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Grass pollen triggers nearly 30% of bronchial allergic asthma cases. While most Q8 research focuses on pollen allergens, pollen lipids may also influence allergic reactions. Previous studies demonstrated that Timothy grass (TG, Phleum pratense) lipids, such as phytoprostanes, can activate immune cells, promoting pro-allergic responses. However, the role of water-insoluble pollen glycolipids in allergic airway inflammation remains unclear. Thus, this study aimed to isolate and characterize glycolipids from TG pollen and evaluate their bioactivity in allergic airway inflammation. Lipids were extracted from the water-insoluble pollen fraction, separated by silica gel, and fractionated by HPLC. GC-MS, HR ESI-MS, and NMR confirmed the presence of di-galactosyldiacylglycerol (DGDG). The biological activity of fractions containing DGDG (DGDG-3 and DGDG-4) and synthetic DGDG variants was tested in vitro in murine and human cell systems and in vivo in mice. Fraction 4 induced strong proliferation of murine NKT cells and upregulated CD69 expression in human NKT cells. Synthetic DGDG variants (DGDG-1, DGDG-2, and DGDG-3) with defined acylation profiles stimulated robust NKT-cell proliferation, with DGDG-2 and DGDG-3 increasing IL-13 production, one of the key Th2 cytokines. In vivo, only these variants caused lung inflammation marked by eosinophil infiltration but did not increase airway resistance. This study reveals for the first time the structure-dependent role of DGDG of TG pollen grains in immune cell recognition in the context of allergic inflammation. Our data may pave the way for therapies targeting lipid components in combination with protein allergens.

The majority of plants are involved in symbioses with arbuscular mycorrhizal fungi (AMF), and these associations are known to have a strong influence on the performance of both plants and insect herbivores. Little is known about the impact of AMF on complex trophic chains, although such effects are conceivable. In a greenhouse study we examined the effects of two AMF species, Glomus intraradices and G. mosseae on trophic interactions between the grass Phleum pratense, the aphid Rhopalosiphum padi, and the parasitic wasp Aphidius rhopalosiphi. Inoculation with AMF in our study system generally enhanced plant biomass (+5.2%) and decreased aphid population growth (-47%), but there were no fungal species-specific effects. When plants were infested with G. intraradices, the rate of parasitism in aphids increased by 140% relative to the G. mosseae and control treatment. When plants were associated with AMF, the developmental time of the parasitoids decreased by 4.3% and weight at eclosion increased by 23.8%. There were no clear effects of AMF on the concentration of nitrogen and phosphorus in plant foliage. Our study demonstrates that the effects of AMF go beyond a simple amelioration of the plants' nutritional status and involve rather more complex species-specific cascading effects of AMF in the food chain that have a strong impact not only on the performance of plants but also on higher trophic levels, such as herbivores and parasitoids.

An in vitro method based on 15N-labelled forage nitrogen (N) was developed to study ruminal N metabolism of soluble N (SN), insoluble N (ISN) and neutral detergent insoluble N (NDIN) fractions of timothy forage. Timothy grass was grown on replicated experimental plots with one plot receiving 15N-labelled and the other unlabelled N fertilizer. Harvested grass was preserved as dried grass or as formic acid treated or untreated silage. The intact forages and their corresponding N fractions were incubated in buffered rumen fluid in vitro to determine degradation parameters based on the 15N fluxes between labelled feed N and ammonia N pools. A high percentage (25-38%) of 15N-labelled ammonia disappeared from ammonia N pool during the first 15 min of incubation. Microbial uptake of dried grass SN fraction was higher than of silage SN fractions. Fractional degradation rates of SN from formic acid treated silage, untreated silage and dried grass during the first 6 hours of incubation were 0.145, 0.125 and 0.115 /h, respectively. By the end of the incubation period (28 h), 69, 66 and 43%, of the SN fraction of formic acid treated silage, untreated silage and dried grass, respectively were recovered as ammonia. The percentage of ISN fractions degraded to ammonia N were 9, 34 and 27%, respectively. Based on the changes in 15N-labelled ammonia N pool in blank incubation and appearance of 15N to ammonia N pool from 15N-labelled NDIN fractions, it was estimated that a significant portion of microbial lysis occurred when incubations were carried out for longer than 20 hours. With dried grass the contribution of ammonia N for microbial N synthesis was greater than with silages. Use of 15N-labelled forages together with this in vitro method is a promising technique for determining soluble N degradation parameters, but it requires further development to be used for determining degradation parameters of insoluble N fractions and work with whole feeds.

Background: Skin prick test results are mostly reported as mean wheal diameter obtained with one concentration of allergen. Differences in technique between personnel causes variation in wheal size. The research question was whether the influence of differences in skin prick test technique among assistants and centers can be reduced by relating the allergen wheal response to that of histamine. Methods: Two methods for estimating skin reactivity, the method of Nordic Guidelines using histamine as a reference and the method of Brighton et al. [Clin Allergy 1979;9:591-596] not using histamine as a reference, were applied to data from two biological standardization trials, using the same batch of freeze-dried timothy pollen preparation. Results: The concentration defining the Nordic biological unit, defined as a concentration of allergen eliciting a wheal of the same size as that of histamine dihydrochloride 10 mg/ml, did not differ between the centers. When not using histamine as a reference, applying the method of Brighton et al., there was a 15-fold difference in the estimate of the biological activity between the trials that was eliminated by adjusting the allergen response to that of the histamine reference. Conclusions: To reduce the influence of differences in test technique among assistants and centers responses to allergen-induced skin prick tests should be compared to that of histamine.

Phleum echinatum Host (2 n  = 2 x  = 10) is an annual Mediterranean species which differs from other representatives of the genus Phleum by reduced chromosome number, asymmetric karyotype and unusually high amount of DNA in the genome. Chromosomes of this plant were studied using conventional acetic-orcein staining and fluorescence in situ hybridization (FISH). FISH showed the major 35S ribosomal DNA (rDNA) site at the secondary constriction of satellite chromosome (3) and the minor 35S rDNA site near 5S rDNA cluster in the monobrachial chromosome 5. Telomeric repeats were detected at all chromosome ends within secondary constriction in satellited chromosome 3 and at the centromeric regions of chromosomes 1 and 2. Intrachromosomally located telomeric repeats are probably traces of chromosomal rearrangements that have shaped P.echinatum genome; they were prone to breakage which was manifested in chromosome fragmentation. The most distinct telomeric signals, suggesting massive amplification of interstitial telomeric sequences (ITRs), were observed at the nucleolar organizer region (NOR) of the third chromosome pair. Double FISH confirmed co-localization of telomeric and 35S rDNA repeats in this locus characterized by the biggest fragility in the karyotype. Fragile sites of P.echinatum , composed of amplified telomeric repeats, may bear a resemblance to metazoan rare fragile sites enriched in microsatellite repeats.

Carbohydrates in forage crops can be divided into neutral detergent-insoluble fiber and neutral detergent-soluble carbohydrates (NDSC); the latter includes organic acids (OA), total ethanol:water-soluble carbohydrates (TESC), starch, and neutral detergent-soluble fiber (NDSF). The accurate and efficient estimation of NDSC in forage crops is essential for improving the performance of dairy cattle. In the present study, visible and near-infrared reflectance spectroscopy (NIRS) were applied to evaluate the feasibility of predicting OA, TESC, starch, NDSF, NDSC, and all related constituents used to calculate these 5 carbohydrate fractions in timothy and alfalfa. Forage samples (n=1,008) of timothy and alfalfa were taken at the first and second harvests at 2 sites in 2007; samples were dried, ground, and then scanned (400 to 2,500nm) using an NIRSystems 6500 monochromator. A calibration (n=60) and a validation (n=15) set of samples were selected for each species and then chemically analyzed. Concentrations of TESC and NDSC in timothy, as well as starch in alfalfa, were successfully predicted, but many other carbohydrate fractions were not predicted accurately when calibrations were performed using single-species sample sets. Both sets of samples were combined to form new calibration (n=120) and validation (n=30) sets of alfalfa and timothy samples. Calibration and validation statistics for the combined sets of alfalfa and timothy samples indicated that TESC, starch, and NDSC were predicted successfully, with coefficients of determination of prediction (RP2) of 0.92, 0.89, and 0.93, and a ratio of prediction to deviation (RPD) of 3.3, 3.1, and 3.6, respectively. The NDSF prediction was classified as moderately successful RP2=0.88,   RPD=2.8. The NIRS prediction of OA was unsuccessful RP2=0.38,   RPD=1.3. All related constituents were predicted successfully RP2>0.90,   RPD>3.0 by NIRS except ethanol-insoluble residual OM, with RP2=0.75   and   RPD=1.9. Our results confirm the feasibility of using NIRS to predict NDSC, its fractions, and other related constituents, except for OA and ethanol-insoluble residual OM, in timothy and alfalfa forage samples.

The current study was undertaken to evaluate the effects of feeding timothy (Phleum pratense L.) hay differing in dietary cation-anion difference (DCAD) on the capability of cows to maintain calcium homeostasis around parturition. We hypothesized that feeding low-DCAD timothy hay during the prepartum period would induce a mild metabolic acidosis prepartum and improve calcium homeostasis postpartum with no effect on dry matter intake. Forty-one dry pregnant Holstein cows entering their second lactation or greater were used in a randomized complete block design. Timothy hay was obtained from an established timothy stand under a pivot irrigation system. Low-DCAD timothy hay was produced by fertilizing the area between the second and third pivot towers at a rate of 224kg of CaCl2/ha, and control timothy hay (high DCAD) was grown on the area between the fourth and fifth pivot towers of the same field. The chloride concentration was 1.07 and 0.15% on a dry matter (DM) basis, and the DCAD was 1.2 and 21.6 mEq/100g of DM for the low- and high-DCAD timothy hay, respectively. Experimental diets, containing timothy hay at 63% of dietary DM, were fed ad libitum starting 30 d before the expected calving date. The DCAD values were 1.6 vs. 14.5 mEq/100g of DM for the low- and high-DCAD timothy-based diets, respectively. At the beginning of the study, urine pH and blood bicarbonate concentration averaged 8.22±0.06 and 28.5±0.3mM, respectively. The low-DCAD timothy diet decreased urine pH compared with the high-DCAD timothy diet on d 21 (7.75 vs. 8.31), d 14 (7.69 vs. 8.22), and d 7 (7.50 vs. 8.19) before calving, and it also decreased the prepartum blood bicarbonate concentration by 2mM. In addition, cows fed the low-DCAD timothy diet had greater blood ionized calcium concentration prepartum (1.22 vs. 1.19mM), greater blood ionized calcium concentration at 0 and 8h after calving, and similar prepartum dry matter intake. These results indicate that timothy hay differing in DCAD affects the acid-base balance of periparturient dairy cows, and that low-DCAD timothy hay improves calcium homeostasis postpartum with no negative effect on dry matter intake.

The timothy grass pollen allergen Phl p 7 assembles most of the IgE epitopes of a novel family of 2 EF‐hand calcium‐binding proteins and therefore represents a diagnostic marker allergen and vaccine candidate for immunotherapy. Here we report the first three‐dimensional structure of a representative of the 2 EF‐hand allergen family, Phl p 7, in the calcium‐bound form. The protein occurs as a novel dimer assembly with unique features: in contrast to well known EF‐hand proteins such as calmodulin, parvalbumin or the S100 proteins, Phl p 7 adopts an extended conformation. Two protein monomers assemble in a head‐to‐tail arrangement with domain‐swapped EF‐hand pairing. The intertwined dimer adopts a barrel‐like structure with an extended hydrophobic cavity providing a ligand‐binding site. Calcium binding acts as a conformational switch between an open and a closed dimeric form of Phl p 7. These findings are interesting in the context of lipid‐ and calcium‐dependent pollen tube growth. Furthermore, the structure of Phl p 7 allows for the rational development of vaccine strategies for treatment of sensitized allergic patients.

Timothy grass has a lower dietary cation-anion difference [DCAD = (Na + K)-(Cl + S)] than other cool-season grass species. Growing timothy on low-K soils and fertilizing it with CaCl2 could further decrease its DCAD. The objective of this study was to evaluate the effects of feeding low-DCAD timothy hay on dry dairy cows. Six nonpregnant and nonlactating cows were used in a replicated 3×3 Latin square. Treatments were as follows: 1) control diet (control; DCAD = 296 mEq/kg of dry matter); 2) low-DCAD diet based on low-DCAD timothy hay (L-HAY; DCAD =-24 mEq/kg of dry matter); and 3) low-DCAD diet using HCl (L-HCl; DCAD =-19 mEq/kg of dry matter). Decreasing DCAD with L-HAY had no effect on dry matter intake (11.8 kg/d) or dry matter digestibility (71.5%). Urine pH decreased from 8.21 to 5.89 when L-HAY was fed instead of the control. Blood parameters that decreased with L-HAY were base excess (− 0.4 vs. 3.8mM) and HCO3− (23 vs. 27mM), and blood parameters that increased were Ca2+ (5.3 vs. 5.1 mg/dL), Cl− (30.5 vs. 29.5 mg/dL), and Na+ (60.8 vs. 60.1 mg/dL). Compared with the control, L-HAY resulted in more Ca in urine (13.4 vs. 1.2 g/d). Comparing L-HAY with L-HCl, cow dry matter intake tended to be higher (11.5 vs. 9.8 kg/d), and blood pH was higher (7.37 vs. 7.31). Urine pH; total dry matter; Ca, K, P, and Mg apparent absorption; and Ca, K, Na, Cl, S, P, and Mg apparent retention were similar. Absorption as a percentage of intake of Na and Cl was lower for L-HAY as compared with L-HCl. In an EDTA-challenge test, cows fed L-HAY regained their initial level of blood Ca2+ twice as quickly as the control treatment (339 vs. 708min); there were no differences between L-HAY and L-HCl. This experiment confirms that feeding low-DCAD hay is an effective means of decreasing the DCAD of rations and obtaining a metabolic response in dry dairy cows.

The objective of this study was to compare the DVE/OEB system (DVE = truly absorbed protein in the small intestine; OEB = degraded protein balance) and the NRC-2001 model in the prediction of supply of protein to dairy cows from selected forages: alfalfa (Medicago sativa L. cv. Pioneer and Beaver) and timothy (Phleum pratense L. cv. Climax and Joliette). Comparisons were made in terms of 1) ruminally synthesized microbial CP, 2) truly absorbed protein in the small intestine, and 3) degraded protein balance. In addition, the effects of variety and cutting stage of the selected forages on the potential nutrient supply to dairy cows were also studied. The results showed that the predicted values from the DVE/OEB system and the NRC-2001 model had significant correlations with high R (>0.96) values. However, using the DVE/OEB system, the overall average microbial protein supply based on available energy was 12% higher, and the truly absorbed protein in the small intestine was 15% lower than that predicted by the NRC-2001 model. The difference was also found in the prediction of the degraded protein balances, which was 11% higher based on data from the NRC-2001 model. These differences are due to considerably different factors used in calculations in the two models, although both are based on similar principles. This indicates that a further refinement is needed for a modern protein evaluation and prediction system. In addition, this study showed that the two alfalfa varieties studied (Pioneer vs. Beaver) had no effect, but cutting stage had a profound influence on ruminally synthesized microbial CP (93, 96, 86 g/kg DM at stage of early bud, late bud, and early bloom, respectively) and truly absorbed intestinal protein predicted by the DVE/OEB system (80, 79, 67 g/kg DM at stage of early bud, late bud, and early bloom, respectively). With timothy, both variety (Climax vs. Joliette) and cutting stage had significant impacts on the potential protein supply predicted by both models. The potential protein supply (DVE or MP) to the dairy cow from Climax timothy was higher than that from Joliette timothy (DVE: 46 vs. 32 g/kg DM; MP: 61 vs. 38 g/kg DM). With increasing stage of cutting, the potential protein supply (DVE or MP) was reduced (DVE: 53, 39, 25 g/kg DM; MP: 62, 51, 36 g/kg DM at stage of joint, prebloom head, and full head, respectively).