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1.
J Clin Invest ; 130(10): 5477-5492, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32634131

ABSTRACT

Transcription infidelity (TI) is a mechanism that increases RNA and protein diversity. We found that single-base omissions (i.e., gaps) occurred at significantly higher rates in the RNA of highly allergenic legumes. Transcripts from peanut, soybean, sesame, and mite allergens contained a higher density of gaps than those of nonallergens. Allergen transcripts translate into proteins with a cationic carboxy terminus depleted in hydrophobic residues. In mice, recombinant TI variants of the peanut allergen Ara h 2, but not the canonical allergen itself, induced, without adjuvant, the production of anaphylactogenic specific IgE (sIgE), binding to linear epitopes on both canonical and TI segments of the TI variants. The removal of cationic proteins from bovine lactoserum markedly reduced its capacity to induce sIgE. In peanut-allergic children, the sIgE reactivity was directed toward both canonical and TI segments of Ara h 2 variants. We discovered 2 peanut allergens, which we believe to be previously unreported, because of their RNA-DNA divergence gap patterns and TI peptide amino acid composition. Finally, we showed that the sIgE of children with IgE-negative milk allergy targeted cationic proteins in lactoserum. We propose that it is not the canonical allergens, but their TI variants, that initiate sIgE isotype switching, while both canonical and TI variants elicit clinical allergic reactions.


Subject(s)
Allergens/genetics , Allergens/immunology , Fabaceae/genetics , Fabaceae/immunology , Frameshifting, Ribosomal , Plant Proteins/genetics , Plant Proteins/immunology , 2S Albumins, Plant/genetics , 2S Albumins, Plant/immunology , Adolescent , Anaphylaxis/etiology , Anaphylaxis/immunology , Animals , Antigens, Plant/genetics , Antigens, Plant/immunology , Arachis/genetics , Arachis/immunology , Cattle , Child , Child, Preschool , Female , Genetic Variation , Humans , Immune Sera/genetics , Immune Sera/immunology , Immunoglobulin E/biosynthesis , Male , Mice , Mice, Inbred BALB C , Milk Hypersensitivity/immunology , Peanut Hypersensitivity/etiology , Peanut Hypersensitivity/immunology , Phaseolus/genetics , Phaseolus/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Glycine max/genetics , Glycine max/immunology , Transcription, Genetic
2.
Vet Dermatol ; 28(6): 589-e143, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28770578

ABSTRACT

BACKGROUND: Commercial hydrolysed diets are used for the diagnosis of food allergy in dogs. The cleaved parent proteins are presumed to be too small to elicit an allergic response by reacting with allergen-specific immunoglobin E (IgE). OBJECTIVES: To evaluate three commercial hydrolysed dog diets for proteins. ANIMALS: Sera were collected from dogs with suspected food allergy. METHODS: Two batches of each hydrolysed diet were examined by electrophoresis and visualized by Coomassie blue, silver nitrate staining and IgE immunoblotting. RESULTS: From two to five proteins, ranging from 21 to 67 kDa, were detected in all three diets evaluated. Circulating IgE antibodies targeting these proteins were detected by immunoblotting of dog sera. Six different carbohydrate proteins were identified by mass spectrometry; maize/potato granule-bound starch synthase-1, soybean glycinin, soybean ß-conglycinin α chain, potato aspartic protease inhibitor, rice glutelin type B1 and soybean sucrose-binding protein. Four of these proteins have been described as allergens in humans. CONCLUSIONS: Some commercial hydrolysed diets contain carbohydrate proteins. Some dogs have circulating IgE antibodies targeting these proteins. The clinical significance of these findings is unknown.


Subject(s)
Allergens/immunology , Animal Feed , Dog Diseases/immunology , Dogs , Food Hypersensitivity/veterinary , Immunoglobulin E/immunology , Animal Feed/adverse effects , Animal Feed/analysis , Animals , Blotting, Western/veterinary , Dogs/immunology , Electrophoresis, Polyacrylamide Gel/veterinary , Food Hypersensitivity/immunology , Mass Spectrometry/veterinary , Proteins/analysis , Proteins/immunology
3.
Vet Dermatol ; 28(2): 189-e42, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28090706

ABSTRACT

BACKGROUND: Food allergy is often suspected in dogs with clinical signs of atopic dermatitis. This diagnosis is confirmed with an elimination diet and a subsequent challenge with regular food. Laboratory tests for the diagnosis of food allergy in dogs are unreliable and/or technically difficult. Cyno-DIAL® is a Western blot method that might assist with the selection of an appropriate elimination diet. HYPOTHESIS/OBJECTIVES: To evaluate the performance of Cyno-DIAL® for the selection of an elimination diet and diagnosis of food allergy. ANIMALS/METHODS: Thirty eight dogs with atopic dermatitis completed an elimination diet. Combining the results of the diet trials and the challenges, 14 dogs were classified as food allergic (FA), 22 as nonfood-allergic and two as ambiguous cases. RESULTS: Amongst all dogs and amongst dogs with a clinical diagnosis of FA, 3% and 7% (respectively) were positive to Royal Canin Anallergenic® , Vet-Concept Kanguru® or Vet-Concept Dog Sana® ; 8% and 7% to Hill's d/d Duck and Rice® ; 8% and 21% to Hill's z/d Ultra Allergen Free® ; 53% and 64% to Eukanuba Dermatosis FP® ; and 32% and 43% to a home-cooked diet of horse meat, potatoes and zucchini. The specificity and sensitivity of Cyno-DIAL® for diagnosing food allergy were 73% and 71%, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Although Cyno-DIAL® was considered potentially useful for identifying appropriate foods for elimination diet trials, it cannot be recommended for the diagnosis of food allergy. The Cyno-DIAL® test performed better than some previously evaluated ELISA-based tests.


Subject(s)
Blotting, Western/veterinary , Dog Diseases/diagnosis , Food Hypersensitivity/veterinary , Animal Feed/analysis , Animals , Diet/veterinary , Dog Diseases/immunology , Dogs , Female , Food Hypersensitivity/diagnosis , Food Hypersensitivity/immunology , Immunoglobulin E/immunology , Male
5.
J Biol Chem ; 283(37): 25650-25659, 2008 Sep 12.
Article in English | MEDLINE | ID: mdl-18644789

ABSTRACT

The lipolysis-stimulated lipoprotein receptor, LSR, is a multimeric protein complex in the liver that undergoes conformational changes upon binding of free fatty acids, thereby revealing a binding site (s) that recognizes both apoB and apoE. Complete inactivation of the LSR gene is embryonic lethal in mice. Here we show that removal of a single LSR allele (LSR(-/+)) caused statistically significant increases in both plasma triglyceride and cholesterol levels, a 2-fold increase in plasma triglyceride changes during the post-prandial phase, and delayed clearance of lipid emulsions or a high fat meal. The longer postprandial lipoprotein clearance time observed in LSR(-/+) mice was further increased in LSR(-/+) mice lacking functional low density lipoprotein (LDL) receptors. LSR(-/+) mice placed on a Western-type diet displayed higher plasma triglycerides and cholesterol levels, increased triglyceride-rich lipoproteins and LDL, and increased aorta lipid content, as compared with control mice on the same diet. Furthermore, a direct correlation was observed between the hyperlipidemia and weight gain but only in the LSR(-/+) mice. Knockdown of LSR expression by small interfering RNA in mouse Hepa1-6 cells led to decreased internalization of both DiI-labeled cyclohexanedione-LDL and very low density lipoprotein in the presence of oleate. These data led us to conclude that LSR contributes to the physiological clearance of atherogenic triglyceride-rich lipoproteins and LDL. We propose that LSR cooperates with the LDL receptor in the final hepatic processing of apoB-containing lipoproteins and represents a novel therapeutic target for the treatment of hyperlipidemia associated with obesity and atherosclerosis.


Subject(s)
Atherosclerosis/metabolism , Hyperlipidemias/metabolism , Lipolysis , Receptors, Lipoprotein/metabolism , Alleles , Animals , Cyclohexanes/chemistry , Heterozygote , Lipoproteins, LDL/chemistry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Biological , Obesity , Weight Gain
6.
Int Arch Allergy Immunol ; 146(3): 212-8, 2008.
Article in English | MEDLINE | ID: mdl-18268389

ABSTRACT

BACKGROUND: A model of peanut food allergy has been developed in mice using a simple sensitization protocol leading to a quantitatively measurable allergic response. METHODS: C3H/HeJ mice received a single intragastric administration of whole peanut (80 mg) without adjuvant. Two weeks later, intraperitoneal challenge with peanut extract led to a severe anaphylaxis. RESULTS: Anaphylactic reaction was evidenced by vascular leakage, severe clinical symptoms, a drop in body temperature, a decrease in breathing rate and also by increased concentrations of serum mouse mast cell protease-1. Sensitization to peanut was demonstrated by positive skin tests (ear swelling test and intradermal skin testing) and increased peanut-specific IgE levels. CONCLUSIONS: Thus, we obtained a model of severe peanut hypersensitivity within 2 weeks following single oral exposure without adjuvant. This model may be useful for further basic and applied studies on peanut allergy.


Subject(s)
Anaphylaxis/immunology , Immunoglobulin E/immunology , Peanut Hypersensitivity/immunology , Animals , Body Temperature/immunology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Immunization , Immunoblotting , Immunoglobulin E/blood , Mice , Mice, Inbred C3H , Respiration/immunology , Skin Tests , Specific Pathogen-Free Organisms
7.
Proc Natl Acad Sci U S A ; 104(18): 7522-7, 2007 May 01.
Article in English | MEDLINE | ID: mdl-17452638

ABSTRACT

Virtually all cancer biological attributes are heterogeneous. Because of this, it is currently difficult to reconcile results of cancer transcriptome and proteome experiments. It is also established that cancer somatic mutations arise at rates higher than suspected, but yet are insufficient to explain all cancer cell heterogeneity. We have analyzed sequence variations of 17 abundantly expressed genes in a large set of human ESTs originating from either normal or cancer samples. We show that cancer ESTs have greater variations than normal ESTs for >70% of the tested genes. These variations cannot be explained by known and putative SNPs. Furthermore, cancer EST variations were not random, but were determined by the composition of the substituted base (b0) as well as that of the bases located upstream (up to b - 4) and downstream (up to b + 3) of the substitution event. The replacement base was also not randomly selected but corresponded in most cases (73%) to a repetition of b - 1 or of b + 1. Base substitutions follow a specific pattern of affected bases: A and T substitutions were preferentially observed in cancer ESTs. In contrast, cancer somatic mutations [Sjoblom T, et al. (2006) Science 314:268-274] and SNPs identified in the genes of the current study occurred preferentially with C and G. On the basis of these observations, we developed a working hypothesis that cancer EST heterogeneity results primarily from increased transcription infidelity.


Subject(s)
Cell Transformation, Neoplastic/genetics , Expressed Sequence Tags , Genetic Variation/genetics , Neoplasms/genetics , Base Sequence , Humans , RNA, Messenger/genetics , Vimentin/genetics
9.
FEBS J ; 273(7): 1362-72, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16689925

ABSTRACT

Benzo[a]pyrene (B[a]P) is a common food pollutant that causes DNA adduct formation and is carcinogenic. The report of a positive correlation between human plasma B[a]P levels and body mass index, together with B[a]P's lipophilicity, led us to test for possible adverse effects of B[a]P on adipose tissue. In ex vivo experiments using primary murine adipocytes, B[a]P rapidly (within minutes) and directly inhibited epinephrine-induced lipolysis (up to 75%) in a dose-dependent manner. Half-maximum inhibition was obtained with a B[a]P concentration of 0.9 mg.L(-1) (3.5 microm). Lipolysis induced by beta(1)-, beta(2)- and beta(3)-adrenoreceptor-specific agonists, as well as ACTH, were also significantly inhibited by B[a]P, whereas forskolin-induced lipolysis was not B[a]P-sensitive. Similar inhibition of catecholamine-induced lipolysis by B[a]P was also seen in isolated human adipocytes; half-maximum inhibition of lipolysis was achieved with a B[a]P concentration of 0.02 mg.L(-1) (0.08 microm). In vivo treatment of C57Bl/6J mice with 0.4 mg.kg(-1) B[a]P inhibited epinephrine-induced release of free fatty acids by 70%. Chronic exposure of mice to B[a]P (0.5 mg.kg(-1) injected i.p. every 48 h) for 15 days also decreased lipolytic response to epinephrine and induced a 43% higher weight gain compared with controls (B[a]P: 2.23 +/- 0.12 g versus control: 1.56 +/- 0.18 g, P < 0.01) due to increased fat mass. The weight gain occurred consistently without detectable changes in food intake. These results reveal a novel molecular mechanism of toxicity for the environmental pollutant B[a]P and introduce the notion that chronic exposure of human population to B[a]P and possibly other polycyclic aromatic hydrocarbons could have an impact on metabolic disorders, such as obesity.


Subject(s)
Adipose Tissue/metabolism , Benzo(a)pyrene/metabolism , Receptors, Adrenergic, beta/metabolism , Weight Gain , Adipose Tissue/cytology , Adipose Tissue/drug effects , Adrenergic beta-Agonists/metabolism , Adrenergic beta-Agonists/pharmacology , Animals , Benzo(a)pyrene/pharmacology , Body Mass Index , Body Weight , Epinephrine/metabolism , Epinephrine/pharmacology , Fatty Acids, Nonesterified/blood , Food Contamination , Humans , Male , Mice , Mice, Inbred C57BL
10.
J Biol Chem ; 281(1): 229-40, 2006 Jan 06.
Article in English | MEDLINE | ID: mdl-16234245

ABSTRACT

A growing body of evidence supports the notion that soluble oligomeric forms of the amyloid beta-peptide (Abeta) may be the proximate effectors of neuronal injuries and death in the early stages of Alzheimer disease. However, the molecular mechanisms associated with neuronal apoptosis induced by soluble Abeta remain to be elucidated. We recently demonstrated the involvement of an early reactive oxygen species-dependent perturbation of the microtubule network (Sponne, I., Fifre, A., Drouet, B., Klein, C., Koziel, V., Pincon-Raymond, M., Olivier, J.-L., Chambaz, J., and Pillot, T. (2003) J. Biol. Chem. 278, 3437-3445). Because microtubule-associated proteins (MAPs) are responsible for the polymerization, stabilization, and dynamics of the microtubule network, we investigated whether MAPs might represent the intracellular targets that would enable us to explain the microtubule perturbation involved in soluble Abeta-mediated neuronal apoptosis. The data presented here show that soluble Abeta oligomers induce a time-dependent degradation of MAP1A, MAP1B, and MAP2 involving a perturbation of Ca2+ homeostasis with subsequent calpain activation that, on its own, is sufficient to induce the proteolysis of isoforms MAP2a, MAP2b, and MAP2c. In contrast, MAP1A and MAP1B sequential proteolysis results from the Abeta-mediated activation of caspase-3 and calpain. The prevention of MAP1A, MAP1B, and MAP2 proteolysis by antioxidants highlights the early reactive oxygen species generation in the perturbation of the microtubule network induced by soluble Abeta. These data clearly demonstrate the impact of cytoskeletal perturbations on soluble Abeta-mediated cell death and support the notion of microtubule-stabilizing agents as effective Alzheimer disease drugs.


Subject(s)
Amyloid beta-Peptides/toxicity , Apoptosis/drug effects , Microtubule-Associated Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Peptide Fragments/toxicity , Animals , Apoptosis/physiology , Calcium/metabolism , Calpain/metabolism , Caspase 3 , Caspase 9 , Caspases/metabolism , Cells, Cultured , Cerebral Cortex/cytology , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Homeostasis/drug effects , Isomerism , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/chemistry , Oxidative Stress/physiology
11.
Eur J Biochem ; 271(15): 3103-14, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15265030

ABSTRACT

The lipolysis stimulated receptor (LSR) recognizes apolipoprotein B/E-containing lipoproteins in the presence of free fatty acids, and is thought to be involved in the clearance of triglyceride-rich lipoproteins (TRL). The distribution of LSR in mice was studied by Northern blots, quantitative PCR and immunofluorescence. In the adult, LSR mRNA was detectable in all tissues tested except muscle and heart, and was abundant in liver, lung, intestine, kidney, ovaries and testes. During embryogenesis, LSR mRNA was detectable at 7.5 days post-coitum (E7) and increased up to E17 in parallel to prothrombin, a liver marker. In adult liver, immunofluorescence experiments showed a staining at the periphery of hepatocytes as well as in fetal liver at E12 and E15. These results are in agreement with the assumption that LSR is a plasma membrane receptor involved in the clearance of lipoproteins by liver, and suggest a possible role in steroidogenic organs, lung, intestine and kidney). To explore the role of LSR in vivo, the LSR gene was inactivated in 129/Ola ES cells by removing a gene segment containing exons 2-5, and 129/Ola-C57BL/6 mice bearing the deletion were produced. Although heterozygotes appeared normal, LSR homozygotes were not viable, with the exception of three males, while the total progeny of genotyped wild-type and heterozygote pups was 345. Mortality of the homozygote embryos was observed between days 12.5 and 15.5 of gestation, a time at which their liver was much smaller than that of their littermates, indicating that the expression of LSR is critical for liver and embryonic development.


Subject(s)
Embryo Loss/genetics , Embryo, Mammalian/metabolism , Gene Deletion , Gestational Age , Receptors, LDL/deficiency , Receptors, LDL/metabolism , Animals , Blotting, Northern , Embryo Loss/pathology , Embryo, Mammalian/abnormalities , Female , Fluorescent Antibody Technique , Genotype , Kidney/embryology , Kidney/metabolism , Liver/cytology , Liver/embryology , Liver/metabolism , Liver/pathology , Mice , Mice, Knockout , Pregnancy , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, LDL/genetics , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
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