Benchmarking of BMDC assay and related QSAR study for identifying sensitizing chemicals.

The Bone-Marrow derived Dendritic Cell (BMDC) test is a promising assay for identifying sensitizing chemicals based on the 3Rs (Replace, Reduce, Refine) principle. This study expanded the BMDC benchmarking to various in vitro, in chemico, and in silico assays targeting different key events (KE) in the skin sensitization pathway, using common substances datasets. Additionally, a Quantitative Structure-Activity Relationship (QSAR) model was developed to predict the BMDC test outcomes for sensitizing or non-sensitizing chemicals. The modeling workflow involved ISIDA (In Silico Design and Data Analysis) molecular fragment descriptors and the SVM (Support Vector Machine) machine-learning method. The BMDC model's performance was at least comparable to that of all ECVAM-validated models regardless of the KE considered. Compared with other tests targeting KE3, related to dendritic cell activation, BMDC assay was shown to have higher balanced accuracy and sensitivity concerning both the Local Lymph Node Assay (LLNA) and human labels, providing additional evidence for its reliability. The consensus QSAR model exhibits promising results, correlating well with observed sensitization potential. Integrated into a publicly available web service, the BMDC-based QSAR model may serve as a cost-effective and rapid alternative to lab experiments, providing preliminary screening for sensitization potential, compound prioritization, optimization and risk assessment.

Identification of the allergenic sensitizing potential of bisphenol A substitutes used in the industry.

BACKGROUND: Bisphenol (BP-)A is a chemical used in Europe to produce polycarbonate plastics and epoxy resin or as colour developer in thermal paper. Due to its toxicity, BPA presence was restricted by European regulations. Therefore, substitute chemicals are replacing BPA. OBJECTIVE: To assess the allergenic sensitizing potential of 27 substitutes to BPA used in the industry. METHODS: The expression of two costimulatory molecules and six cytokines were analysed by flow cytometry in mouse bone marrow-derived dendritic cells (BMDCs) exposed to the chemicals. RESULTS: All substances except one induced overexpression of at least one receptor and were thus identified as having allergenic sensitizing potential. Based on the BMDC model, they were classified as extreme (1 out of 27), strong (20 out of 27) and moderate (5 out of 27) sensitizers. BPA was classified as a moderate sensitizer and BPF was the only substitute classified as a non-sensitizer. The more potent substitutes induced more than 2-fold secretion of CCL3, CCL4 and/or CCL5 by dendritic cells. CONCLUSION: Most of the BPA substitutes tested in this study have an allergenic sensitizing potential; 24 of them being more potent than BPA itself. Only BPE, BPF and 2,4-BPS appeared to be weaker sensitizers than BPA.

Transcriptional frameshifts contribute to protein allergenicity.

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.

Anaphylaxis to Beluga caviar.

Fish roe is an extremely rare cause of anaphylaxis and although its consumption has increased in recent years. We described the case of a 59-year-old man, who experienced an anaphylactic reaction after consuming caviar. Skin prick-test were performed with Beluga caviar, salmon caviar, cod, salmon, hen egg yolk and egg white, ovalbumin, ovomucoid, shrimp and mold. Only SPT to Beluga caviar was positive. The absence of sensitization to fish and hen egg was confirmed by undetectable specific IgEs to cod, parvalbumin (Gad c 1 and Cyp c 1), egg yolk and egg white, ovalbumin and ovomucoid. An immunoblot was also performed and showed an IgE-reactive band indicated that the patient was sensitized to a 26 kDa protein in Beluga caviar. In the present case, immunoblotting of the patient's serum revealed a single IgE-reactive band at 26 kDa band, which does not appear to correspond to the previous cases.

A pilot study of total and allergen-specific IgE serum levels during anestrous, estrous and pregnancy in healthy female dogs.

BACKGROUND: Allergen-specific IgE serology is used for the determination of sensitization status in dogs with atopic dermatitis; the influence of the female reproductive cycle on the results of such methods has not been studied in dogs. OBJECTIVES: To compare the total and allergen-specific IgE of healthy bitches during anestrous, estrous and pregnancy. ANIMALS: Eight privately owned, healthy bitches. METHODS: Total and allergen-specific IgE levels were determined in eight bitches at three different time-points of their reproductive cycle: anestrous, estrous and pregnancy. RESULTS: Total IgE was significantly decreased (median: 74%) in female dogs during pregnancy when compared to anestrous. In 14 of 216 (6%), allergen-specific IgE test results were variably positive and negative at different stages of the reproductive cycle. This variation, however, was not related to changes in total serum IgE levels. CONCLUSIONS: Total IgE serum levels are reduced during pregnancy in female dogs. However, results of one allergen-specific IgE test did not appear to be markedly altered by the reproductive cycle in healthy bitches.

Detection of IgE-reactive proteins in hydrolysed dog foods.

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.

Probable walnut-induced anaphylactic reaction in a dog.

BACKGROUND: Anaphylaxis due to nuts is frequent in humans; to the best of the authors' knowledge, it has not been reported previously in dogs. CASE REPORT: A 5-year-old female, intact, Vizsla dog was presented with acute diarrhoea, vomiting, respiratory distress and erythematous wheals. The dog had eaten walnuts, which she had been fed in small amounts for years, hours before the onset of clinical signs. A diagnosis of generalized anaphylaxis was made. Skin testing and Western blotting revealed positive results with walnuts and hazelnuts. CONCLUSIONS AND CLINICAL IMPORTANCE: This case report illustrates the need for a thorough food history and for recognition that a dog may experience severe allergic reactions to unusual and regularly fed food items. It also shows that allergen specific tests may help to confirm the diagnosis and help in planning the dog's future dietary regime.

Western blot analysis of sera from dogs with suspected food allergy.

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.

Role of RNA structure and protein factors in the control of HIV-1 splicing.

Alternative splicing plays a key role in the production of numerous proteins by complex lentiviruses such as HIV-1. The study of HIV-1 RNA splicing has provided useful information not only about the physiology of the virus, but also about the general mechanisms that regulate mammalian pre-mRNA alternative splicing. Like all retroviruses, a fraction of HIV-1 transcripts remains intact to serve as genomic RNA and to code for Gag and Gag-Pol protein precursors. In addition, splicing is important for controlling the production of some viral proteins, which could otherwise have a negative effect on the infected cell. Here, we summarize how the utilization of HIV-1 splicing sites is limited by the binding of nuclear factors to cis-acting silencer elements, taking into account the role of RNA secondary structure in these mechanisms. We also describe how the poorly efficient HIV-1 acceptor sites are nevertheless activated by serine/arginine-rich proteins. Finally, we discuss how nuclear factors that interact with both the transcription and splicing machineries also participate in the control of HIV-1 RNA splicing.

A single oral sensitization to peanut without adjuvant leads to anaphylaxis in mice.

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.

Nonrandom variations in human cancer ESTs indicate that mRNA heterogeneity increases during carcinogenesis.

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.

Predictive value of skin prick tests using recombinant allergens for diagnosis of peanut allergy.

BACKGROUND: Current diagnosis of peanut allergy relies on natural extracts that lack standardization. Recombinant DNA technology allows production of pure biochemically characterized proteins. Their usefulness for peanut allergy diagnosis is not established. OBJECTIVE: This study aimed to evaluate the diagnostic value of the 3 major recombinant peanut allergens. METHODS: Recombinant (r) Ara h 1, rAra h 2, and rAra h 3 were produced according to the recommendations of good manufacturing practice for recombinant allergens. Skin prick tests (SPTs) and IgE ELISA assays were performed in 30 patients with peanut allergy and 30 control subjects without food allergy: 15 nonatopic and 15 sensitized to birch pollen. Disease severity was graded by clinical scoring. RESULTS: All patients with peanut allergy showed positive SPT results to rAra h 2; 40% reacted with rAra h 1 and 27% with rAra h 3. No control subjects reacted with any of the recombinant allergens. Monosensitization to rAra h 2 was observed in 53% of patients. Neither SPT size nor levels of specific IgE were correlated with the disease severity. However, patients with monosensitization to rAra h 2 had a significantly lower severity score than polysensitized subjects and a lower level of specific IgE against peanut extract and rAra h 2. CONCLUSION: Skin prick tests to individual recombinant peanut allergens appear to be a safe and effective diagnostic tool. Cosensitization to rAra h 2 and rArah 1 and/or rAra h 3 is predictive of more severe reactions. CLINICAL IMPLICATIONS: Recombinant peanut allergens can be used by SPTs for diagnosis and evaluation of allergy severity.

Benzo[a]pyrene impairs beta-adrenergic stimulation of adipose tissue lipolysis and causes weight gain in mice. A novel molecular mechanism of toxicity for a common food pollutant.

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.

Dual effect of the SR proteins ASF/SF2, SC35 and 9G8 on HIV-1 RNA splicing and virion production.

In HIV-1 infected cells transcription of the integrated provirus generates the single full length 9 kb viral RNA, a major fraction of which is spliced to produce the single-spliced 4 kb RNAs and the multiple-spliced 2 kb RNAs. These spliced RNAs are the messengers for the Env glycoproteins and the viral regulatory factors. The cellular SR and hnRNP proteins were shown in vitro to control alternative splicing by binding cis-regulatory elements on the viral RNA. To better understand in vivo the role of the SR proteins on HIV-1 genomic RNA splicing and virion production, we used a human cell line expressing high levels of complete HIV-1 and either one of the ASF/SF2, SC35, and 9G8 SR proteins. Results show that over-expressing SR proteins caused a large reduction of genomic RNA and that each SR protein modified the viral 9 kb RNA splicing pattern in a specific mode. In fact, ASF/SF2 increased the level of Vpr RNA while SC35 and 9G8 caused a large increase in Tat RNA. As expected, overexpressing SR proteins caused a strong reduction of total Gag made. However, we observed by immuno-confocal microscopy an accumulation of Gag at the plasma membrane and in intracellular compartments while there is a dramatic reduction of Env protein made in most cells. Due to the negative impact of the SR proteins on the levels of genomic RNA and HIV-1 structural proteins much less virions were produced which retained part of their infectivity. In conclusion, SR proteins can down-regulate the late steps of HIV-1 replication.

The chaperoning and assistance roles of the HIV-1 nucleocapsid protein in proviral DNA synthesis and maintenance.

In the following three sections, we will briefly review the seminal roles of the HIV-1 nucleocapsid protein p7 (NCp7) in the fate of the HIV-1 full length RNA from genomic RNA in a dimeric form to the proviral DNA. Emphasis will be given to the mechanisms of NC-directed assistance to the genomic RNA and reverse transcriptase (RT) in the course of proviral DNA synthesis and to DNA integrity at the end of the polymerization process, and to the NC-assisted repair and recombination reactions fueling the viability and variability of the virus.

Differential effects of the SR proteins 9G8, SC35, ASF/SF2, and SRp40 on the utilization of the A1 to A5 splicing sites of HIV-1 RNA.

Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites.

The chaperoning and assistance roles of the HIV-1 nucleocapsid protein in proviral DNA synthesis and maintenance.

In the following three sections we will briefly review the seminal roles of the HIV-1 nucleocapsid protein NCp7 in the fate of the HIV-1 full length RNA from genomic RNA in a dimeric form to the proviral DNA. Emphasis will be given to the mechanisms of NC-directed assistance to the genomic RNA and reverse transcriptase in the course of proviral DNA synthesis and to DNA integrity at the end of the polymerization process, and to the NC-assisted repair and recombination reactions fueling the viability and variability of the virus.