L1 (cPD-L1) have not been commercially available. Here, we developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties in multiple assays. We also evaluated the therapeutic efficacy of cPD-L1 antibodies in our unique caninized PD-L1 mice. Together, these in vitro and in vivo data, which include an initial safety profile in laboratory dogs, support development of this cPD-L1 antibody as an immune checkpoint inhibitor for studies in dogs with naturally occurring cancer for translational research. Our new therapeutic antibody and caninized PD-L1 mouse model will be essential translational research tools in raising the success rate of immunotherapy in both dogs and humans. Significance: Our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population.

Growth and Nutritional Biomarkers in Brazilian Infants with Cow's Milk Allergy at Diagnosis and 18-Month Follow-Up: A Prospective Cohort Study.

Purpose: This study aimed to describe the growth, body protein status, and micronutrient biomarkers of Brazilian infants with cow's milk allergy (CMPA) at baseline and at 18 months of follow-up in comparison with their healthy peers. Methods: Thirty infants with CMPA younger than six months of age were included in this longitudinal study, and their nutritional status was compared with that of 24 non-allergic age-matched children. Anthropometric measurements were used to assess growth, and blood and urine samples were analyzed for protein and micronutrient status. Mixed linear models adjusted for birth weight, socioeconomic status, infant feeding at baseline, weight-for-age, C-reactive protein, serum albumin, micronutrient dietary supplementation, and salt consumption were employed to evaluate the evolution of nutritional parameters throughout the follow-up period. Results: Overall, the mean age of the children at enrolment was 2.9 (standard deviation 1.7) months, and 29 children (53.7%) were male. Infants with CMPA showed a higher prevalence of functional iron depletion (transferrin saturation <20) (p=0.027), lower serum ferritin (p=0.009), and lower urinary iodine (p=0.034) levels than non-allergic children at baseline. Patients with CMPA showed a higher increment in weight-for-age and length-for-age over time than those in the control group (p<0.01). Mixed linear analyses showed a significantly lower increase in serum vitamin B12 (s-B12) (p=0.001) and urinary iodine (p<0.001) concentrations over time compared to the control group. Conclusion: Infants with CMPA on a cow's milk elimination diet had a higher weight and length at 18 months of follow-up but showed signs of inadequate iron, iodine, and B-12 vitamin status.

Multi-Omics Approach Reveals Dysregulation of Protein Phosphorylation Correlated with Lipid Metabolism in Mouse Non-Alcoholic Fatty Liver.

Obesity caused by overnutrition is a major risk factor for non-alcoholic fatty liver disease (NAFLD). Several lipid intermediates such as fatty acids, glycerophospholipids and sphingolipids are implicated in NAFLD, but detailed characterization of lipids and their functional links to proteome and phosphoproteome remain to be elucidated. To characterize this complex molecular relationship, we used a multi-omics approach by conducting comparative proteomic, phoshoproteomic and lipidomic analyses of high fat (HFD) and low fat (LFD) diet fed mice livers. We quantified 2447 proteins and 1339 phosphoproteins containing 1650 class I phosphosites, of which 669 phosphosites were significantly different between HFD and LFD mice livers. We detected alterations of proteins associated with cellular metabolic processes such as small molecule catabolic process, monocarboxylic acid, long- and medium-chain fatty acid, and ketone body metabolic processes, and peroxisome organization. We observed a significant downregulation of protein phosphorylation in HFD fed mice liver in general. Untargeted lipidomics identified upregulation of triacylglycerols, glycerolipids and ether glycerophosphocholines and downregulation of glycerophospholipids, such as lysoglycerophospholipids, as well as ceramides and acylcarnitines. Analysis of differentially regulated phosphosites revealed phosphorylation dependent deregulation of insulin signaling as well as lipogenic and lipolytic pathways during HFD induced obesity. Thus, this study reveals a molecular connection between decreased protein phosphorylation and lipolysis, as well as lipid-mediated signaling in diet-induced obesity.

Local and Systemic Changes in Lipid Profile as Potential Biomarkers for Canine Atopic Dermatitis.

Lipids play a critical role in the skin as components of the epidermal barrier and as signaling and antimicrobial molecules. Atopic dermatitis in dogs is associated with changes in the lipid composition of the skin, but whether these precede or follow the onset of dermatitis is unclear. We applied rapid lipid-profiling mass spectrometry to skin and blood of 30 control and 30 atopic dogs. Marked differences in lipid profiles were observed between control, nonlesional, and lesional skin. The lipid composition of blood from control and atopic dogs was different, indicating systemic changes in lipid metabolism. Female and male dogs differed in the degree of changes in the skin and blood lipid profiles. Treatment with oclacitinib or lokivetmab ameliorated the skin condition and caused changes in skin and blood lipids. A set of lipid features of the skin was selected as a biomarker that classified samples as control or atopic dermatitis with 95% accuracy, whereas blood lipids discriminated between control and atopic dogs with 90% accuracy. These data suggest that canine atopic dermatitis is a systemic disease and support the use of rapid lipid profiling to identify novel biomarkers.

Developmental atrazine exposure in zebrafish produces the same major metabolites as mammals along with altered behavioral outcomes.

Atrazine (ATZ) is the second most commonly applied agricultural herbicide in the United States. Due to contamination concerns, the U.S. EPA has set the maximum contaminant level in potable water sources at 3 parts per billion (ppb; µg/l). Depending on the time of year and sampling location, water sources often exceed this limit. ATZ is an endocrine disrupting chemical in multiple species observed to target the neuroendocrine system. In this study the zebrafish vertebrate model was used to test the hypothesis that a developmental ATZ exposure generates metabolites similar to those found in mammals and alters morphology and behavior in developing larvae. Adult AB zebrafish were bred, embryos were collected, and exposed to 0, 0.3, 3, or 30 ppb ATZ from 1 to 120 h post fertilization (hpf). Targeted metabolomic analysis found that zebrafish produce the same major ATZ metabolites as mammals: desethyl atrazine (DEA), deisopropyl atrazine (DIA), and diaminochloroatrazine (DACT). The visual motor response test at 120 hpf detected hyperactivity in larvae in the 0.3 ppb treatment group and hypoactivity in the 30 ppb treatment group (p < 0.05). Further analysis into behavior during the dark and light phases showed zebrafish larvae exposed to 0.3 ppb ATZ had an increase in total distance moved in the first light phase and time spent moving in the first dark and light phases (p < 0.05). Alternatively, a decrease in total distance moved was observed in the second and third dark phases in zebrafish exposed to 30 ppb ATZ (p < 0.05). No significant differences were observed for any of the morphological measurements following ATZ exposure from 1 to 120 hpf (p > 0.05). These findings suggest that a ATZ exposure during early development generates metabolite profiles similar to mammals and leads to behavioral alterations supporting ATZ as a neurodevelopmental toxicant.

Protective Effects of the Hydroethanolic Extract of Fridericia chica on Undifferentiated Human Neuroblastoma Cells Exposed to α-Zearalenol (α-ZEL) and ß-Zearalenol (ß-ZEL).

Fridericia chica (Bignoniaceae) is a traditional medicinal plant. The aim of this research was to determine the protective effects of the hydroethanolic extract from the F. chica leaves (HEFc) against the cytotoxicity of zearalenone (α-ZEL) and ß-ZEL on SH-SY5Y cells. Free radical scavenging activity of HEFc was evaluated using the DPPH method. The cytotoxicity of both zearalenone metabolites and HEFc was examined using MTT test, as was the cytoprotective effects of the HEFc on cells treated with these mycotoxins. The chemical composition of HEFc was determined using UPLC-QTOF-MS/MS. HEFc elicited good DPPH radical scavenging activity following a concentration-dependent relationship. Cells exposed to α-ZEL exhibited a viability ˂50% after 48 h of treatment (25 and 50 µM), while those exposed to ß-ZEL showed viability ˂50% (100 µM) and ˂25% (25-100 µM) after 24 and 48 h of exposure, respectively. HEFc showed a significant increase in cell viability after exposure to α-ZEL (25 and 50 µM) and ß-ZEL (6-100 µM) (p < 0.05). UPLC-QTOF-MS/MS analyses allowed the identification of 10 phytochemical components in the HEFc. In short, the hydroethanolic extract of F. chica grown in Colombian Caribbean can protect against the effects of mycotoxins and it is a valuable source of compounds with antioxidant properties.

Allergic sensitization pattern of patients in Brazil.

OBJECTIVE: Allergic sensitization is one of the key components for the development of allergies. Polysensitization seems to be related to the persistence and severity of allergic diseases. Furthermore, allergic sensitization has a predictive role in the development of allergies. The aim of this study was to characterize the pattern of sensitization of atopic patients treated at different pediatric allergy referral centers in Brazil. METHODS: A nation-wide transversal multicenter study collected data on patients attended in Brazil. Peripheral blood samples were collected to determine the serum levels of allergen-specific IgE. If allergen-specific IgE was higher than 0.1 kUA/L, the following specific components were quantified. RESULTS: A total of 470 individuals were enrolled in the study. Mite sensitization was the most frequent kind in all participants. A high frequency of sensitization to furry animals and grasses featured in the respiratory allergies. Regarding components, there was a predominance of sensitization to Der p 1 and Der p 2. It has been verified that having a food allergy, atopic dermatitis, or multimorbidity are risk factors for the development of more severe allergic disease. CONCLUSION: Studies on the pattern of allergic sensitization to a specific population offer tools for the more effectual prevention, diagnosis, and treatment of allergic diseases. Sensitization to dust mites house was the most prevalent in the evaluated sample. High rates of sensitization to furry animals also stand out. Patients with food allergy, atopic dermatitis, or multimorbidity appear to be at greater risk for developing more severe allergic diseases.

Phase I/II Trial of Vemurafenib in Dogs with Naturally Occurring, BRAF-mutated Urothelial Carcinoma.

BRAF-targeted therapies including vemurafenib (Zelboraf) induce dramatic cancer remission; however, drug resistance commonly emerges. The purpose was to characterize a naturally occurring canine cancer model harboring complex features of human cancer, to complement experimental models to improve BRAF-targeted therapy. A phase I/II clinical trial of vemurafenib was performed in pet dogs with naturally occurring invasive urothelial carcinoma (InvUC) harboring the canine homologue of human BRAF V600E The safety, MTD, pharmacokinetics, and antitumor activity were determined. Changes in signaling and immune gene expression were assessed by RNA sequencing and phosphoproteomic analyses of cystoscopic biopsies obtained before and during treatment, and at progression. The vemurafenib MTD was 37.5 mg/kg twice daily. Anorexia was the most common adverse event. At the MTD, partial remission occurred in 9 of 24 dogs (38%), with a median progression-free interval of 181 days (range, 53-608 days). In 18% of the dogs, new cutaneous squamous cell carcinoma and papillomas occurred, a known pharmacodynamic effect of vemurafenib in humans. Upregulation of genes in the classical and alternative MAPK-related pathways occurred in subsets of dogs at cancer progression. The most consistent transcriptomic changes were the increase in patterns of T lymphocyte infiltration during the first month of vemurafenib, and of immune failure accompanying cancer progression. In conclusion, the safety, antitumor activity, and cutaneous pharmacodynamic effects of vemurafenib, and the development of drug resistance in dogs closely mimic those reported in humans. This suggests BRAF-mutated canine InvUC offers an important complementary animal model to improve BRAF-targeted therapies in humans.