IgG1+ B-cell immunity predates IgE responses in epicutaneous sensitization to foods.

BACKGROUND: The generation of IgE-mediated food allergy in humans is silent and only diagnosed upon manifestation of clinical symptoms. While experimental models have been used to investigate some mechanisms of allergic sensitization, the generation of humoral immunity and memory remains to be elucidated. Here, we defined the evolution of allergen-specific B-cell responses during epicutaneous sensitization to foods. METHODS: Wild-type and genetic knockout animals, and drug or antibody strategies for cell depletion and immunoglobulin signaling blockade were used to investigate epicutaneous sensitization and disease progression; we analyzed allergen-specific germinal centers and IgG1+ memory B cells by flow cytometry, evaluated humoral responses, and determined clinical reactivity (anaphylaxis). RESULTS: Epicutaneous sensitization caused microscopic skin damage, inflammation, and recruitment of activated dendritic cells to the draining lymph nodes. This process generated allergen-specific IgG1+ germinal center B cells, serum IgG1, and anaphylaxis that was mediated by the alternative pathway. Whether we used peanut and/or ovalbumin from the egg white for sensitization, the allergen-specific IgG1+ memory compartment predominantly exhibited an immature, pro-germinal center phenotype (PDL-2- CD80- CD35+ CD73+ ). Subsequent subclinical exposures to the allergen induced IgE+ germinal center B cells, serum IgE, and likely activated the classical pathway of anaphylaxis. CONCLUSIONS: Our data demonstrate that IgG1+ B-cell immunity against food allergens in epicutaneous sensitization precedes the generation of IgE responses. Therefore, the assessment of allergen-specific cellular and humoral IgG1+ immunity may help to identify individuals at risk of developing IgE-mediated food allergy and hence provide a window for therapeutic interventions.

Comprehensive metabolomics identifies the alarmin uric acid as a critical signal for the induction of peanut allergy.

BACKGROUND: Food allergy, in particular peanut allergy, is a growing concern in Western countries. The prevalence of allergy to peanut, which currently stands at 1.4%, nearly tripled between 1997 and 2008. Allergic sensitization is a particularly difficult process to study as it is clinically silent. We sought to identify key pathways and mediators critically involved in the induction of allergic sensitization to peanut. METHODS: Comprehensive metabolomics analysis with liquid chromatography-mass spectrometry was used to detect metabolite changes in mice (C57BL/6) undergoing sensitization. Loss-of-function and gain-of-function studies were performed in mice subjected to two models of peanut sensitization and anaphylaxis that involved either oral or epicutaneous sensitization. Flow cytometric analyses on dendritic cells (DCs) in vitro and in vivo were used to investigate the mechanisms of immune activation. RESULTS: Elevated levels of uric acid (UA) were detected in mice undergoing sensitization as well as in peanut-allergic children who were not challenged with peanut. In mice, the depletion of UA during sensitization prevented the development of peanut-specific immunoglobulins IgE and IgG1 as well as anaphylaxis while exogenous delivery of UA crystals (monosodium urate, MSU) restored the allergic phenotype. Monosodium urate enhanced CD86 and OX40L expression on DCs, independent of Toll-like receptors 2 and 4, the NLRP3 inflammasome, and IL-1ß, via a PI3K signaling pathway. CONCLUSION: Overproduction of the UA alarmin in the local microenvironment plays a critical role in the induction of peanut-allergic sensitization, likely due to its ability to activate DCs. These finding suggest that cellular damage or tissue injury may be an essential requisite for the development of allergic sensitization to foods.

T helper cell IL-4 drives intestinal Th2 priming to oral peanut antigen, under the control of OX40L and independent of innate-like lymphocytes.

Intestinal T helper type 2 (Th2) immunity in food allergy results in IgG1 and IgE production, and antigen re-exposure elicits responses such as anaphylaxis and eosinophilic inflammation. Although interleukin-4 (IL-4) is critically required for allergic sensitization, the source and control of IL-4 during the initiation of Th2 immunity in vivo remains unclear. Non-intestinal and non-food allergy systems have suggested that natural killer-like T (NKT) or γδ T-cell innate lymphocytes can supply the IL-4 required to induce Th2 polarization. Group 2 innate lymphoid cells (ILCs) are a novel IL-4-competent population, but their contribution to initiating adaptive Th2 immunity is unclear. There are also reports of IL-4-independent Th2 responses. Here, we show that IL-4-dependent peanut allergic Th2 responses are completely intact in NKT-deficient, γδ T-deficient or ILC-deficient mice, including antigen-specific IgG1/IgE production, anaphylaxis, and cytokine production. Instead, IL-4 solely from CD4(+) Th cells induces full Th2 immunity. Further, CD4(+) Th cell production of IL-4 in vivo is dependent on OX40L, a costimulatory molecule on dendritic cells (DCs) required for intestinal allergic priming. However, both Th2 cells and ILCs orchestrated IL-13-dependent eosinophilic inflammation. Thus, intestinal Th2 priming is initiated by an autocrine/paracrine acting CD4(+) Th cell-intrinsic IL-4 program that is controlled by DC OX40L, and not by NKT, γδ T, or ILC cells.

Effects of added chelated trace minerals, organic selenium, yeast culture, direct-fed microbials, and Yucca schidigera extract in horses. Part I: Blood nutrient concentration and digestibility.

The objective of this study was to test the hypothesis that feed additives such as chelated minerals, organic Se, yeast culture, direct-fed microbials, and Yucca schidigera extract would improve nutrient digestibility when included in an equine diet. Horses (Quarter Horse geldings 4.5 to 16 yr of age; mean BW 522 kg ± 46 kg) were acclimated to 100% pelleted diets formulated with (ADD) and without (CTRL) commercially available sources of the aforementioned additives followed by a 14-d collection period of feces and urine. Chelated sources of Cu, Zn, Mn and Co were utilized versus sulfated forms, at a 100% replacement rate. No significant differences among apparent the digestibility of DM, ADF, or NDF (P= 0.665, P = 0.866, P = 0.747, respectively) were detected between dietary treatments. Likewise, no differences in apparent digestibility of Cu (P = 0.724), Zn (P = 0.256), Mn (P = 0.888), Co (P = 0.71), or Se (P = 0.588) were observed. No differences were observed in serum Cu, Mn, or Co concentrations between ADD and CTRL at acclimation or collection time points (P > 0.05). While no difference in serum Zn concentrations were observed between ADD and CTRL groups at acclimation (P > 0.05), they were statistically higher at the collection time period for horses consuming CTRL (P < 0.0001). Whole blood Se concentration was greater in the CTRL group versus the ADD group both at acclimation (P = 0.041) and collection (P = 0.005) time periods. In reference to time, serum Cu concentrations increased (P = 0.012) for animals consuming CTRL, but not ADD (P > 0.05). Serum Zn concentrations of horses consuming both ADD (P = 0.021) and CTRL (P < 0.0001) increased over time from acclimation to collection time points. No time differences (P > 0.05) were observed in serum Mn concentrations. Serum Co concentrations increased over time in horses consuming both ADD (P = 0.001) and CTRL (P = 0.021). From acclimation to collection, whole blood Se concentration increased for horses consuming CTRL (P = 0.01) but not for ADD (P > 0.05). The results of this study indicate no effect on nutrient digestibility due to the inclusion of chelated minerals, organic Se, yeast culture, direct-fed microbials, and Yucca schidigera extract for horses at maintenance.

Effects of added chelated trace minerals, organic selenium, yeast culture, direct-fed microbials, and Yucca schidigera extract in horses: II. Nutrient excretion and potential environmental impact.

The objective of this study was to test the hypothesis that an equine diet formulated with chelated trace minerals, organic selenium, yeast culture, direct-fed microbials (DFM) and Yucca schidigera extract would decrease excretion of nutrients that have potential for environmental impact. Horses were acclimated to 100% pelleted diets formulated with (ADD) and without (CTRL) the aforementioned additives. Chelated sources of Cu, Zn, Mn, and Co were included in the ADD diet at a 100% replacement rate of sulfate forms used in the CTRL diet. Additionally, the ADD diet included organic selenium yeast, DFM, and Yucca schidigera extract. Ten horses were fed the 2 experimental diets during two 42-d periods in a crossover design. Total fecal and urine collection occurred during the last 14 d of each period. Results indicate no significant differences between Cu, Zn, Mn, and Co concentrations excreted via urine (P > 0.05) due to dietary treatment. There was no difference between fecal Cu and Mn concentrations (P > 0.05) based on diet consumed. Mean fecal Zn and Co concentrations excreted by horses consuming ADD were greater than CTRL (P < 0.003). Differences due to diet were found for selenium fecal (P < 0.0001) and urine (P < 0.0001) excretions, with decreased concentrations found for horses consuming organic selenium yeast (ADD). In contrast, fecal K (%) was greater (P = 0.0421) for horses consuming ADD, whereas concentrations of fecal solids, total N, ammonia N, P, total ammonia, and fecal output did not differ between dietary treatments (P > 0.05). In feces stockpiled to simulate a crude composting method, no differences (P > 0.05) due to diet were detected for particle size, temperature, moisture, OM, total N, P, phosphate, K, moisture, potash, or ammonia N (P > 0.05). Although no difference (P = 0.2737) in feces stockpile temperature due to diet was found, temperature differences over time were documented (P < 0.0001). In conclusion, the addition of certain chelated mineral sources, organic Se yeast, DFM, and Yucca schidigera extract did not decrease most nutrient concentrations excreted. Horses consuming organic selenium as part of the additive diet had lower fecal and urine Se concentrations, as well as greater fecal K concentrations.

Developmental changes in the concentrations of glutamine and other amino acids in plasma and skeletal muscle of the Standardbred foal.

Glutamine is concentrated within skeletal muscle, where it has been proposed to play a regulatory role in maintaining protein homeostasis. The work presented here addressed the hypothesis that glutamine would be the most abundant free alpha-AA in plasma and skeletal muscle in the foal during the first year of life. Glycine, however, was the most abundant free alpha-AA in plasma at birth and between 3 and 12 mo of age. The concentration of glutamine, the second most abundant AA at birth, increased through the first 7 d (P < 0.05) and then returned to values similar to those at birth. This resulted in glutamine being the most abundant free alpha-AA in plasma from 1 d through 1 mo of age. The most abundant free alpha-AA in skeletal muscle at birth was glutamine, but the concentration fell by more than 50% by d 15 and continued to decrease, reaching about one-third of the original values by 1 yr of age (P < 0.05). Glutamine synthetase was barely detectable in skeletal muscle at birth, but the abundance increased rapidly within 15 d of birth. The concentration of glycine, the second most abundant alpha AA in muscle at birth, decreased by about 40% by d 15 (P < 0.05) and then stabilized at this value throughout the year. In contrast, glutamate, alanine, and serine concentrations, the third, fourth, and fifth most abundant free alpha-AA in muscle at birth, respectively, increased to new stable concentrations between 3 and 6 mo of age (P < 0.05). This resulted in alanine being the most abundant free alpha-AA in skeletal muscle at 12 mo of age, followed by glutamate, glutamine, and glycine. The decrease in intramuscular glutamine content, particularly during the first 2 wk after birth, is not compatible with a regulatory role for glutamine in muscle protein synthesis because it occurred at the time of maximum growth in these animals. The findings that, at certain times of development, glutamine was not the most abundant free alpha-AA in the foal is novel and signifies that intramuscular glutamine may have functions specific to muscle type and mammalian species.

Changes in glutamine metabolism indicate a mild catabolic state in the transition mare.

Glutamine is the most abundant free alpha-AA in the mammalian body, and large amounts of glutamine are extracted by both the fetus during pregnancy and the mammary gland during lactation. The work presented here addressed the hypothesis that there would be major changes in glutamine metabolism in the mare during the transition period, the time between late gestation, parturition, and early lactation. Eight foals were born to Standardbred mares provided with energy and protein at 10% above NRC recommendations, and foals remained with mares for 6 mo. During lactation, lean body mass decreased by 1.5% (P < 0.05), whereas fat mass was unchanged throughout gestation and lactation. There was a sharp increase in the concentration of most plasma metabolites and hormones after birth, which was due in part to hemoconcentration because of fluid shifts at parturition. Plasma glutamine concentration, however, was maintained at greater concentrations for up to 2 wk postpartum but then began to decrease, reaching a nadir at approximately 6 wk of lactation. Skeletal muscle glutamine content did not change, but glutamine synthetase expression was decreased at the end of lactation (P < 0.05). Free glutamine was highly abundant in milk early in lactation, but the concentration decreased by more than 50% after 3 mo of lactation and paralleled the decrease in plasma glutamine concentration. Thus, lactation represents a mild catabolic state for the mare in which decreased glutamine concentrations may compromise the availability of glutamine to other tissues such as the intestines and the immune system.

Apoptosis and antioxidant status are influenced by age and exercise training in horses.

Eight mature (12 +/- 2 yr; MAT) and 5 older (22 +/- 2 yr; OLD) Standardbred mares were used to test the hypothesis that aging and exercise training would alter apoptosis in white blood cells and antioxidant status. The horses were housed indoors overnight (16 h/d) in 3 m x 3 m stalls and were turned out in a drylot during the day. They were fed a diet consisting of total mixed ration, hay cubes fed ad libitum or an equine senior diet plus grass hay. Horses were trained for 20 to 30 min/d, 3 to 5 d/wk for 8 wk at a submaximal work intensity between 60 to 70% of maximal heart rate. A graded exercise test (GXT; stepwise test until exhaustion) was performed before (GXT1) and after (GXT2) the 8 wk of training. During the GXT, blood samples and heart rate were taken at rest, 6 m/s, fatigue, and at 5 and 60 min postfatigue. Fatigue plasma lactate concentration was greater in MAT (19.3 +/- 1.5 at 10 m/s) compared with the OLD (10.9 +/- 1.2 mmol/L at 9 m/s; P = 0.008) horses. There was no effect of age or training on plasma lipid hydroperoxide (LPO) concentration. However, there was a positive correlation between LPO and plasma lactate concentration (r = 0.27, P = 0.006) during acute exercise. There was a greater concentration of total glutathione after GXT1 than after GXT2 (111.8 +/- 5.0 vs. 98.6 +/- 3.4 microM, respectively; P = 0.0002) for both age groups. Apoptosis was less (P = 0.002) in white blood cells of the MAT vs. the OLD group. These results demonstrate that older horses are under similar amounts of oxidative stress, measured by LPO, and have similar levels of glutathione in their systems compared with mature horses. The observation that more glutathione was needed during GXT1 for both groups of horses indicates that training helps horses adapt their system for the intense post-training exercise tests. The greater level of white blood cell apoptosis also indicates that older horses may be immune-compromised during exercise. However, research still needs to be performed regarding dietary supplementation in the aged horse.

Oral and intravenous carbohydrate challenges decrease active ghrelin concentrations and alter hormones related to control of energy metabolism in horses.

This study tested the hypothesis that grain and intravenous dextrose challenges would alter plasma concentrations of active ghrelin, adiponectin, leptin, glucose, insulin, and cortisol in Standardbred mares. To deliver 0.5 g of glucose (dextrose solution for the intravenous test)/kg of BW, mares received intravenous dextrose (50% solution) or oral grain administration in 2 trials. In response to the oral grain challenge, plasma glucose and insulin concentrations increased (P < 0.001) by 56 and 802%, respectively. Plasma ghrelin concentration initially decreased (P < 0.001) by 40%, then subsequently increased (P < 0.05) from its nadir by 259%. Plasma leptin concentration decreased (P = 0.002) 17% compared with baseline. There was no change (P = 0.34) in plasma adiponectin concentration in response to oral grain challenge; however, plasma cortisol concentrations decreased (P < 0.001) by 24%. In response to the intravenous dextrose challenge, plasma glucose and insulin concentrations increased (P < 0.001) by 432 and 395%, respectively. Plasma active ghrelin concentration initially decreased (P < 0.001) by 56%, then subsequently increased (P < 0.001) from its nadir by 314%. Plasma leptin concentration also increased (P < 0.001) by 33% compared with baseline. There was no change (P = 0.18) in plasma adiponectin concentration throughout the dextrose challenge. Plasma cortisol concentration increased (P = 0.027) by 20%. Hence, oral grain and intravenous nutrient challenges have the ability to alter variables potentially related to energy metabolism in mares, with acute changes in glucose and insulin possibly modulating changes in ghrelin and leptin.

Interval exercise alters feed intake as well as leptin and ghrelin concentrations in standardbred mares.

REASONS FOR PERFORMING STUDY: Horses in training tend to become inappetant; however, the mechanism responsible for this training-induced inappetance is not known. HYPOTHESIS: Training and/or ulcers alter the feed intake (FI) and hormonal and/or biochemical (active ghrelin, leptin, glucose, insulin and cortisol) responses to acute high intensity exercise. METHODS: Eight Standardbred mares underwent 3 interval exercise tests (IET) and 3 parallel control tests (CON) before (IET1) and after 8 weeks of training (IET2) and after treatment for gastric ulcers (IET3). Plasma samples were taken before (0 min), during (last 10 sec of velocities eliciting 40, 100 and 20% VO2max), and after (30 min, 60 min, 24 h) exercise (EX) or CON tests for RIA and colorimetric measurement of the concentrations of the above parameters. Samples were also collected before and after feeding. Horses were trained at a work intensity of 70% HRmax for 30 min/day, 5 days per week with FI measured daily. RESULTS: There were no changes (P>0.05) in any variable during the parallel control trials. However, there was a mismatch between FI and digestible energy (DE) requirements (P<0.05) with EX horses not meeting their DE requirements during the post training IETs. During all IETs, ghrelin, glucose and cortisol increased (P<0.05) during EX. Leptin only increased (P<0.05) during EX in the post training IETs. Insulin remained low during EX, but increased (P<0.05) post EX. CONCLUSION: High intensity exercise appeared to be associated with decreases in FI and alterations of leptin and ghrelin. POTENTIAL RELEVANCE: More research is needed to determine if there is a relationship between alterations of these hormones and changes in FI in horses that lose weight while in training.

Effect of omeprazole on markers of performance in gastric ulcer-free standardbred horses.

REASONS FOR PERFORMING STUDY: A large percentage of performance horses develop gastric ulcers and many of those horses are treated with omeprazole. Unfortunately, no data have been published on the effects of the drug on markers of performance in animals without ulcers. HYPOTHESIS: Omeprazole would alter markers of aerobic and anaerobic performance. METHODS: Ten unfit, healthy, ulcer free, Standardbred mares were administered either control (CON; oral apple sauce, 20 ml) or omeprazole (OP; oral paste, 4 mg/kg bwt s.i.d.) in a random crossover fashion with the investigators blind to the treatment. Treatments were administered for 7 days prior to performing an incremental exercise test (GXT) on a high-speed treadmill. Endoscopic examinations were performed just prior to the trial to verify that the mares were ulcer-free. During the GXT, the mares ran on a treadmill up a 6% grade to measure maximal oxygen consumption (VO2max), run time (RT), velocity at VO2max, maximal velocity (Vmax), packed cell volume (PCV), plasma lactate concentration (LA) and plasma protein concentration (TP). Measurements were recorded at rest, at the end of each 1 min step of the GXT and at 2 and 5 min post GXT. Data were analysed using ANOVA for repeated measures and t tests for paired comparisons. RESULTS: There was no effect (P>0.05) of omeprazole on VO2max; velocity at VO2max; RT; Vmax; 2 min recovery plasma LA. Nor were there any changes (P>0.05) in the relationship between treadmill speed and VO2, PCV, TP, or plasma LA. CONCLUSIONS: Omeprazole does not appear to improve physiological markers of performance in healthy, ulcer free horses. POTENTIAL RELEVANCE: These data may benefit various authorities responsible for deciding administration and timing policies of omeprazole as well as clinicians and horse owners.

Diurnal variation of ghrelin, leptin, and adiponectin in Standardbred mares.

Twelve Standardbred mares underwent blood sampling for 24 h to test the hypothesis that there is diurnal variation of humoral mediators of peripheral energy balance including active ghrelin, adiponectin, leptin, glucose, insulin, and cortisol. The experiment was conducted under acclimated conditions. Grass hay and pelleted grain were provided at 0730 and 1530. Plasma concentrations of active ghrelin and leptin concentrations both peaked (47.3 +/- 6.5 pg/ mL and 5.9 +/- 1.1 ng/mL, respectively; P < 0.05) at 1550, 20 min after feeding. Active ghrelin decreased (P < 0.05) to 28.9 +/- 4.5 pg/mL overnight. The nadir of leptin (4.6 +/- 0.9 ng/mL) occurred at 0650. Neither hormone showed variation (P > 0.05) after the morning feeding. Plasma glucose and insulin concentrations increased (P < 0.05) in response to feeding; however, the morning responses (glucose = 96.9 +/- 2.6 mg/dL; insulin = 40.6 +/- 7.3 uIU/mL) were greater (P < 0.05) than the afternoon responses (glucose = 89.9 +/- 1.8 mg/dL; insulin = 23.2 +/- 4.3 uIU/mL at 180 and 60 min after feeding, respectively). Cortisol concentrations increased (P < 0.05) during the morning hours, but did not respond to feeding, whereas adiponectin concentrations remained stable throughout the study. Hence, active ghrelin and leptin may be entrained to meal feeding in horses, whereas adiponectin seems unaffected. We concluded that there seems to be a diurnal variation in glucose and insulin response to a meal in horses. Furthermore, elevated glucose and insulin concentrations resulting from the morning feeding may be responsible for the increase in leptin concentration in the afternoon.

Efficacy and safety of the ProTek (Vifilcon A) therapeutic soft contact lens after photorefractive keratectomy.

PURPOSE: To test the ProTek (Vifilcon A) therapeutic soft contact lens in the alleviation of post-photorefractive keratectomy pain, its effect on epithelial healing, and its safety. METHODS: Forty-seven consecutive eligible patients undergoing unilateral excimer laser photorefractive keratectomy for myopia were randomly assigned to receive standard postoperative care with or without the use of a ProTek soft contact lens. Patients prospectively graded a self-administered 5-point scale for pain and a 4-point scale for abnormal sensations at 4, 8, 12, 16, and 20 hours after surgery. They also recorded the type and dose of all medications taken during that time period. All patients were examined on the first and third days after surgery. The lenses were worn continuously for 3 days. RESULTS: The soft contact lens group (n = 24) disclosed a statistically significant (P < .05) reduction in pain intensity and abnormal sensations that was greatest at 8, 12, 16, and 20 hours postoperatively. Compared with control patients (n = 23), the soft contact lens group showed significant decreased dependence on most pain medications after the 12th hour (P < .05) and faster epithelial healing (P = .03). However, one case of bacterial keratitis, two cases of subepithelial infiltrates, and seven cases of contact lens intolerance were present in the soft contact lens group. CONCLUSIONS: The ProTek therapeutic soft contact lenses were effective in decreasing pain and other related abnormal sensations after excimer photorefractive keratectomy. They decreased dependence on pain medications and hastened epithelial healing but were not well tolerated in some patients.

A comprehensive algorithm for determining whether a run-in strategy will be a cost-effective design modification in a randomized clinical trial.

In randomized clinical trials, poor compliance and treatment intolerance lead to reduced between-group differences, increased sample size requirements, and increased cost. A run-in strategy is intended to reduce these problems. In this paper, we develop a comprehensive set of measures specifically sensitive to the effect of a run-in on cost and sample size requirements, both before and after randomization. Using these measures, we describe a step-by-step algorithm through which one can estimate the cost-effectiveness of a potential run-in. Because the cost-effectiveness of a run-in is partly mediated by its effect on sample size, we begin by discussing the likely impact of a planned run-in on the required number of randomized, eligible, and screened subjects. Run-in strategies are most likely to be cost-effective when: (1) per patient costs during the post-randomization as compared to the screening period are high; (2) poor compliance is associated with a substantial reduction in response to treatment; (3) the number of screened patients needed to identify a single eligible patient is small; (4) the run-in is inexpensive; (5) for most patients, the run-in compliance status is maintained following randomization and, most importantly, (6) many subjects excluded by the run-in are treatment intolerant or non-compliant to the extent that we expect little or no treatment response. Our analysis suggests that conditions for the cost-effectiveness of run-in strategies are stringent. In particular, if the only purpose of a run-in is to exclude ordinary partial compliers, the run-in will frequently add to the cost of the trial. Often, the cost-effectiveness of a run-in requires that one can identify and exclude a substantial number of treatment intolerant or otherwise unresponsive subjects.