Sesame allergy threshold dose distribution.

BACKGROUND: Sesame is a relevant food allergen in France. Compared to other allergens there is a lack of food challenge data and more data could help sesame allergy risk management. The aim of this study is to collect more sesame challenge data and investigate the most efficient food challenge method for future studies. METHOD: Records of patients at University Hospital in Nancy (France) with objective symptoms to sesame challenges were collected and combined with previously published data. An estimation of the sesame allergy population threshold was calculated based on individual NOAELs and LOAELs. Clinical dosing schemes at Nancy were investigated to see if the optimal protocol for sesame is currently used. RESULTS: Fourteen patients (10 M/4 F, 22 ± 14.85 years old) with objective symptoms were added to previously published data making a total of 35 sesame allergic patients. The most sensitive patient reacted to the first dose at challenge of 1.02 mg sesame protein. The ED05 ranges between 1.2 and 4.0 mg of sesame protein (Log-Normal, Log-Logistic, and Weibull models) and the ED10 between 4.2 and 6.2 mg. The optimal food challenge dosing scheme for sesame follows semi-log dose increases from 0.3 to 3000 mg protein. CONCLUSION: This article provides a valuable update to the existing clinical literature regarding sesame NOAELs and LOAELs. Establishment of a population threshold for sesame could help in increasing the credibility of precautionary labelling and decrease the costs associated with unexpected allergic reactions. Also, the use of an optimal dosing scheme would decrease time spent on diagnostic and thereafter on the economic burden of sesame allergy diagnosis.

A novel immunoassay using recombinant allergens simplifies peanut allergy diagnosis.

BACKGROUND: Double-blind placebo-controlled food challenge (DBPCFC) is currently considered the gold standard for peanut allergy diagnosis. However, this procedure that requires the hospitalization of patients, mostly children, in specialized centers for oral exposure to allergens may cause severe reactions requiring emergency measures. Thus, a simpler and safer diagnosis procedure is needed. The aim of this study was to evaluate the diagnostic performance of a new set of in vitro blood tests for peanut allergy. METHODS: The levels of IgE directed towards peanut extract and recombinant peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 7, and Ara h 8 were measured in 3 groups of patients enrolled at 2 independent centers: patients with proven peanut allergy (n=166); pollen-sensitized subjects without peanut allergy (n=61), and control subjects without allergic disease (n=10). RESULTS: Seventy-nine percent of the pollen-sensitized patients showed IgE binding to peanut, despite their tolerance to peanut. In contrast, combining the results of specific IgE to peanut extract and to recombinant Ara h 2 and Ara h 6 yielded a peanut allergy diagnosis with a 98% sensitivity and an 85% specificity at a positivity threshold of 0.10 kU/l. Use of a threshold of 0.23 kU/l for recombinant Ara h 2 increased specificity (96%) at the cost of sensitivity (93%). CONCLUSION: A simple blood test can be used to diagnose peanut allergy with a high level of precision. However, DBPCFC will remain useful for the few cases where immunological and clinical observations yield conflicting results.

Interest of ImmunoCAP system to recombinant omega-5 gliadin for the diagnosis of exercise-induced wheat allergy.

BACKGROUND: omega-5 gliadin is a major allergen in exercise-induced wheat allergy (EIWA), but it is also implicated in immediate-type reactions to wheat. An ImmunoCAP assay to measure omega-5 gliadin-specific IgE has become available. This study aimed to evaluate this new biological test in wheat allergy diagnosis and to also determine if it was able to discriminate EIWA from other types of wheat allergy. METHODS: Sixty-one patients with wheat allergy were divided into 3 groups as a function of their symptoms (EIWA, immediate-type reactions and atopic dermatitis). These patients underwent skin prick tests with purified omega gliadins and ImmunoCAP to wheat flour, gluten and recombinant omega-5 gliadin. RESULTS: The experimental data showed that 78% of EIWA patients had a positive skin prick test to natural omega-5 gliadin and the same proportion had detectable specific IgE to recombinant omega-5 gliadin, indicating that omega-5 gliadin is the main allergen, but not the only one, in our population. Additionally, we showed that this detection was not EIWA specific since omega-5 gliadin-specific IgE was detected in 30% of other patients who had a wheat allergy. These results lead to a positive predictive value of 37.5% and to a negative predictive value of 91%. CONCLUSIONS: Although not specific to EIWA, the new ImmunoCAP omega-5 gliadin is an important biological test because of its negative predictive value. In case of food-dependent exercise-induced allergy, the absence of omega-5 gliadin-specific IgE will almost completely exclude the implication of wheat.

A murine model of cow's milk protein-induced allergic reaction: use for safety assessment of hidden milk allergens.

BACKGROUND: Masked allergens in processed food products can lead to severe allergic reactions following unintentional ingestion. We sought to develop a murine model for the detection of hidden cow's milk proteins (CMP). This study aimed to induce cow's milk allergy in mice, to characterize the anaphylaxis induced by CMP in this model, and to validate its reliability using three margarines manufactured with (A) or without (B, C) milk, sharing the same production line. MATERIALS AND METHODS: Three-week-old BALB/c mice were sensitized intragastrically with CMP plus cholera toxin and boosted 6 times at weekly intervals. CMP-sensitization status was monitored by skin tests, and measurement of CMP-specific IgE and IgG1 levels. On day 44, the minimal threshold of clinical reactivity to CMP in terms of anaphylaxis was determined by performing a dose response of intraperitoneal CMP challenge. Under the same conditions, anaphylaxis was evaluated in CMP-sensitized mice after challenge with protein extracts of margarines A, B or C. RESULTS: Sensitization to CMP was demonstrated by positive skin tests and increased CMP-specific IgE and IgG1. The minimal clinical reactivity threshold corresponding to 0.1 mg CMP elicited detectable anaphylaxis evidenced by clinical symptoms, a decrease in breathing frequency, and increased plasma histamine upon challenge. Similarly, challenges with margarine A containing CMP demonstrated anaphylaxis, whereas those with B or C did not elicit any detectable allergic reaction. CONCLUSION: This study shows that our murine model of CMP-induced anaphylaxis is useful for investigating the allergenic activity and the assessment of margarines with respect to milk.

Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice.

Adipocyte complement-related protein (30 kDa) (Acrp30), a secreted protein of unknown function, is exclusively expressed in differentiated adipocytes; its mRNA is decreased in obese humans and mice. Here we describe novel pharmacological properties of the protease-generated globular head domain of Acrp30 (gAcrp30). Acute treatment of mice with gAcrp30 significantly decreased the elevated levels of plasma free fatty acids caused either by administration of a high fat test meal or by i.v. injection of Intralipid. This effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle. As a result, daily administration of a very low dose of gAcrp30 to mice consuming a high-fat/sucrose diet caused profound and sustainable weight reduction without affecting food intake. Thus, gAcrp30 is a novel pharmacological compound that controls energy homeostasis and exerts its effect primarily at the peripheral level.

Acquisition of lipoproteins in the procyclic form of Trypanosoma brucei.

The procyclic form of Trypanosoma brucei binds and internalizes bovine high density lipoprotein (HDL) particles in a saturable process; the binding and uptake of (125)I-labeled HDL are inhibited by excess unlabeled HDL. We calculated that each procyclic trypanosome exposes approximately 1.0 x 10(6) binding sites for bovine HDL, with an equilibrium dissociation constant (Kd) of approximately 1.26 x 10(-7) M. Uptake of HDL particles does not occur at 4 degrees C. At 28 degrees C, a significant amount of the internalized HDL particles were efficiently degraded through a process that is sensitive to the presence of 50 microM chloroquine. These results suggested that the uptake of HDL particles in procyclic T. brucei may occur via receptor mediated endocytosis, leading to proteolytic degradation of the particles in an acidic and endocytic compartment.

Molecular cloning of a lipolysis-stimulated remnant receptor expressed in the liver.

The lipolysis-stimulated receptor (LSR) is a lipoprotein receptor primarily expressed in the liver and activated by free fatty acids. Antibodies inhibiting LSR functions showed that the receptor is a heterotrimer or tetramer consisting of 68-kDa (alpha) and 56-kDa (beta) subunits associated through disulfide bridges. Screening of expression libraries with these antibodies led to identification of mRNAs derived by alternate splicing from a single gene and coding for proteins with molecular masses matching that of LSR alpha and beta. Antibodies directed against a synthetic peptide of LSR alpha and beta putative ligand binding domains inhibited LSR activity. Western blotting identified two liver proteins with the same apparent molecular mass as that of LSR alpha and beta. Transient transfections of LSR alpha alone in Chinese hamster ovary cells increased oleate-induced binding and uptake of lipoproteins, while cotransfection of both LSR alpha and beta increased oleate-induced proteolytic degradation of the particles. The ligand specificity of LSR expressed in cotransfected Chinese hamster ovary cells closely matched that previously described using fibroblasts from subjects lacking the low density lipoprotein receptor. LSR affinity is highest for the triglyceride-rich lipoproteins, chylomicrons, and very low density lipoprotein. We speculate that LSR is a rate-limiting step for the clearance of dietary triglycerides and plays a role in determining their partitioning between the liver and peripheral tissues.

The lipolysis stimulated receptor: a gene at last.

The lipolysis stimulated receptor is a lipoprotein receptor that was initially described in 1992. In the presence of free fatty acids, the lipolysis stimulated receptor recognizes either apolipoprotein B or apolipoprotein E, and as a consequence, leads to the internalization and degradation of the lipoprotein particles. Its affinity is highest for those lipoproteins most susceptible to lipolysis, triglyceride-rich lipoproteins. Since the initial biochemical identification and description of the lipolysis stimulated receptor, several reports have been published by our group that provide circumstantial evidence for its role in vivo for the clearance of triglyceride-rich lipid particles. In this review, we bring the readers up-to-date on the evidence for the role of the lipolysis stimulated receptor in lipoprotein metabolism, as well as the recent developments in its molecular characterization.

Selectivity of the major histocompatibility complex class II presentation pathway of cortical thymic epithelial cell lines.

Major histocompatibility complex (MHC) restriction of the immune response is established during positive selection of T cells in the thymus. This occurs mainly through interactions of T cell receptor of developing thymocytes with MHC/peptide ligands on cortical thymic epithelial cells (TEC). An ongoing controversy concerns the origin and the role of peptides involved in the positive selection of thymocytes. Evidence provided here shows that processing of MHC class II complexes in cortical TEC differs from that of medullary TEC. Removal of the invariant chain associated with MHC class II complexes was rapid and complete in medullary TEC which present peptides from both exogenous and cytosolic origin. In cortical TEC, a large fraction of class II dimers remained associated with a 10-12-kDa fragment of invariant chain (Ii). Incomplete removal of Ii correlated with the inability of cortical TEC to present peptides from exogenous origin. However, presentation of peptides from cytosolic proteins by cortical TEC remained possible. Thus, most peptides from exogenous proteins may be excluded from participating in positive selection of CD4+ T cells by a mechanism limiting Ii breakdown.

Inhibitory effects of specific apolipoprotein C-III isoforms on the binding of triglyceride-rich lipoproteins to the lipolysis-stimulated receptor.

ApoC-III overexpression in mice results in severe hypertriglyceridemia due primarily to a delay in the clearance of triglyceride-rich lipoproteins. We have, in primary cultures of rat hepatocytes, characterized a lipolysis-stimulated receptor (LSR). The apparent number of LSR that are available on rat liver plasma membranes is negatively correlated with plasma triglyceride concentrations measured in the fed state. We therefore proposed that the primary physiological role of the LSR is to contribute to the cellular uptake of triglyceride-rich lipoproteins. We have now tested the effect of apoC-III on the binding of triglyceride-rich lipoproteins to LSR. Supplementation of 125I-very low density lipoprotein (VLDL) with apoC-III inhibited the LSR-mediated binding, internalization, and degradation of 125I-VLDL in primary cultures of rat hepatocytes. Studies using isolated rat liver plasma membranes showed that enrichment of human VLDL and chylomicrons with synthetic or purified human apoC-III decreased their binding to the LSR by about 40%. Supplementation of triglyceride-rich lipoproteins under the same conditions with human apoC-II had no such inhibitory effect, despite the fact that this apoprotein bound as efficiently as apoC-III to these particles. Preincubation of LDL with apoC-III did not modify its binding to LSR. Partitioning studies using 125I-apoC-III showed that this lack of effect was due to apoC-III's inability to efficiently associate with LDL. Purified human apoC-III1 was as efficient as the synthetic nonsialylated form of apoC-III in inhibiting binding of VLDL to LSR. However, despite a 2-fold greater binding of apoC-III2 to VLDL, this isoform was a less efficient inhibitor of the binding of VLDL to LSR than apoC-III1 or nonsialylated apoC-III. Desialylation of apoC-III2 by treatment with neuraminidase increased the inhibition of VLDL binding to LSR to a level similar to that observed with apoC-III1 and nonsialylated apoC-III. We propose that apoC-III regulates in part the rate of removal of triglyceride-rich particles by inhibiting their binding to the LSR, and that the level of inhibition is determined by the degree of apoC-III sialylation.

Blockade of the alpha 2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein on rat liver parenchymal cells by the 39-kDa receptor-associated protein leaves the interaction of beta-migrating very-low-density lipoprotein with the lipoprotein remnant receptor unaffected.

The nature of the liver binding site which is responsible for the initial recognition and clearance of chylomicron-remnants and beta-migrating very-low-density lipoprotein (beta-VLDL) is under active dispute. We have investigated the effect of the 39-kDa receptor-associated protein (RAP) on the recognition site for activated alpha 2-macroglobulin and beta-VLDL on rat liver parenchymal cells in vivo and in vitro in order to analyze whether both substrates are recognized and internalized by the same receptor system. Radiolabelled trypsin-activated alpha 2-macroglobulin (alpha 2M-T) was cleared rapidly by the liver (maximal uptake of 80.8 +/- 1.0% of the injected dose). Prior injection of 5, 15, or 50 mg gluthathione-S-transferase-linked RAP (GST-RAP)/kg rat reduced the liver uptake to 62.2 +/- 2.3%, 59.3 +/- 1.1%, or 2.9 +/- 0.1% of the injected dose, respectively. Concurrently the serum decay was strongly delayed after injection of 50 mg GST-RAP/kg rat but this did not affect the serum decay and liver uptake of 125I-beta-VLDL. Binding studies with isolated liver parenchymal cells in vitro demonstrated that the binding of 125I-alpha 2M-T was 98% inhibited by GST-RAP with an IC50 of 0.3 microgram/ml (4.2 nM), whereas the binding of 125I-beta-VLDL and 125I-beta-VLDL + recombinant apolipoprotein E (rec-apoE) was unaffected by GST-RAP up to 50 micrograms/ml (700 nM). Also, the cell association and degradation of alpha 2M-T was blocked by RAP, while the association and degradation of beta-VLDL and beta-VLDL + rec-apoE were not influenced. The inhibitory effect of RAP on the cell association and degradation of alpha 2M-T lasted for 1-2 h of incubation at 37 degrees C. The binding of the radioiodinated RAP to isolated liver parenchymal cells was highly efficiently coupled to lysosomal degradation. Upon in vivo injection into rats, 125I-labeled RAP is rapidly cleared from the serum and taken up by the liver, which is also coupled to efficient degradation. Since RAP blocks binding of all known ligands to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (the alpha 2Mr/LRP) and at high concentrations the binding to the LDL receptor, we conclude that the initial binding and internalization of beta-VLDL by rat liver parenchymal cells is not mediated by the alpha 2Mr/LRP. The properties of binding of beta-VLDL to rat liver parenchymal cells points to an apoE-specific recognition site for lipoprotein remnants which differs from the alpha 2Mr/LRP, proteoglycans and the LDL receptor and is tentatively called the lipoprotein remnant receptor.

Mechanism of activation and functional significance of the lipolysis-stimulated receptor. Evidence for a role as chylomicron remnant receptor.

In cultured human and rat cells, the lipolysis-stimulated receptor (LSR), when activated by free fatty acids (FFA), mediates the binding of apoprotein B- and apoprotein E-containing lipoproteins and their subsequent internalization and degradation. To better understand the physiological role of LSR, we developed a biochemical assay that optimizes both the activation and binding steps and, thus, allows the estimation of the number of LSR binding sites expressed in the livers of living animals. With this technique, a strong inverse correlation was found in rats between the apparent number of LSR binding sites in liver and the postprandial plasma triglyceride concentration (r = -0.828, p < 0.001, n = 12). No correlation existed between the number of LSR and plasma triglycerides measured in the same animals after 24 h of fasting. The same membrane binding assay was used to elucidate the mechanism by which FFA induce lipoprotein binding to LSR. The LSR activation step was mediated by direct interaction of FFA with LSR candidate proteins of apparent molecular masses of 115 and 90 kDa and occurred independently of the membrane lipid environment. The FFA-induced conformational shift that revealed the lipoprotein binding site remained fully reversible upon removal of the FFA. However, occupancy of the site by the apoprotein ligand stabilized the active form of LSR. Comparison of the effect of different FFA alone or in combination indicated that the same binding site is revealed by different FFA and that the length and saturation of the FFA monomeric carbon chain are critical in determining the potency of the FFA activating effect. We propose that the LSR pathway represents a limiting step for the cellular uptake of intestinally derived triglyceride-rich lipoproteins and speculate that FFA liberated by lipolysis initiate this process by altering the conformation of LSR to reveal the lipoprotein binding site.

Inhibitory effect on the lipolysis-stimulated receptor of the 39-kDa receptor-associated protein.

Adenovirus vector-mediated transfer of the receptor-associated protein (RAP) gene into low density lipoprotein (LDL) receptor-deficient mice was shown to achieve plasma concentrations ranging between 20 and 200 micrograms/ml and to result in the accumulation of remnant lipoproteins (Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J. (1994) Science 264, 1471-1474). Both this finding and the observation that in addition to various other members of the LDL receptor gene family, RAP binds to a yet unidentified protein of apparent molecular mass of 105 kDa prompted us to examine the effect of high concentrations of RAP on the lipolysis-stimulated receptor (LSR). LSR is a receptor distinct from the LDL receptor and the LDL receptor-related protein and is capable of binding apoB and apoE when activated by free fatty acids. Data reported here show that in fibroblasts isolated from a subject homozygous for familial hypercholesterolemia, RAP fusion protein inhibited LSR-mediated binding of 125I-LDL and the subsequent internalization and degradation of the particles. Studies on the interaction of RAP with LSR in isolated rat liver membranes revealed that at concentrations > or = 10 micrograms/ml, RAP inhibited in a dose-dependent manner the binding of LDL to LSR; half-maximum inhibition was obtained with 20 micrograms/ml RAP. Ligand blotting studies revealed that RAP bound directly to two rat liver membrane proteins of apparent molecular masses identical to those that bind 125I-LDL after preincubation with oleate. However, unlike LDL, binding of 125I-RAP to LSR did not require preincubation with oleate. Preincubation of nitrocellulose membranes with an excess of unlabeled RAP fusion protein decreased oleate-induced binding of 125I-LDL to LSR candidate proteins, whereas preincubation with excess unlabeled LDL was unable to prevent the subsequent binding of 125I-RAP to the LSR proteins. Both the latter data and analysis of the mechanism of inhibition were consistent with the RAP inhibitory effect on LSR being achieved by interference with a site distinct from the oleate-induced LDL binding site. In conclusion, this study shows that at concentrations reported to delay chylomicron remnant removal in LDL receptor-deficient mice, RAP exerted a significant inhibitory effect on LSR.

Lipolysis-stimulated receptor: a newcomer on the lipoprotein research scene.

It has been widely accepted that the remnants of the intestinally-derived lipoprotein chylomicrons, i.e., chylomicron remnants (CMR), are cleared from the circulation by a receptor genetically distinct from the well-known LDL-receptor. This second receptor was initially considered as a receptor specific for apo E, in contrast to the LDL-receptor, which binds both apo B and apoE. This article critically examines the current dogma of the putative CMR receptor, as well as both supporting and conflicting evidence for the recently-proposed identity of this receptor with the LDL-receptor related protein (LRP). Next, we introduce the lipolysis-stimulated receptor, LSR, which bears all the biochemical characteristics of the CMR receptor. In addition, the apparent number of LSR expressed in the liver is inversely correlated with nonfasting levels of plasma triglycerides. A change in LSR expression and parallel inverse change in plasma triglycerides is observed in rats treated with hyperlipidemic (retinoic acid) or hypolipidemic (fish oil in MaxEPA) agents, indicating that LSR represents a definite target for pharmacological management of hyperlipidemia. In support of this notion is the observation that MaxEPA, which causes an increase in LSR expression, also reduces both plasma triglyceride and cholesterol levels in the thus far intractable homozygous Watanabe heritable hyperlipidemic rabbit.

[Liver mechanisms for the elimination of lipoproteins of intestinal origin]. / Mécanismes hépatiques d'élimination des lipoprotéines d'origine intestinale.

This article critically examines the concept of the putative chylomicron remnant receptor (CMR). The molecular nature of this second lipoprotein receptor remains disputed. Indeed, two proteins, the low density lipoprotein receptor-related protein (LRP) and the lipolysis stimulated receptor (LSR) have been proposed as candidates for this function. The LRP bears significant structural homology with the LDL receptor and mediates the internalisation of beta-VLDL enriched with apo E. In addition, LRP binds several ligands not related to the lipoprotein system. Thus, LRP's contribution to the clearance of CMR has been questioned. The precise biochemical structure of LSR remains unclear. However, a series of observations support the hypothesis that LSR is the CMR receptor. LSR, which is activated by free fatty acis (FFA), the products of lipolysis, is present in primary cultures of rat hepatocytes. It displays the highest affinity for triglyceride-rich lipoproteins and is inhibited by lactoferrin. The existence of a strong inverse correlation in rats between the apparent number of hepatic LSR and the plasma triglyceride concentration measured in the post-prandial state, indicate that LSR represents a rate-limiting step for the removal of triglyceride-rich lipoproteins. Moreover, the ability of MAXEPA to enhance the expression of LSR in parallel with its well documented hypotriglyceridemic effect indicates that, contrary to popular belief, the putative CMR receptor represents a target for pharmacological management of hyperlipidemia.

Asymptomatic homozygous hypobetalipoproteinemia associated with apolipoprotein B45.2.

Familial hypobetalipoproteinemia is caused by apolipoprotein (apo) B gene mutations and is frequently associated with a truncated apo-B protein in the plasma. Homozygosity for mutations yielding a truncated apo-B is extremely rare; fewer than five true homozygotes have been described in the world's literature. These patients typically have normal levels of triglycerides and virtually absent low density lipoprotein (LDL) cholesterol. The clinical status of these patients is variable, ranging from asymptomatic in two homozygotes who synthesized a truncated apo-B (apo-B87) to severe neurological disease resulting from vitamin E deficiency in a homozygote who synthesized a shorter apo-B (apo-B50). In this report, we describe a 48-year-old female homozygous for a nonsense mutation resulting in an even shorter apo-B, apo-B45.2. Although this individual had virtually no LDL cholesterol, she was asymptomatic and had normal plasma levels of vitamin E. This case demonstrates that homozygosity for an apo-B mutation associated with a relatively short apo-B truncation can be completely asymptomatic.