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.

Mechanism of plasma cholesteryl ester transfer in hypertriglyceridemia.

Plasma net cholesteryl ester (CE) transfer and optimum cholesteryl ester transfer protein (CETP) activity were determined in primary hypertriglyceridemic (n = 11) and normolipidemic (n = 15) individuals. The hypertriglyceridemic group demonstrated threefold greater net CE transfer leading to enhanced accumulation of CE in VLDL. This increased net transfer was not accompanied by a change in CETP activity. In normolipidemia, but not in hypertriglyceridemia, net CE transfer correlated with VLDL triglyceride (r = 0.92, P less than 0.001). In contrast, net CE transfer in hypertriglyceridemia, but not in normolipidemia, correlated with CETP activity (r = 0.73, P less than 0.01). Correction of hypertriglyceridemia with bezafibrate reduced net CE transfer towards normal and restored the correlation with VLDL triglyceride (r = 0.90, P less than 0.005) while suppressing the correlation with CETP activity. That net CE transfer depends on VLDL concentration was confirmed by an increase of net CE transfer in normolipidemic plasma supplemented with purified VLDL. Supplementation of purified CETP to normolipidemic plasma did not stimulate net CE transfer. In contrast, net CE transfer was enhanced by addition of CETP to both plasma supplemented with VLDL and hypertriglyceridemic plasma. Thus, in normal subjects, VLDL concentration determines the rate of net CE transfer. CETP becomes rate limiting as VLDL concentration increases, i.e., in hypertriglyceridemia.

Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei.

We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma brucei and Trypanosoma equiperdum. Unlike other membrane proteins of T. brucei, which are distributed throughout the cell surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis has been documented. Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization. CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid cytoplasmic extension. A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich 12-amino-acid repeat. CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural similarities with eucaryotic cell surface receptors. The most striking homology of CRAM is to the human low-density-lipoprotein receptor. We propose that CRAM functions as a cell surface receptor of different trypanosome species.

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.

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.

Changes in apolipoprotein and lipids in patients after surgery.

Plasma levels of apoproteins A-I, A-II and B and apoprotein/lipid ratios were determined in 43 patients (14 male and 29 female) on the second to the sixth day after gastrointestinal surgery. During this period, all the patients received i. v. 5% dextrose solutions only. Apoprotein levels (A-I, A-II and B), total cholesterol, HDL-cholesterol and phospholipid were significantly decreased after surgery but there was no sex differences. Patients undergoing major surgery showed much lower values than the ones undergoing minor surgery. Both the extent of the trauma and the caloric restriction resulting from sole carbohydrate intravenous feeding may be responsible for these changes. Decreased hepatic synthesis combined to a lack of intestinal supply might result in apo A-I and A-II reduction. Repeated determinations of apoprotein levels are proposed as simple means of follow-up for evaluating the degree of recovery of patients undergoing gastrointestinal surgery.

[Activities of the emergency department of a suburban hospital in 1984]. / Activité du service de garde d'un hôpital de Faubourg en 1984.

This is a review of the population coming to the outpatients department along June 1984. Most of the people are coming between noon and midnight. 20% of the patients are driven by ambulance and 59% of them are admitted, while only 23% of the total population and 11% of surgical cases are hospitalized. 56% of the patients are living in the neighbourhood of the hospital and 38% in the other suburbs of Brussels' urban area. Only 26% of the patients are referred by the family physician and 86% of them are admitted, while only 14% are requesting for a specific examination.

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.

Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia.

This paper describes a mechanism for degradation of low-density lipoprotein (LDL) in fibroblasts unable to synthesize the LDL receptor. In this cell line, long-chain free fatty acids (FFA) activated 125I-LDL uptake; unsaturated FFA were the most efficient. The first step of this pathway was the binding of LDL apoB to a single class of sites on the plasma membrane and was reversible in the presence of greater than or equal to 10 mM suramin. Binding equilibrium was achieved after a 60-90-min incubation at 37 degrees C with 1 mM oleate; under these conditions, the apparent Kd for 125I-LDL binding was 12.3 micrograms/mL. Both cholesterol-rich (LDL and beta-VLDL) and triglyceride-rich (VLDL) lipoproteins, but not apoE-free HDL, efficiently competed with 125I-LDL for this FFA-induced binding site. After LDL bound to the cell surface, they were internalized and delivered to lysosomes; chloroquine inhibited subsequent proteolysis of LDL and thereby increased the cellular content of the particles. A physiological oleate to albumin molar ratio, i.e., 1:1 (25 microM oleate and 2 mg/mL albumin), was sufficient to significantly (p less than 0.01) activate all three steps of this alternate pathway: for example, 644 +/- 217 (25 microM oleate) versus 33 +/- 57 (no oleate) ng of LDL/mg of cell protein was degraded after incubation (2 h, 37 degrees C) with 50 micrograms/mL 125I-LDL. We speculate that this pathway could contribute to the clearance of both chylomicron remnants and LDL.

Fat emulsions are more than energy suppliers.

The type of triglycerides in exogenous fat emulsions as well as the phospholipid: triglyceride ratio influences the plasma clearance. The plasma clearance cannot be used for indicating the utilization of fat emulsions as energy substrate. Introducing exogenous fat in replacement of part of the calories provided by glucose reduces a series of complications associated with large glucose intake. There are exchanges of triglycerides, esterified cholesterol, phospholipids and apoproteins between exogenous lipids and endogenous lipoproteins, depending on the composition of exogenous lipid emulsions. These interactions can significantly modify the composition of both, exogenous particles and endogenous lipoproteins. In future it will become essential to determine the removal site of 'exogenous remnants'. Future progress will allow a better understanding of the influence of these modifications on the metabolism of endogenous lipoproteins and the utilization of exogenous fat emulsions.

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.

Interactions between exogenous fat and plasma/lipoproteins.

Fat emulsions are essentially composed of triglycerides and phospholipids. Their elimination from the plasma--which is generally rapid--is influenced by the amount and the composition of both these components. During their short stay in the vascular compartment, exogenous particles undergo major compositional changes. They acquire various apolipoproteins--namely C-II, C-III, E and A-IV--by transfer from HDL. They also acquire esterified cholesterol from HDL and LDL and transfer exogenous triglycerides and phospholipids to these endogenous lipoproteins. These exchanges are affected by the type of triglyceride fatty acids and the amount of phospholipids present in fat emulsions, as well as by the infusion rate. Some 10% of emulsions--with a high phospholipid: triglyceride ratio--contain a huge phospholipid excess which can be isolated as a separate fraction from the triglyceride-rich particles. These phospholipids markedly interfere with the metabolism of cholesterol and the plasma lipoprotein profile.

Plasma lipid and plasma lipoprotein concentrations in low birth weight infants given parenteral nutrition with twenty or ten percent lipid emulsion.

Because 10% and 20% intravenously administered lipid emulsions (intralipid preparations) differ in their phospholipid/triglyceride ratio (0.12 and 0.06, respectively), 28 low birth weight infants requiring parenteral nutrition for at least 1 week were selected at random to receive either emulsion to determine the effects on plasma lipids and lipoproteins. Triglyceride intake was progressively increased to reach 2 gm/kg per day between days 4 and 7. During that period, all plasma lipids in samples taken 6 hours after infusion were higher in the 10% intralipid group. In comparison with day 0 values, triglyceride concentrations decreased (63 +/- 7 to 45 +/- 4 mg/dl; p less than 0.05) in the 20% group. Cholesterol levels increased in both groups, but the rise was more than twofold higher in the 10% group. Phospholipid increase was approximately 25% in the 20% group but more than 125% in patients receiving the 10% emulsion (p less than 0.005). The changes in plasma cholesterol and phospholipid levels were almost entirely in low-density lipoproteins. After 7 days, eight infants from each group were given the alternate emulsion, which resulted in a reversal of lipid patterns in each patient. We conclude that the higher phospholipid intake in 10% than in 20% intralipid is associated with higher plasma triglyceride concentrations and leads to accumulation of cholesterol and phospholipids in low-density lipoproteins. Emulsions with lower phospholipid content may be preferable for low birth weight infants and perhaps other patient populations with impaired removal of parenteral fat emulsions.

Fatty acid control of lipoprotein lipase: a link between energy metabolism and lipid transport.

Lipoprotein lipase (LPL) catalyzes the flux-generating step in transport of fatty acids from lipoprotein triacylglycerols into tissues for use in metabolic reactions. In vitro studies have shown that fatty acids can bind to the enzyme and impede its other interactions. In this study we have searched for evidence of fatty acid control of LPL in vivo by rapid infusion of a triacylglycerol emulsion to healthy volunteers. During infusion the activity of LPL but not of hepatic lipase increased in plasma, but to different degrees in different individuals. The time course for the increase in LPL activity differed from that for triacylglycerols but followed the plasma levels of free fatty acids. This was true during infusions and when the emulsion was given as a bolus injection. In particular there were several instances when plasma triacylglycerol levels were very high but free fatty acids and LPL activity remained low. Model studies with bovine LPL showed that fatty acids displace the enzyme from heparin-agarose. We suggest that in situations when fatty acids are generated more rapidly by LPL than they are used by the local tissue, they cause dissociation of the enzyme from its binding to endothelial heparin sulfate and are themselves released into circulation.

Unesterified fatty acids inhibit the binding of low density lipoproteins to the human fibroblast low density lipoprotein receptor.

Micromolar concentrations of oleate were found to inhibit reversibly the binding of low density lipoprotein (LDL) to the human fibroblast LDL receptor. The decrease in LDL binding caused a parallel reduction of both 125I-LDL uptake and degradation at 37 degrees C. At 4 degrees C, oleate was also found to displace 125I-LDL already bound to the LDL receptor. The effect of oleate was rapid, reaching 70-80% of maximum displacement with 5-10 min of incubation, and was closely correlated to oleate-albumin molar ratios. Partition analysis of unesterified fatty acids between cells and LDL showed that the inhibitory effect of oleate resulted mainly from an interaction of unesterified fatty acids with the cell surface rather than with the LDL particles. Using different unesterified fatty acids and fatty acid analogs, we found that the inhibitory effect was modulated by both the length and the conformation of the monomeric carbon chain and was directly dependent on the presence of a negative charge on the carboxylic group. At 4 degrees C, the inhibitory effect of oleate never exceeded half of maximum binding capacity. This limitation was associated with the ability of oleate to interact only with part of the population of LDL receptors which spontaneously recycles in the absence of ligand, as demonstrated by the fact that oleate did not induce any reduction of LDL binding after cell treatment with monensin in the absence of LDL. Our results indicate that unesterified fatty acids could participate in the control of LDL catabolism in vivo by direct modulation of the ability of LDL receptor to bind LDL.