Evaluation of an in vitro mast cell degranulation test in the context of food allergy to wheat.

BACKGROUND: Antigenic profiles obtained by ELISA with IgE from patients with wheat food allergy (WFA) established that major allergens are albumins/globulins (AG) for children suffering from atopic eczema/dermatitis syndrome (AEDS), omega5-gliadins for adults suffering from wheat-dependent exercise-induced anaphylaxis (WDEIA), anaphylaxis or urticaria and low-molecular-weight (LMW) glutenin subunits for patients with anaphylaxis. We aimed to characterize a new mast cell transfectant for its ability to degranulate with wheat proteins and patient sera and compare these results to those obtained by ELISA. METHODS: Thirty sera from patients with WFA were tested: 14 with AEDS (group 1) and 16 with WDEIA, anaphylaxis or urticaria (group 2). An IgE Fc receptor (FcepsilonRI) humanized rat RBL-2H3 line was established by transfection with cDNAs encoding alpha-, beta- and gamma-subunits for the human IgE receptor. RESULTS: A humanized RBL clone was selected for its capacity to express mRNA alpha-, beta- and gamma-subunits of FcepsilonRI, to bind allergen-specific human IgE and to degranulate. In group 1, sera induced enhanced degranulation with AG extract, but rarely reacted with gliadins and glutenins. In group 2, half of the sera showed degranulation with LMW glutenins whereas the AG fraction and lipid transfer proteins were rarely positive. omega5-Gliadins did not appear as a major allergen in degranulation assays, although functional allergen-specific IgE was measurable in appreciable amounts. CONCLUSION: Our data demonstrate that in wheat food allergen evaluation, correlation exists between mast cell degranulation and IgE measurements, depending on the type of allergen. Therefore, the biological activity of some allergen types may also be affected by other parameters.

MUC genes are differently expressed during onset and maintenance of inflammation in dextran sodium sulfate-treated mice.

Colonic mucosal protection is provided by mucous gel, mainly composed of secreted (Muc2) and membrane-bound (Muc1, Muc3, Muc4) mucins. Our aim was to determine the expression profile of secreted and membrane-bound mucins in experimental dextran sulfate sodium (DSS)-induced colitis. Acute colitis was induced in Balb/C mice by oral administration of 1.0% DSS (5 days) and chronic colitis was maintained by subsequent 0.15% DSS treatment (28 days). Clinical symptoms (mortality, weight gain), stool scores, and MPO activity confirmed the inflammatory state in the two phases of colitis. Muc2 gene expression was not modified by colitis, whereas Muc3 gene expression was increased (x2) only in the cecum and the distal colon of mice after acute colitis. Muc1 and Muc4 mRNA levels were more significantly increased in the cecum (x8-10) than in colonic segments (x4) after acute colitis. TFF3 involved in mucosal repair was up-regulated during colitis induction. These results indicate that Muc and TFF3 genes are regulated early in inflammation and suggest that their mRNA levels could be used as early markers of inflammation.

Butyrate specifically modulates MUC gene expression in intestinal epithelial goblet cells deprived of glucose.

The mucus layer covering the gastrointestinal mucosa is considered the first line of defense against aggressions arising from the luminal content. It is mainly composed of high molecular weight glycoproteins called mucins. Butyrate, a short-chain fatty acid produced during carbohydrate fermentation, has been shown to increase mucin secretion. The aim of this study was to test 1) whether butyrate regulates the expression of various MUC genes, which are coding for protein backbones of mucins, and 2) whether this effect depends on butyrate status as the major energy source of colonocytes. Butyrate was provided at the apical side of human polarized colonic goblet cell line HT29-Cl.16E in glucose-rich or glucose-deprived medium. In glucose-rich medium, butyrate significantly increased MUC3 and MUC5B expression (1.6-fold basal level for both genes), tended to decrease MUC5AC expression, and had no effect on MUC2 expression. In glucose-deprived medium, i.e., when butyrate was the only energy source available, MUC3 and MUC5B increase persisted, whereas MUC5AC expression was significantly enhanced (3.7-fold basal level) and MUC2 expression was strikingly increased (23-fold basal level). Together, our findings show that butyrate is able to upregulate colonic mucins at the transcriptional level and even better when it is the major energy source of the cells. Thus the metabolism of butyrate in colonocytes is closely linked to some of its gene-regulating effects. The distinct effects of butyrate according to the different MUC genes could influence the composition and properties of the mucus gel and thus its protective function.

Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins.

Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

Polymorphism and signalling of melatonin receptors.

Melatonin receptors belong to the superfamily of G protein-coupled receptors. Cloning of Mel1c receptors expressed in Xenopus skin revealed the existence of a polymorphism for these receptors. Heterologous expression of the two allelic isoforms, called Mel1c(alpha) and Mel1c(beta), indicated functional differences in their signalling properties. Both isoforms are coupled to the cAMP and cGMP pathways. However, the alpha isoform is preferentially coupled to the cAMP pathway, whereas the beta isoform couples preferentially to the cGMP pathway. Coupling differences may be explained by the fact that five of the six amino acid substitutions between the two isoforms are localized within intracellular receptor regions potentially involved in G protein coupling. Allelic isoforms were also observed for Mel1a receptors expressed in ovine pars tuberalis, suggesting that polymorphism is a general feature of the melatonin receptor family. We also evaluated the potential of the two human melatonin receptor subtypes, Mel1a and Mel1b, to modulate the cGMP pathway. Melatonin inhibited intracellular cGMP levels in a dose-dependent manner in HEK293 cells transfected with the human Mel1b receptor. This was not the case for HEK293 cells transfected with the human Mel1a receptor. In conclusion, our results indicate that the expression of receptor subtypes and isoforms may permit differential signalling between melatonin receptors.

Differential signaling of human Mel1a and Mel1b melatonin receptors through the cyclic guanosine 3'-5'-monophosphate pathway.

Cyclic guanosine 3'-5'-monophosphate (cGMP) has recently been shown to constitute a second messenger for Xenopus laevis melatonin Mel1c receptors. To verify whether cGMP levels are also modulated by mammalian melatonin receptors, we cloned the genes encoding the human Mel1a and Mel1b receptor subtypes and expressed them in human embryonic kidney cells. Pharmacological profiles and inhibition of forskolin-stimulated adenosine 3'-5'-cyclic monophosphate levels by melatonin confirmed functional expression of high-affinity melatonin receptors. Mel1b receptor-transfected cells modulated cGMP levels in a dose-dependent manner via the soluble guanylyl cyclase pathway. In contrast, Mel1a receptors had no effect on cGMP levels. These results demonstrate that mammalian melatonin receptors modulate cGMP levels and reveal for the first time differences in signaling between melatonin receptor subtypes, which may explain the necessity to express different receptor subtypes.

The human copper-zinc superoxide dismutase gene (SOD1) proximal promoter is regulated by Sp1, Egr-1, and WT1 via non-canonical binding sites.

Human copper-zinc superoxide dismutase (Cu,Zn-SOD) participates in the control of reactive oxygen intermediate intracellular concentration. In this study, we show that phorbol 12-myristate 13-acetate (PMA) increases Cu,Zn-SOD mRNA expression within 30 min. The sequence between nucleotides -71 and -29 is essential for both basal and PMA-induced gene expression. This region includes an Sp1-binding site that is also recognized by a possible Sp1-like protein and by Egr-1 in a PMA-inducible manner. Egr-1 and two splicing variants of the Egr-related protein WT1 were able to transactivate the SOD1 promoter in co-transfection experiments. Sp1 and the possible Sp1-like proteins bind to two overlapping, but distinct sequences. However, Egr-1 and Sp1 seem to interact with two sites that are either identical or very close to each other. None of these sites fit the consensus sequences previously reported for these proteins. Analysis of various mutants of the SOD1 proximal promoter revealed that the region that binds Sp1 and Egr-1 is required for both basal and Egr-1-driven expression. Interplay between different members of the Sp1 family, Egr-1, and different splicing variants of WT1 in the SOD1 proximal promoter may provide clues about the physiological function of Cu,Zn-SOD.

[Structure and function of melatonin receptors]. / Structure et fonction des récepteurs de la mélatonine.

Melatonin receptors belong to the super-family of G protein-coupled receptors. They modulate a large spectrum of physiological functions including regulation of circadian rhythms and seasonal reproduction. Pharmacological evidence suggests the expression of two types of receptors, called Mel1 and Mel2. So far, only Mel1 receptors have been cloned and classified into three subtypes (Mel1A, Mel1B, Mel1C). Mel1 receptors are expressed in the brain, the retina and several other peripheral tissues. All Mel1 subtypes show comparable pharmacological profiles including inhibition of adenylyl cyclase. Cloning and expression of two allelic isoforms of the Mel1 receptor from Xenopus laevis has revealed another signalling pathway, inhibition of cGMP levels via the soluble guanylyl cyclase pathway. The two isoforms are differentially coupled to the cAMP and cGMP pathways indicating the existence of functional differences between melatonin receptors. Future research topics will include cloning of the Mel2 receptor, receptor regulation and the elucidation of melatonin receptor's function in peripheral tissues.

Desensitization of the beta-adrenergic response in human brown adipocytes.

Activation of adenylyl cyclase by beta-adrenergic receptors (betaARs) plays a major role in adipose tissue homeostasis. The increase in cAMP promotes lipolysis in white adipose tissue, activates both thermogenesis and lipolysis in brown adipose tissue (BAT), and induces BAT hypertrophy. Previous studies indicated that among the three betaAR subtypes present in adipose tissue, beta3AR could be a potential target for antiobesity treatments in humans. We studied immortalized human brown adipocytes (PAZ6 adipocytes) as a model of beta-adrenergic response in human BAT. PAZ6 adipocytes and freshly isolated mature human brown adipocytes display the same proportions of betaAR subtypes, with beta3AR being the most abundant (approximately 80% of the total). However, beta3AR was poorly coupled to the adenylyl cyclase pathway in PAZ6 cells, contributing to only 10% of the isoproterenol-induced accumulation of cAMP, whereas 20% and 70% of the signal depended on beta1- and beta2-subtypes, respectively. Upon isoproterenol stimulation, beta1- and beta2AR down-regulated with a half-life of about 3 h and the beta3AR with a half-life of 30-40 h. Long term stimulation with both saturating (micromolar) and nonsaturating (nanomolar) concentrations of beta-adrenergic agonists caused a complete desensitization of the beta-adrenergic response at the adenylyl cyclase level and loss of stimulated protein kinase A activity and CREB phosphorylation. These results suggest that cAMP-dependent processes will be desensitized upon permanent treatment with beta3AR agonists. Further studies should establish whether the beta3AR is coupled to other signaling pathways in human brown adipocytes and whether these may contribute to BAT hypertrophy and/or thermogenesis.

Novel isoforms of Mel1c melatonin receptors modulating intracellular cyclic guanosine 3',5'-monophosphate levels.

Two cDNAs encoding novel isoforms of Xenopus laevis melatonin receptors were cloned using PCR primers specific for the X. laevis-melanophore Mel1c melatonin receptor described in a recent publication. The novel isoforms were highly homologous to the described frog Mel1c cDNA, although the C-terminal tail of both was shorter by 65 amino acid residues. Nucleotide sequences of these novel isoforms, called Mel1c(alpha) and Mel1c(beta), differed from each other by only 35 nucleotides and six amino acid residues. Studies on several animals of various Xenopus species indicate that Mel1c(alpha) and Mel1c(beta) receptors may correspond to allelic variants of the same locus. Studies on cells transfected with both receptor cDNAs showed the expression of high-affinity 2-[125I]iodomelatonin binding sites. Agonist stimulation of Mel1c(alpha) receptor was associated with the inhibition of cAMP accumulation stimulated by forskolin (IC50 approximately 10(-10) M) in HeLa, Ltk-, and human embryonic kidney 293 (HEK 293) cells. Mel1c(beta) receptor modulated cAMP in HeLa and HEK 293 cells but not in Ltk- cells. Both receptors inhibited, in a dose-dependent manner, cGMP accumulation in all three cell lines incubated with a phosphodiesterase inhibitor. This effect was localized upstream of soluble guanylyl cyclase and was blocked by pertussis toxin treatment. However, IC50 values (approximately 10(-10) M for Mel1c(beta) and 10(-9) to 10(-7) M for Mel1c(alpha)) and maximal inhibition levels showed that Mel1c(alpha) receptors are much less efficiently coupled to the cGMP pathway. Coupling differences may be explained by the fact that five of the six amino acid substitutions between Mel1c(alpha) and Mel1c(beta) receptors are located within cytoplasmic regions potentially involved in signal transduction. The existence of coupling differences is in agreement with the observation that expression of both receptors is evolutionally conserved in native tissue. In conclusion, two novel, potentially allelic, isoforms of Xenopus Mel1c melatonin receptors display identical ligand-binding characteristics, but different potencies in modulating cAMP and cGMP levels through G(i)/G(o)-dependent pathways. Furthermore, to our knowledge, this study provides the first data on the modulation of intracellular cGMP levels by cloned melatonin receptors.

Human glutathione S-transferase M1 null genotype is associated with a high inducibility of cytochrome P450 1A1 gene transcription.

We investigated the transcriptional regulation of cytochrome P450 1A1 (CYP1A1) gene in human lymphoblastoid B cells and report that a high inducibility of CYP1A1 gene transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin is associated with glutathione S-transferase M1 (GSTM1) null genotype, whereas the presence of at least one GSTM1 allele is correlated with induction of only low levels of CYP1A1 mRNA by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These data underline the major importance of the CYP1A1 inducibility phenotype associated with the homozygous GSTM1 null genotype in chemically induced cancers.