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Abstract The prevalence of hypersensitivities towards wheat has increased in the last decades. Apart from celiac disease these include allergic and other inflammatory reactions summarized under the term non-celiac wheat sensitivity. One suspected trigger is the family of amylase/trypsin-inhibitors (ATIs), non-gluten proteins that are prominent wheat allergens and that activate the toll-like receptor 4 on intestinal immune cells to promote intestinal and extra-intestinal inflammation. We therefore quantified 13 ATIs in 60 German hexaploid winter wheat cultivars originating from 1891 to 2010 and harvested in three years by targeted liquid chromatography-tandem mass spectrometry combined with stable isotope dilution assay using specific marker peptides as internal standards. The total ATI content and that of the two major ATIs 0.19 and CM3 did not change from old cultivars (first registered from 1891 to 1950) to modern cultivars (1951–2010). There were also no significant changes in ATI distribution.

Background: Western lifestyle has been associated with an increase in relapsing–remitting multiple sclerosis (RRMS). In mice, dietary wheat amylase–trypsin inhibitors (ATIs) activate intestinal myeloid cells and augment T cell-mediated systemic inflammation. Objective: The aim of this study was to assess whether a wheat- and thus ATI-reduced diet might exert beneficial effects in RRMS patients with modest disease activity. Methods: In this 6-month, crossover, open-label, bicentric proof-of-concept trial, 16 RRMS patients with stable disease course were randomized to either 3 months of a standard wheat-containing diet with consecutive switch to a > 90% wheat-reduced diet, or vice versa. Results: The primary endpoint was negative, as the frequency of circulating pro-inflammatory T cells did not decrease during the ATI-reduced diet. We did, however, observe decreased frequencies of CD14 + CD16 ++ monocytes and a concomitant increase in CD14 ++ CD16 − monocytes during the wheat-reduced diet interval. This was accompanied by an improvement in pain-related quality of life in health-related quality of life assessed (SF-36). Conclusion: Our results suggest that the wheat- and thus ATI-reduced diet was associated with changes in monocyte subsets and improved pain-related quality of life in RRMS patients. Thus, a wheat (ATI)-reduced diet might be a complementary approach accompanying immunotherapy for some patients. Registration: German Clinical Trial Register (No. DRKS00027967).

Wheat (Triticum aestivum ssp. aestivum) contributes to 20% of the human protein supply, delivers essential amino acids and is of fundamental importance for bread and pasta quality. Wheat proteins are also involved in adverse human reactions like celiac disease (CD), wheat allergy (WA) and non-celiac wheat sensitivity (NCWS). Using liquid chromatography-mass spectrometry (LC-MS)-based label-free quantitative (LFQ) proteomics of aqueous flour extracts, we determined 756 proteins across 150 wheat cultivars grown in three environments. However, only 303 proteins were stably expressed across all environments in at least one cultivar and only 89 proteins thereof across all 150 cultivars. This underlines the large influence of environmental conditions on the expression of many proteins. Wheat cultivars varied largely in their protein profile, shown by high coefficients of variation across different cultivars. Heritability (h2) ranged from 0–1, with 114 proteins having h² > 0.6, including important proteins for baking quality and human health. The expression of these 114 proteins should be amenable to targeted manipulation across the wheat supply chain by varietal choice and breeding for designing healthier wheat with better quality. Further technical development is urgently required to assign functions to identifiable proteins labeled yet uncharacterized in databases and speeding up detection methods to routinely use proteomics in wheat supply chains.

BackgroundMacrophages are a heterogeneous cell population which in response to the cytokine milieu polarize in either classically activated macrophages (M1) or alternatively activated macrophages (M2). This plasticity makes macrophages essential in regulating inflammation, immune response and tissue remodeling and a novel therapeutic target in inflammatory diseases such as atherosclerosis. The aim of the study was to describe the transcriptomic profiles of differently polarized human macrophages to generate new hypotheses on the biological function of the different macrophage subtypes.Methods and resultsPolarization of circulating monocytes/macrophages of blood donors was induced in vitro by IFN-γ and LPS (M1), by IL-4 (M2a), and by IL-10 (M2c). Unstimulated cells (RM) served as time controls. Gene expression profile of M1, M2a, M2c and RM was assessed at 6, 12 and 24h after polarization with Whole Human Genome Agilent Microarray technique. When compared to RM, M1 significantly upregulated pathways involved in immunity and inflammation, whereas M2a did the opposite. Conversely, decreased and increased expression of mitochondrial metabolism, consistent with insulin resistant and insulin sensitive patterns, was seen in M1 and M2a, respectively. The time sequence in the expression of some pathways appeared to have some specific bearing on M1 function. Finally, canonical and non-canonical Wnt genes and gene groups, promoting inflammation and tissue remodeling, were upregulated in M2a compared to RM.ConclusionOur data in in vitro polarized human macrophages: 1. confirm and extend known inflammatory and anti-inflammatory gene expression patterns; 2. demonstrate changes in mitochondrial metabolism associated to insulin resistance and insulin sensitivity in M1 and M2a, respectively; 3. highlight the potential relevance of gene expression timing in M1 function; 4. unveil enhanced expression of Wnt pathways in M2a suggesting a potential dual (pro-inflammatory and anti-inflammatory) role of M2a in inflammatory diseases.

We investigated whether changes in the dopamine transporter in the nucleus accumbens or striatum are involved in cocaine-induced behavioral sensitization by using in vivo electrochemistry to monitor the clearance of locally applied dopamine in anesthetized rats. Rats were injected with cocaine-HCl (10 mg/kg i.p.) or saline daily for 7 consecutive days and then withdrawn for 7 days. Pressure ejection of a finite amount of dopamine at 5-min intervals from a micropipette adjacent to the electrochemical recording electrode produced transient and reproducible dopamine signals. After a challenge injection of cocaine (10 mg/kg i.p.), the signals in the nucleus accumbens of cocaine-treated animals became prolonged and the clearance rate of the dopamine decreased, indicating significant inhibition of the dopamine transporter. In contrast, simultaneous measurements in the dorsal striatum indicated a transient increase in both the amplitude of the signals and the clearance rate of the dopamine. The signals in both brain regions in the saline-treated animals given the cocaine challenge were similar to those in untreated animals given an acute injection of cocaine (10 mg/kg i.p.) or saline. Behaviorally, not all of the cocaine-treated animals were sensitized; however, both sensitized and nonsensitized animals displayed similar changes in dopamine clearance rate. Quantitative autoradiography with [3H]mazindol revealed that the affinity of the dopamine transporter for cocaine and the density of binding sites were similar in cocaine- and saline-treated rats. The decrease in dopamine clearance rate observed in the nucleus accumbens of the cocaine-treated rats after a challenge injection of cocaine is consistent with increased dopaminergic transmission, but does not appear to be sufficient in itself for producing behavioral sensitization.

Acute cocaine administration preferentially increases extracellular dopamine levels in nucleus accumbens as compared with striatum. To investigate whether a differential effect of cocaine on dopamine uptake could explain this observation, we used in vivo electrochemical recordings in anesthetized rats in conjunction with a paradigm that measures dopamine clearance and diffusion without the confounding effects of release. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible increases in dopamine levels were detected. In response to 15 mg/kg of cocaine-HCl (i.p.), these signals increased in nucleus accumbens, indicating significant inhibition of the dopamine transporter. The time course of the dopamine signal increase paralleled that of behavioral changes in unanesthetized rats receiving the same dose of cocaine. In contrast, no change in the dopamine signal was detected in dorsal striatum; however, when the dose of cocaine was increased to 20 mg/kg, enhancement of the dopamine signal occurred in both brain areas. Quantitative autoradiography with [3H]mazindol revealed that the affinity of the dopamine transporter for cocaine was similar in both brain areas but that the density of [3H]mazindol binding sites in nucleus accumbens was 60% lower than in dorsal striatum. Tissue dopamine levels in nucleus accumbens were 44% lower. Our results suggest that a difference in dopamine uptake may explain the greater sensitivity of nucleus accumbens to cocaine as compared with dorsal striatum. Furthermore, this difference may be due to fewer dopamine transporter molecules in nucleus accumbens for cocaine to inhibit, rather than to a higher affinity of the transporter for cocaine.

The greater sensitivity of long-sleep (LS), as compared with short-sleep (SS), mice to ethanol is due in part to differences in GABAA receptor function in specific brain regions. To determine if differences in subunit composition of GABAA receptors contribute to this differential sensitivity, we measured alpha 1 and gamma 2 subunit mRNAs with Northern analysis and in situ hybridization and gamma 2S, gamma 2L and alpha 6 subunit mRNAs with polymerase chain reaction (PCR) amplification. No differences in mRNAs in whole brain were apparent by Northern analysis. In situ hybridization revealed that alpha 1 and gamma 2 subunit mRNAs were co-localized in many brain regions but that they still had distinct patterns of hybridization. However, the few differences observed between LS and SS mice in the levels of hybridization for these subunits did not show a regional distribution consistent with ethanol sensitivity differences. Similar ratios of gamma 2L, and gamma 2S subunit mRNAs were found in LS and SS mouse cerebral cortex and hippocampus, and both mouse lines expressed essentially only gamma 2L subunit mRNA in cerebellum. mRNA for the alpha 6 subunit was detected only in cerebellum and also was qualitatively similar between LS and SS mice. Studies of muscimol-stimulated 36Cl- uptake by cortical membrane vesicles confirmed earlier findings that ethanol does not enhance function of GABAA receptors in SS mice when assayed at 30 degrees C. However, at 34 degrees C ethanol did increase this function in SS mice although the enhancement remained greater in LS mice. These functional results, together with the results showing similar levels of alpha 1, gamma 2S, gamma 2L and alpha 6 subunits in LS and SS mice, suggest that the ethanol-insensitivity of SS mouse GABAA receptors cannot be due solely to lack of subunits required for ethanol action and further suggest that differences in catalytic mechanisms affecting post-translational processing may account for some genetic differences in ethanol sensitivity of GABAA receptors.

Potential age-related differences in cardiovascular responsiveness and receptor regulation induced by short-term administration of a selective beta 2-adrenergic receptor agonist were investigated. Young (age range, 23 to 31 years) and elderly (age range, 64 to 73 years) healthy subjects were treated with terbutaline (5 mg, three times daily) for 5 days. Similar plasma terbutaline concentrations were achieved in the two age groups. The elderly group had higher baseline plasma norepinephrine concentrations and mean arterial pressures, neither of which were altered by terbutaline administration. During terbutaline treatment, heart rate increased in both age groups while subjects were supine but consistently increased only in the young group while subjects were standing. In both age groups, the density of beta 2-adrenergic receptors on polymorphonuclear leukocyte membranes was reduced by 50% during terbutaline administration and returned to baseline values at similar rates after drug administration was stopped. Isoproterenol-stimulated cyclic adenosine monophosphate accumulation in polymorphonuclear leukocytes from elderly subjects was regulated similarly. These findings suggest that the ability of terbutaline to increase standing heart rate is selectively impaired in the elderly, whereas the ability of polymorphonuclear leukocyte beta 2-adrenergic receptors to down-regulate and to return to baseline values is not.

Hippocampal tissue transplanted into the anterior chamber of the eye offers a unique system in which development can be studied in the absence of the noradrenergic innervation. This system was used to determine the extent to which noradrenergic innervation regulates the development of adrenergic receptors. In addition to examining single denervated transplants, transplants grown with innervation from the superior cervical ganglia of the host rat or from locus coeruleus cotransplants were also examined to determine whether the source of norepinephrine and extent of innervation in oculo regulate the development and density of adrenergic receptors. In vitro autoradiographic analysis of ligand binding to both alpha 1- and beta-adrenergic receptors with 125I-BE 2254 and 125I-pindolol, respectively, was used to characterize adrenergic receptors in the intraocular transplants. Quantitative analysis of the receptors showed an up-regulation of both alpha 1- and beta-adrenergic receptors in tissue grown in the absence of norepinephrine, but in general there was not a high degree of correlation between norepinephrine content and receptor density. Although high-performance liquid chromatography (HPLC) analysis of catecholamines revealed higher than normal amounts of norepinephrine in hippocampal transplants innervated by the superior cervical ganglia or a locus coeruleus cotransplant, the density of alpha 1 and beta receptors was quite comparable with values found in the literature for normal adult hippocampus. These results suggest that the relationship between receptor number and density of innervation may differ significantly from what is observed in response to pharmacological manipulation of norepinephrine systems in the adult brain.

Behavioral sensitization involving the nigrostriatal dopaminergic tract is manifested after treatment with only a single dose of cocaine and is augmented following repeated treatment. One neurochemical change observed that is consistent with behavioral sensitization is the increase in amphetamine-induced 3H-DA release from striatal slices seen after one injection of cocaine. One day after repeated administration of cocaine, however, the increase is no longer evident. It is possible that transient compensatory changes, such as increased D-2 autoreceptor inhibition, may obscure this effect when it is measured at relatively short times after the repeated administration has been terminated. One day after cessation of repeated cocaine administration, D-2 autoreceptors in both striatum and substantia nigra compacta were upregulated consistent with a compensatory mechanism and the development of behavioral tolerance rather than sensitization. In contrast, DA content, neuronal DA uptake, and postsynaptic D-2 DA receptors in striatum were not regulated by this treatment. Likewise, D-1 DA receptors in striatum and substantia nigra were unaffected. In the mesolimbic system, both the pre- and postsynaptic receptor changes are consistent with sensitization. Amphetamine-stimulated release from nucleus accumbens has not yet been measured in cocaine-sensitized animals. It is possible that changes similar to those seen in striatum may occur in this area. It is interesting that, in general, presynaptic parameters associated with the DA neuron, with the notable exception of the uptake pump, appear to be more sensitive to regulation by cocaine administration than do postsynaptic parameters. The long-lasting effects of a single moderate dose of cocaine are also surprising. It will be important to determine the molecular mechanisms underlying this regulation and whether or not similar changes are induced in mesolimbic dopaminergic systems by single and repeated administration of cocaine.

A new quantitative staining technique for the assay of protein in tissue sections using the dye Coomassie brilliant blue G 250 is discussed. This densitometric procedure uses commercially available hardware and software employed in the quantitation of receptor autoradiographs. Rather than assuming a homogeneous distribution of protein in the tissue section, regional levels of protein are measured in the same tissue slice used to produce the autoradiograph. Additionally, the process of staining tissue with Coomassie blue for protein is reversible; the tissue can be destained after the measurement of protein is complete and then restained with a standard histological stain such as Cresyl violet. This technique is a more reliable method for normalizing receptor densities by tissue protein levels and allows for a more accurate comparison between QAR and membrane binding techniques.