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.

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.

In vitro allergenicity of peanut after hydrolysis in the presence of polysaccharides.

BACKGROUND: Peanut is a major allergenic product. Manufacturing processes used in food industries to improve the physicochemical properties of food-based peanut (stabilization, texturization), could cause a modification of the digestibility of peanut proteins and, consequently, their allergenicity. OBJECTIVE: This study aimed at examining the influence of polysaccharides, i.e., gum arabic, low methylated pectin (LMP) and xylan, on the in vitro hydrolysis of peanut protein isolate (PPI) and the in vitro allergenicity of the digestion products. METHODS: PPI was hydrolysed during a two-step in vitro hydrolysis by pepsin, followed by a trypsin/chymotrypsin (T/C) mixture performed in dialysis bags with molecular weight cut-offs (MWCO) of 1000 or 8000 Da. SDS-PAGE electrophoresis and immunoblotting were assessed on the peptic and T/C digestion products in (retentates) and out of the dialysis bags (dialysates). RESULTS: Hydrolysis by all of the digestive enzymes showed retention of some proteins in the dialysis bags in the presence of gum arabic and xylan. The retentates were recognized by IgG and IgE, particularly peptides <20 kDa. The IgE binding with peptides of retentate containing xylan from the dialysis bag with an MWCO of 1000 Da was reduced. The immunoreactivity of hydrolysis products in dialysates was considerably reduced by polysaccharides, regardless of the dialysis bag. CONCLUSION: Reduction of PPI hydrolysis was probably due to non-specific interactions between polysaccharides and peptides. In retentates, IgE-binding epitopes were reduced by digestion and the presence of xylan. In dialysates, they were reduced by all of the polysaccharides. This work highlights the possibility of modulating this food allergy through optimized formulation.

[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.

[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.

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.

Prospective study of mustard allergy: first study with double-blind placebo-controlled food challenge trials (24 cases).

BACKGROUND: Mustard allergy accounts for 1.1% of food allergies in children. However, double-blind placebo-controlled food challenge trials (DB PCFCs) have not yet been proposed. OBJECTIVE: To carry out DB PCFCs to determine the real frequency of mustard allergy in patients sensitized to mustard. METHODS: A prospective study was conducted in 30 subjects aged 3-20 years presenting positive prick tests to ground mustard seeds (Brassica nigra), mustard flour (B. juncea), metabisulfite-free strong mustard seasoning (B. juncea) and a commercialized allergenic extract (B. nigra). Twenty-seven subjects were screened for mustard-specific immunoglobulin E (IgE). PCFCs were carried out either DB or single blind (SB) with up to 1340 mg of metabisulfite-free seasoning. RESULTS: The mean diameter of the wheal induced by prick tests with the allergenic extract was lower (n.s.) than that induced by the native mustard products: 5.8 mm (1.5-15) vs 6.9 mm (0.5-18) for B. nigra ground seeds, 7.8 mm (1-20) for B. juncea flour and 9.7 mm (3-20) for the strong mustard seasoning. The diameter of the wheal induced by the allergenic extract was significantly different from that induced by the mustard seasoning (P < 0.005). The mean of mustard specific-IgE values was 8.7 KU/l (0.35-72.4). Seven of 30 food challenges were considered positive. Mean prick test results in the positive and negative PCFC subgroups were 5.5 mm vs 5.9 mm for the commercialized extract, 10.9 mm vs 5.8 mm for B. nigra ground seeds (P < 0.01), 9.9 mm vs 7.1 mm for B. juncea flour (n.s. P > 0.25) and 11.5 mm vs 9.1 mm for the metabisulfite-free mustard seasoning (n.s. P > 0.1). Mean specific IgE values determined by CAP system radioallergosorbent test (Phadebas Pharmacia) were higher but not significantly so (P > 0.25) in the subgroup with mustard allergy (12.3 K/l vs 7.6 KU/l). CONCLUSIONS: About 23.3% of the sensitized subjects were allergic to a routine dose of mustard. Positive prick tests and the presence of specific IgE were not predictive. SB PCFC or DB PCFC is required before recommending avoidance diets.

Effects of different levels of gum arabic, low methylated pectin and xylan on in vitro digestibility of beta-lactoglobulin.

Plant hydrocolloids used in the food industry to improve texture and stability of food, such as dairy products, can reduce protein digestibility and, consequently, modify the bioavailability of amino acids. We studied the in vitro hydrolysis at 37 degrees C of beta-lactoglobulin (beta-lg) in mixed dispersions containing either gum arabic or low-methylated pectin or xylan at levels of 0, 1, 10, 20, 30, and 50% weight. Proteolysis used either pepsin alone by progressive reduction of pH during proteolysis or pepsin followed by trypsin and chymotrypsin in two different dialysis bags with a molecular weight (MW) cutoff of 1000 or 8000 Da. Results showed that beta-lg was almost resistant to pepsin digestion and that the three plant hydrocolloids inhibited significantly beta-lg digestibility as determined using dialysis bag with a 1000-Da MW cutoff. Among the three polysaccharides used, xylan showed a digestibility decrease greater than that obtained with gum arabic and low-methylated pectin. On the other hand, no significant effect of polysaccharides on the in vitro beta-lg digestibility was detected using the dialysis bag with an 8000 Da MW cutoff. This mainly suggests that peptides with MW in the range 1000 to 8000 Da may interact with polysaccharides more than peptides and proteins with a greater molecular weight to decrease the protein digestibility, and that the nature of the polysaccharides plays a role in the interaction.

Allergenicity of some isoforms of white sesame proteins.

BACKGROUND: Allergy to sesame seeds is often associated with particularly severe reactions, with a high risk of anaphylaxis. The increase in reports of allergic reactions to sesame is probably due to the growing use of sesame seeds or sesame oil in food. OBJECTIVE: To determine the molecular weights of the proteins in three variety of sesame seeds and to study the isoelectric points and the allergenicity of white sesame proteins. METHODS: Extracts of white, brown and black sesame seeds were prepared. The white sesame extract, mostly used in bakery, was run on SDS-PAGE and two dimensional electrophoresis. Six sera from patients sensitized or symptomatic to sesame seed were used for Western blotting. RESULTS: The protein patterns of the white, brown and black sesame extracts showed major quantitative differences. The white extract had the higher protein concentration and contained 15 proteins of 12-79 kDa, some of them having several acidic isoelectric points. The lowest isoelectric point was 4.9 and the highest was 6.4, giving 35 isoforms. Ten of the 15 proteins (12-57.5 kDa) were recognized by specific IgE. The 12-13 kDa and 22-33 kDa proteins could correspond to the main allergens. CONCLUSION: White sesame seeds contain at least 10 allergenic proteins with acidic isoelectric points. In accordance with previous results, two of them seem to contain the major allergens.

[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.

[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.

Latex hypersensitivity in spinal cord injured adult patients.

Latex hypersensitivity is a major cause of anaphylaxis during anaesthesia. Patients with spina bifida, health care or rubber industry workers have been considered at risk for latex sensitization. By analogy, the existence of other at-risk subsets of patients with latex exposure due to frequent surgical procedures has been suggested. The aim of this study was to evaluate the prevalence of latex sensitization in a cohort of adult patients with spinal cord injury and repeated latex exposure. Forty-two adult patients with spinal cord injury were studied and retrospectively compared to a group of 30 children with spina bifida evaluated using a similar protocol. Patients were administered a questionnaire concerning history of latex hypersensitivity, atopy, and surgical procedures. Latex sensitivity was investigated by skin prick-tests and latex-specific IgE assay. The search for atopy was based on in vivo and in vitro tests against a panel of environmental allergens. No chronic spinal cord injured patient had a history of latex allergy. When compared with spina bifida, the number of surgical procedures was not statistically different. Although not significantly different, the prevalence of atopy was higher in spina bifida patients. The high level of latex sensitization in spina bifida patients contrasted sharply with the absence of sensitization observed on both skin and in vitro tests in patients with spinal cord injury (P<0.0001). This study confirms that adult patients with chronic neurologic defects resulting from spinal cord injury exhibit a low risk of latex sensitization. These results suggest that considering adult patients with repeated surgical procedures as a group at risk for latex sensitization because of a high degree of latex exposure should be re-examined.

No correlation between wine intolerance and histamine content of wine.

BACKGROUND: Histamine is thought to be the main cause of adverse reactions to wines. OBJECTIVE: The purpose of this study was to test the hypothesis that the level of histamine in wine affects the tolerance to wine in 16 subjects with wine intolerance. METHODS: We performed a study to examine the effects of wine histamine content in 16 adults with wine intolerance. Each subject underwent 2 double-blind provocation tests with wine: 1 with a wine poor in histamine (0.4 mg/L), and 1 with a wine rich in histamine (13.8 mg/L). Blood was collected for histamine and methylhistamine RIAs at 0, 10, 30, and 45 minutes after ingestion of the wine. Methylhistamine and methylimidazolacetic acid (gas chromatography and mass spectrometry) were measured in urine 5 hours before and 5 hours after ingestion. RESULTS: No significant differences in the occurrence of adverse reactions were noted after ingestion of either of the wines (McNemar test). At 10 minutes, a significant increase was observed in plasma histamine with histamine-poor wine. No significant changes (Wilcoxon test) were observed in the methylhistamine and methylimidazolacetic acid levels after ingestion of either histamine-poor or histamine-rich wine. CONCLUSION: This study demonstrates that there is no correlation between the histamine content of wine and wine intolerance. The increase of plasma histamine levels at 10 minutes with histamine-poor wine suggested the role of a histamine-releasing substance. The role of acetaldehyde is discussed.

Influence of refining steps on trace allergenic protein content in sunflower oil.

BACKGROUND: Although allergy to sunflower seed and oil is a relatively rare occurrence, several cases of sunflower seed allergy have been observed, and we have already described one case of anaphylaxis after eating sunflower oil and margarine. OBJECTIVE: The aim of our study was to determine and characterize the allergens from sunflower oil at the different steps of the refining process: crude pressed oil (step A), acidification and neutralization (step B), pregumming by centrifugation (step C), washing (step D), bleaching (step E), gumming by filtration (step F), and deodorization (step G). METHODS: A sample of oil from each step of the process (steps A to G) was heat extracted with PBS. The protein concentration of each extract was evaluated by using the micro-Bradford assay. Samples were run on SDS-PAGE. The immunoblot was performed with the serum of a patient sensitized to sunflower seed and oil. RESULTS: The extracts obtained after each step reveal a decrease in total protein concentration from 13.6 microg/mL to 0. 22 microg/mL. The result of SDS-PAGE shows 5 bands, from 67 kd to 145 kd, with the most abundant being the 67-kd protein. The amount of this protein decreases after each step of the process. It is, however, still present in trace amounts in the refined oil. The 67-kd protein, which is mainly present in the crude oil and slightly in the refined oil, has been shown to be allergenic. CONCLUSION: Because of the presence of allergenic proteins, refined sunflower oil may pose a threat to people highly sensitized to sunflower seeds.

The effect of 677C-->T and 1298A-->C mutations on plasma homocysteine and 5,10-methylenetetrahydrofolate reductase activity in healthy subjects.

We have studied the effect of common mutations (677C-->T and 1298A-->C) of the methylenetetrahydrofolate reductase (MTHFR) gene in sixty-six healthy French subjects, aged 27-47 years. Serum folate, vitamin B12, and plasma total homocysteine were measured as well as the specific activity of MTHFR in lymphocytes. The frequency of subjects homozygous for the 677TT genotype was 18%, and that of those homozygous for the 1298CC genotype was 12.5%. The frequency of individuals heterozygous for both mutations was 23.5%. The 1298A-->C mutation was associated with decreased MTHFR specific activity in subjects with both 677CC and 677CT genotypes. This activity was 60% for the 677CC/1298AC genotype and 52% for the 677CC/1298CC genotype when compared with the MTHFR specific activity of the 677CC/1298AA genotype. Heterozygotes for both mutations (677CT/1298AC genotype) had 36% of the reference specific activity. Although homocysteine levels in 677TT and 1298CC genotype subjects were higher than for other genotypes, no significant differences were observed among different genotypes. This may be due to high serum folate level in our samples, and suggests that folate therapy may be useful to prevent hyperhomocysteinaemia in homozygous mutant subjects.