Prospective study of sensitization and food allergy to flaxseed in 1317 subjects.

BACKGROUND: Foods containing flaxseed proteins rich inpolyunsaturatedfatty acids are new on the market. OBJECTIVES: In a population of patients attending the allergology department, we evaluated the frequency of sensitization to flaxseed, characterized allergens and looked for modifications related to industrial processing. METHODS: Natural, heated and extruded flaxseeds were tested using prick-in-prick tests (PIP using the fresh seed), SDS PAGE, immunoblots, immunoblot inhibition and Fourier Transform Infrared (FTIR) spectroscopy. RESULTS: PIP tests to natural flaxseed were positive in 5.8% of the 1317 patients. 73 of 77 PIP-positive patients were atopic. There was cross-reactivity with five seeds. peanut, soybean, rapeseed, lupine and wheat, and with rape pollen. Immunoblot inhibition by bromelain confirmed the presence of specific IgE to cross-reactive carbohydrate determinants (CCD). 0.15% of this population presented with food allergy to flaxseed and positive PIP to heated and extruded flaxseed. Two sera showed that clinically relevant allergens in industrial products had MW between 25 and 38 kDa. Sensitization to processed flaxseed characterized only the allergic subjects. FTIR spectroscopy showed major modifications in 3 and alpha structures following industrial processing. CONCLUSION: Positive prick tests to natural flaxseed were mainly due to cross-reactions. Flaxseed allergy is rare and could be detected by PIP to heated extruded flaxseed. Increasing consumption callsfor monitoring of clinical risk.

Studying cytokinesis and midbody remnants using correlative light/scanning EM.

Cytokinesis is an essential step of cell proliferation leading to the physical separation of the dividing cells. Cytokinesis relies on both large scale and local scale cell shape changes, and terminates with the final abscission cut that requires close apposition of the plasma membrane. While furrow ingression is a prominent feature of the early phase of cytokinesis and is easy to visualize in all models, from dividing eggs to culture cells, the later steps of cytokinesis until abscission can be much more difficult to visualize. One key issue is to combine live-cell imaging over several hours and detailed, structural analysis of the cell shape changes in 3D, in particular at the time of cytokinetic abscission. Here, we describe the methodologies that we recently developed for studying cytokinetic abscission in human culture cells using live-cell phase-contrast microscopy, combined with correlative scanning electron microscopy. This allows us to determine the membrane surface and underlying cytoskeleton of the intercellular bridge with unprecedented precision and to determine the fate of the midbody remnant after abscission.

Electron microscope radioautographic evidence of in vivo androgen-binding protein internalization in the rat epididymis principal cells.

The androgen-binding protein (ABP) has been purified from rat testes with a yield of 14% using four steps of HPLC and was subsequently iodinated to a specific activity of 0.1 mCi/mg protein. Using a micromanipulator, [125I] iodo-ABP-dihydrotestosterone was injected intraluminally into the proximal caput of the rat epididymis. Epididymides were sampled from 3 to 120 min after the injection of the tracer and processed for transmission electron microscopy autoradiography. Our results showed the accumulation of detectable radioactive sources in the apical cytoplasm of only one of the epithelial cell type lining the ductus, the principal cells. In the interval from 3 to 120 min, the iodinated ABP was mainly present in the supranuclear region and was especially concentrated over coated structures, endosomes, multivesicular bodies, and over the Golgi apparatus. The same pattern was obtained using [3H]dihydrotestosterone-ABP complex instead of iodinated ABP. In addition, there was a negative correlation between the log time and the distribution of the silver grains in the luminal border and in the compartment of the apical vesicles. On the contrary, there was a positive correlation between the log time and the distribution of the silver grains in the Golgi apparatus. These results provide, for the first time, direct histological evidence of the in vivo ABP internalization by the principal cells. Since horseradish peroxidase, a fluid-phase endocytosis marker, when injected under the same conditions was internalized in both apical and principal cells, since labeled radioactive ABP appeared to be bound to the membrane of the endocytic apparatus rather than to its content, and since this binding and uptake could be prevented in the presence of an excess of unlabeled ABP, it is concluded that the internalization of ABP could not be a nonspecific fluid-phase endocytosis but should be dependent on its interaction with the apical plasma membrane of the principal cell. It still remains to be determined if these mechanisms involve the binding of ABP to a specific membrane receptor.

Purification of androgen-binding protein from rat testis using high-performance liquid chromatography and physicochemical properties of the iodinated molecule.

The androgen-binding protein (ABP) has been purified 87,500-fold from rat testis using 4 steps of HPLC, with a yield of 14%. The molecule was 99% pure with a specific activity estimated to 16,600 pmol/mg protein. The iodinated molecule was eluted in 2 peaks in Sephacryl S300 gel filtration with a molecular mass estimated to be 92,600 +/- 3300 and 50,300 +/- 4000 Da. The column isoelectrofocusing of 125I-ABP demonstrated 3 isoproteins isoelectric at pH 4.7, 4.9 and 5.3 and the sedimentation coefficient was estimated to be 4.7 S in sucrose gradient ultracentrifugation. The 125I-ABP had similar physiochemical properties to the non-labelled ABP of epididymis.

Photoaffinity labelled rat androgen-binding protein and human sex hormone steroid-binding protein bind specifically to rat germ cells.

A specific receptor with high affinity for rat androgen-binding protein (rABP) was identified in isolated adult rat germ cells and in the corresponding plasma membrane-enriched preparations. Binding was reversible and time-dependent, with maximum relative binding after 40 min at 4 degrees C; it was pH-dependent, with maximum binding at pH 6-8. Unlabelled rABP and human sex steroid-binding protein (hSBP), but not lactotransferrin, serotransferrin, asialofetuin, fetuin or bovine serum albumin, competed with labelled rABP for binding sites on isolated germ cells. Scatchard analysis revealed a single class of binding site with apparent dissociation constant (Kd) values of 0.78 +/- 0.04 nM and 0.97 +/- 0.05 nM in intact germ cells and plasma membrane preparations respectively. A Kd of 1.72 +/- 0.12 nM for hSBP showed that the receptor binding site was effective for both androgen-carrier molecules. Labelled rABP incubated with solubilized germ cell membrane fractions at pH 7 formed a complex excluded from Superose 6B mini-gels; this complex was not formed at pH 3. The receptor complex was also abolished in the presence of a 100-fold excess of either unlabelled rABP or unlabelled hSBP, or in the presence of 20 mM EDTA. These results suggest that the plasma membrane of rat germ cells contains a receptor which selectively binds rABP and hSBP.

Evidence that androgen-binding protein endocytosis in vitro is receptor mediated in principal cells of the rat epididymis.

We have studied the binding of [125I-iodo]androgen-binding protein (ABP) and of [3H]delta 6-testosterone photoaffinity-labelled ABP to receptors in the plasma membrane of rat epididymal cells in three ways: ABP binding to a Triton X-100-solubilized membrane extract, ABP binding to isolated epithelial cells in suspension and autoradiography of segments of dissected epididymides after in-vitro intraluminal injection of labelled ABP. The binding of iodinated ABP to the receptor was similar to that of photoaffinity-labelled ABP in gel filtration. The ABP-receptor complex was eluted from Superose 6 gels as an aggregate, with a molecular mass of 2000 kDa. It was separated into two peaks by sucrose gradient ultracentrifugation, with respective sedimentation coefficients of 18.4 and 9.0 s. The activity of the receptor (ABP-binding capacity/mg protein) was tenfold higher in the caput than in the cauda. The binding of ABP to the receptor was pH dependent, being almost abolished at pH less than 4. The binding at 4 degrees C of photoaffinity-labelled ABP to epithelial cells corresponded to two types of binding sites. The numbers of high-affinity and low-affinity sites per cell were 1600 and 7700 respectively; the association constants of these sites were 67.9 and 2.8 litres/nM respectively. The binding was decreased by treatment of the cells with trypsin or incubation in the presence of EDTA. The binding in vitro of labelled ABP to the epididymis epithelium reached a maximum after about 20 min at 4 degrees C. In the autoradiographic study the tracer was found to be closely associated with coated pits, coated vesicles, endosomes and pale multivesicular bodies. Treatment of rats with cycloheximide significantly reduced the uptake of the tracer. Perfusion in vitro of epididymides with chloroquine produced a fourfold increase of the tracer in endosomes and multivesicular bodies.

The plasma membrane of epididymal epithelial cells has a specific receptor which binds to androgen-binding protein and sex steroid-binding protein.

The binding of [3H] delta 6-testosterone photoaffinity-labelled rat androgen-binding protein (rABP) has been studied in an enriched fraction of plasma membranes of epithelial epididymal cells in immature (15 days) and adult rats (40 days). The binding was maximal in less than 30 min and more rapid at 4 degrees C than at 34 degrees C. It was calcium and pH dependent. Scatchard plots of the binding data gave curvilinear plots with two types of binding sites corresponding to a K(ass1) of 18.2 nM-1 and K(ass2) of 1.6 nM-1 (2.2 x 10(11) sites/mg protein and 5.4 x 10(11) sites/mg protein, respectively). In adult rats, only one type of binding site was found, with a K(ass) of 3.7 nM-1 (4.5 x 10(11) sites/mg protein). The number of receptors was 5-fold lower in the cauda than in the caput of the epididymis. The pretreatment of the isolated intact cells with streptozotocin induced a 45% reduction of the binding. Only unlabelled rABP and hSBP (human sex steroid-binding protein) but not other proteins (lactotransferrin, serotransferrin, asialofetuine, fetuine and bovine serum albumin) competed with the labelled ligand to bind plasma membranes. The membrane fraction was solubilized by triton X-100. Its incubation with labelled rABP and hSBP provoked the elution of the tracer as an aggregate into the void volume fraction of superose 6B mini-gel filtration columns. Structural homology between hSBP and rABP could be responsible for the common behaviour of the steroid-carrier molecules for the ABP receptor of rat epididymal epithelial cells.

Laboratory tests for diagnosis of food allergy: advantages, disadvantages and future perspectives.

Numerous biological tests point to the diagnosis of food sensitization: detection of specific IgEs by Rast techniques, multi-detection assays, immunoblotting, screening of basophil activation (BAT or FAST), assays for leukotriene LTC4 release (CAST), measurement of plasma histamine, serum tryptase, serum ECP, urinary EDN, completed by mannitol-lactulose test evaluating intestinal permeability, assay of fecal IgEs, Rast for specific IgG4. Primary screening for anti-food IgEs by multi-detection assays seeks justification from insufficient clinical data and false positive tests are common in patients sensitized to pollens or latex, on account of in vitro cross reactivities (CR). Multiple CR explain positive Rast to vegetal food allergens in such patients. Biological tests should not be performed as the first line of diagnosis. In vivo sensitisation is assessed by positive prick-tests, demonstrating the bivalence of allergens, as well as the affinity of specific IgEs, two conditions necessary to bridge membrane bound specific IgEs, leading to the release of mediators. Prick-tests are closer to clinical symptoms than biological tests. However, the diagnosis of food allergy is based on standardised oral challenges. Exceptions are high levels of specific IgEs to egg (> 6 kUl/l), peanut (> 15 kUl/l), fish (> 20 kUl/l) and milk (> 32 kUl/l), reaching a 95% predictive positive value. Rast inhibition tests are useful to identify masked allergens in foods. Research developments will have impact on the development of new diagnostic tools: allergen mixes reinforcing a food extract by associated recombinant major allergens, multiple combination of recombinant allergens (chips) or tests with synthetic epitopes aimed a the prediction of recovery. Laboratory tests take place in the decision free for the diagnosis for the food allergy and the follow-up of the levels specific IgEs is a tool to assess outcome and contributes to predict recovery or persistent allergy. Up to now the significance of positive laboratory tests showing the implication of IgEs is at the crossroads of the allergist's and biologist's expertise.

[Food allergy: effect of proteins-lipids and proteins-polysaccharides interactions]. / Allergénicité alimentaire: importance des interactions protéines-lipides et protéines-polysaccharides.

The most widely used ingredients in food formulation are proteins, lipids and polysaccharides. Proteins-lipids and proteins-polysaccharides interactions play a key role in the structure, stability, sensorial and nutritional properties of formulated foods. The objective of the present study is to highlight the importance of proteins-lipids and proteins-polysaccharides interactions, on the immuno-reactivity of allergenic proteins. Two models have been studied, on the one hand refined and not refined oils (soya and sunflower) and soya lecithin, on the other hand mixtures based on peanut proteins and polysaccharides (arabic gum, pectin, xylan). STUDY OF OILS: We have extracted proteins, using a PBS buffer, from refined and not refined oils from soya, sunflower and from soya lecithin, determined protein concentrations and identified allergenic proteins using SDS-PAGE electrophoresis and immuno-blotting. Phospholipids are determined by atomic absorption spectrometry. The protein determination and SDS-PAGE show the presence of a higher amount of proteins in not refined oils and lecithin as compared to refined oils. An important amount of proteins associated to phospholipids are eliminated by degumming on the form of lecithin. On the other hand, residual proteins from refined oils are accompanied by phospholipids. Immuno-blots reveal the presence of a 56 kDa allergen in oils issued from soya seeds and soya lecithin, and the presence of a 67 kDa allergen in oils issued from sunflower seeds. We conclude that the presence or elimination of proteins, especially allergens from oils is linked to amphiphilic association to phospholipids. STUDY OF PEANUT PROTEINS-POLYSACCHARIDES MIXTURES: We have digested in vitro proteins in a dialysis bag using a multi-enzymatic method and characterized proteins and peptides using SDS-PAGE electrophoresis and immuno-blotting. Our results confirm that peanut proteins alone are digested by proteases and that a number of large peptides still have epitopes recognized by anti-peanut proteins antibodies. Our results also show that the presence of polysaccharides changes the peptidic profile after digestion and that, depending on the polysaccharide type, smaller or larger peptides can be obtained in the dialysis bag. Smaller peptides are obtained using pectin whereas larger peptides are obtained using arabic gum and xylan. In the latter case, an increasing amount of peptides reacts to antibodies. Our first observations clearly show the need to better understand modifications of proteins allergenicity induced by the presence of other ingredients such as polysaccharides and lipids, in relation to technological treatments.

[Biological exploration of food allergy]. / Exploration biologique de l'allergie alimentaire.

Most of the time, food allergies are the consequence of an immediate IgE mediated hypersensitivity. In France, the frequency of food allergies is of 3.24%, it raises 8% in children. Their number as been multiplied by 2 in 5 years. The biological approach of food allergy is based essentially on the detection and the measurement of specific IgE. To date, a high number of tests are marketed and it is important to determine which of them are of high quality, using analytical or clinical-biological evaluations. Some other ways are available to study thoroughly and to characterize the allergens responsible of clinical reactions: the detection of hidden allergens, the study of crossed reaction between food or between food and pollen, the modification of the allergens by food processing.

[Free radicals and HIV infection]. / Radicaux libres et infection par le VIH.

In HIV infected patients, the increase of the concentration of free radicals is related to: a depletion of protective system (glutathione peroxidase, superoxide dismutase, vitamin E, selenium ...), and an increased production of free radicals (superoxide anion, hydrogen peroxide, hydroxil radical) consecutive to the activation of lymphocytes and phagocyting cells, the chronic inflammation, the increased polyinsatured fatty acids concentration and lipoperoxidation, and direct or indirect effect of several pathologic agents including Mycoplasma sp. This free radical excess could impair cell membranes and generate apoptosis, the main cause of lymphocytes CD4+ depletion. After a brief review of the free radicals synthesis pathway, their potential deleterious effects and the protective systems, the role of free radicals in the pathogenesis of HIV infection are discussed in regard to data reported in the literature.

[Influence of visible interferences on biochemical assays realised on Au 5231, AU 5223 (Olympus) and CL 7200 (Shimadzu)]. / Influence des interférences visibles sur les dosages de biochimie réalisés sur AU 5231, AU 5223 (Olympus) et CL 7200 (Shimadzu).

The impact of the major interferents (hemolysis, bilirubin, turbidity), on the quality of biochemical tests, was evaluated on multiparametric analysers (CL 7200 Shimadzu, Japan/Ciba-Corning, France; AU 5231 and AU 5223 Olympus, Japan/bioMérieux, France), according to the SFBC instructions. Interferences were detected in 33 cases upon 165 tests realized, that is to say 20% of the performed analysis. Turbidity was the most frequent cause of interference (7.8%), followed by hemolysis (8.5%) and bilirubin (3.6%). The use of a sample blank, a bireagent, the change of reagent, the change of the secondary wavelength or the modification of the measurement times, allowed us to reduce more than 80% of the interferences. Only three interferences remained: hemolysis upon the measurement of TGO and potassium, and bilirubin upon the measurement of creatinine. For these parameters, a suitable note using the Olympus factors (semi quantitative expression of the importance of the three interferents) is reported on the answer sheet.

[Delayed diagnosis of homocystinuria by major deficiency in cystathionine beta synthase]. / Diagnostic tardif d'homocystinurie par déficit majeur en cystathionine beta synthase.

INTRODUCTION: Homocystinuria due to cystathionine beta synthase (CBS) deficiency is a special type of hyperhomocysteinemia because of its clinical expression (thrombotic events, ectopic lens and mental retardation). It's a rare, hereditary recessive autosomic disease generally diagnosed during childhood. EXEGESIS: Thrombophilia examination in a 50-year-old man found a dramatically increase homocysteinemia. Homocystinuria, profile of plasmatic amino acids and reduced CBS activity, (0.05 microkat/kg protein; N = 1.5 +/- 0.8) confirmed homocystinuria's diagnosis. Family study demonstrates that three siblings suffer from homocystinuria. Vitamin enriched diet with pyridoxin, vitamin B12 and folates induced reducing hyperhomocysteinemia and homocystinuria. CONCLUSION: This case report, original because of the diagnosis age, suggests a hyperhomocysteinemia's screening in patients with recurrent thrombotic events.

[Allergenicity of oils]. / Allergénicité des huiles.

Cases of allergy to the oils of groundnut, sunflower, soya and sesame have been described in the literature. In parallel, other authors have affirmed that these oils are not allergenic. The objective of this article is to make the point on this question, to cite the procedures to which the seeds are submitted to extract the oil, to remember that the oils are not composed only of triglycerides and to describe the results of our work. Allergy of oils is a subject that is constantly submitted to controversy and the bibliography does not cease to give contradictory examples. This may be explained by the variations in extraction procedures used by the manufactures, as well as by the conditions of extraction of the proteins in the laboratory.

The use of two multitests fx5 and fx10 in the diagnosis of food allergy in children: regarding 42 cases.

The multitests Cap RAST fx5 and fx10 (Pharmacia, Cap System) are used for a rapid biological diagnosis of food allergy. These tests were assessed in 29 children who presented 42 food allergies (FA) documented by prick tests, specific IgE and labial or oral provocation tests (single blind placebo controlled food challenges). When the multitests were positive, the search for specific IgE to the corresponding allergens was performed (Cap RAST, Pharmacia). The theoretical coverage of FA could be estimated according to the frequency of food allergens involved in the children. It reaches 85% for Cap RAST fx5 and 29% for Cap RAST fx10. The sensitivity of Cap RAST fx5 is 89% and 50% for Cap RAST fx10. Even in the case when the child had a single food allergy, the detail of specific IgE showed multiple positivities to several allergens included in the multitest. Consequently, the positive predictive value of Cap RAST was only 32%. Prick tests to the same allergens were more rarely positive, gaining thus a better positive predictive value. The authors propose the use of Cap RAST fx5, eventually completed by a Cap RAST to beef for the first approach of food allergy in children. They stress the point that prick tests have to be carried on subsequently, in order to select properly allergens responsible for food allergy.

[Measurement of levels of specific IgE by the Efficient New Enzymatic Allergy (ENEA) System II (CIS bio)]. / Evaluation du dosage des IgE spécifiques par l'ENEA System II (CIS bio).

The diagnosis of IgE dependant food allergy relies on the demonstration of specific IgE by prick tests or in vitro tests. The ENEA System II (CIS bio international) is a new automatic assay analyser of specific IgE, that uses allergens coupled to a solid phase and a urease marked anti-IgE antibody. This study aims to compare the performance of the ENEA System II to that of Pharmacia CAP System for the assay of food specific IgE (milk, eggs, peanuts) by means of unit tests and multitests. Sixty three patients were included: 10 non atopic controls, 19 egg-allergic patients, 10 patients allergic to cow's milk, and 24 patients allergic to peanuts. The food allergy was proved by means of a double blind oral, labial or bronchial challenge and/or effective avoidance of the food. For both systems, the specificity of unit tests was 100%. Sensitivity was 60% and 100% with both systems, using milk and peanuts respectively. However, using eggs, it was only 74% with ENEA System II versus 95% with Pharmacia CAP System. The intra-trial variation coefficients were comparable. In contrast, inter-trial variation coefficient was very high for the ENEA System II (20.3% versus 7.3%). The multitest named "children's food" showed an important inter-set variability. In conclusion, the ENEA System II is a rapid automatic tester whose performance has to be improved. The actual thermostatically control of the system was shown to achieve quality assay. The conservation of the solid phase, recently perfected, is expected to suppress the inter-set variability.

Histamine content does not influence the tolerance of wine in normal subjects.

Histamine has been incriminated as having a responsibility for intolerance reaction to wines. We have made a study by double blind oral provocation test to find the effect of ingestion of a histamine-rich (22.8 mg.l-1) and a histamine free wine in eight healthy subjects. Blood samples were taken at 0, 10, 30 and 45 minutes after ingestion of the wine for measurement of plasma histamine and methylhistamine. Urines were collected 5 hours before and 5 hours after ingestion for measurement of urinary methylhistamine. No subject presented a reaction of intolerance after ingestion of wine rich or poor in histamine. No change in plasma histamine and plasma and urinary methylhistamine was seen. This study shows that the amount of histamine in wine has no clinical or biological effect in healthy subjects, and this emphasized the efficiency in man of the systems for degradation of histamine that is absorbed by the alimentary tract.