Junctional Epidermolysis Bullosa in the German shorthaired pointer: a spontaneous model for Junctional Epidermolysis Bullosa in man

A Junctional Epidermolysis Bullosa (JEB), described in the German shorthaired pointer, is a remarkable spontaneous canine model for Junctional Epidermolysis Bullosa in man. In the German shorthaired pointer, JEB is caused by a homozygous substitution of the 1514C-T nucleotide. This variation in sequence results in a non-conservative change of 14 amino acids (505T –I) in domain I of laminin-5 α3. This non-conservative substitution modifies the hydrophobicity profile of the peptidic fragment, and therefore alters the stability of the binding between the α3 chain and the β3γ2 heterotrimer, which could result in a breakdown of the muted polypeptide. A fraction of muted α3 could be incorporated in laminin-5, modifying its function and hindering the extracellular cleavage of the γ2 chain. Genetic complementation studies were carried out. An MMLV retrovirus expressing the whole cDNA, muted and wild, of the α3 chain was developed to be transducted in the keratinocytes of JEB dogs (KJEB). This phenotypic transversion was successful and brought back a3 chain secretion, and thus the production of functional laminin-5 molecules. In addition, the adhesion capabilities of these transducted KJEB in cultures also returned. Finally, the reconstitution of canine JEB epithelia expressing a hybrid laminin-5 is now possible, and is currently used in studies on the fate of these ex vivo grafts in JEB dogs.Une Epidermolyse Bulleuse Jonctionnelle (EBJ) est décrite chez le Braque allemand et constitue un remarquable modèle canin de l'épidermolyse bulleuse jonctionnelle de l'Homme. Chez le Braque allemand, cette maladie est causée par une substitution homozygote du nucléotide 1514 C-T chez les chiens EBJ. Cette variation de séquence induit un changement non conservatif de 14 acides aminés (505T-I) dans le domaine I de la laminine a3. Cette substitution non conservative modifie le profil d'hydrophobicité du fragment peptidique et altère donc la stabilité d'association de la chaîne a3 avec l'hétérotrimère β3γ2. Ceci pourrait aboutir à une dégradation du polypeptide muté. Une fraction d'a3 mutée pourrait s'incorporer dans la laminine 5, en altérer le fonctionnement et entraver le clivage extra-cellulaire de la chaîne y2. Des expériences de complémentation génétique ont donc été réalisées. Pour cela, un rétrovirus MMLV exprimant l'ADNc entier muté et sauvage de la chaîne a3 a été construit pour être transduit dans les kératinocytes des chiens EBJ (KEBJ). Cette transversion phénotypique a été réussie et a permis aux KEBJ de sécréter de nouveau la chaîne a3 et ainsi, de produire des molécules de laminine 5 fonctionnelles. Par ailleurs, ces KEBJ transduits ont montré aussi la restauration de leur capacité d'adhésion en culture. Enfin, la reconstitution d'épithéliums canins EBJ exprimant une laminine 5 hybride est désormais possible et actuellement à l'origine d'études sur le devenir de ces greffes ex vivo chez des chiens atteints d'EBJ

Etablissement d'un modèle animal pour la thérapie génique des épidermolyses bulleuses jonctionnelles non létales

L'objectif de mon étude a été de mettre au point un modèle animal pour la thérapie génique cutanée. Nous avons identifié des chiens souffrant d'épidermolyse bulleuse jonctionnelle (EBJ), un groupe de génodermatoses autosomiques récessives causées par un défaut d'expression des composants des structures d'ancrage de l'épiderme au derme. Chez ces chiens, la maladie est causée par un défaut d'expression de la chaîne a3 de la laminine-5, le principal ligand d'adhésion des kératinocytes basaux. Une insertion intronique dans le gène codant pour la laminine a3 est à l'origine de la maladie, et aboutit à la production d'ARNm aberrants et à une diminution de la production de laminine-5 sauvage. La réversion phénotypique des kératinocytes canins a été réalisée par transduction rétrovirale de l'ADNc codant pour la chaîne a3 de type sauvage. Les kératinocytes transduits montrent une restauration de la capacité d'adhésion, de prolifération et de la clonogénicité. Des peaux artificielles générées in vitro à partir des kératinocytes transduits montrent une expression durable de la laminine-5 localisée à la jonction dermo-épidermique après la greffe sur des souris SCID. La greffe des épithéliums transgéniques autologues sur les chiens EBJ immunocompétents permettra d'évaluer l'éventuelle réponse immunitaire dirigée contre le produit du transgène. La laminine-5 et en particulier sa chaîne ?2 étant également impliquées dans la migration et l'invasion tumorale, nous avons procédé à la localisation chromosomique des gènes codant pour les trois chaînes de la laminine-5 chez le chien, et nous avons isolé et séquencé l'ADNc codant pour la chaîne ?2 canine.To prove the feasability and safety of gene therapy of mechanobullous diseases, we have characterized a breed of dogs suffering from a mild form of JEB, a devastating, untreatable inherited skin disease characterized by blistering and erosions of the skin. These dogs exhibit the clinical hallmarks of the human condition, associated with reduced expression and secretion of laminin-5, an heterotrimeric a3ß3?2 glycoprotein that is the principal adhesion ligand of basal keratinocytes. An intronic insertion was identified in the gene coding for the a3 chain of laminin-5 and lead to the synthesis of an aberrant messenger carrying a downstream PTC. Consequently, the JEB dog keratinocytes secrete low amounts of wild-type laminin-5. We achieved sustained expression of the transgene product by retroviral-mediated transduction of epidermal stem cells. The transduced keratinocytes displayed enhanced adhesion, proliferation and clonogenic potential, and deposited immunoreactive laminin-5 efficiently and permanently at the dermal-epidermal junction of in vitro reconstructed skin grafted into SCID mice. Our results set the basis for preclinical gene therapy on the first immunocompetent large animal model for an inherited skin disease. Expression of laminin-5 and particularly of its ?2 chain are also deregulated in number of human cancers. Because dog models of human cancers provide the opportunity to clarify the relationship between laminin-5 and tumor malignancy, we have isolated and characterized the cDNA of dog g2 chain. We have also determined the synthenic location of the dog laminin-5 loci on CFA7.NICE-BU Sciences (060882101) / SudocSudocFranceF

Technical variability of 2D gel electrophoresis – Application to soybean allergens

Two-dimensional gel electrophoresis (2-DE) technique is used as a performing technique to assess the variability of protein expression in crops, and especially soybean endogenous food allergens, which are a subset of proteins of interest for assessing whether genetically modified (GM) soybean has a different allergenic profile compared to its non-GM counterpart. On top of the biological variability of the 2-DE, which has already been studied by several laboratories, technical variability has to be evaluated. In this study, several sources of variability (number of gel replicates, protein extracts, study timings and operators) were assessed qualitatively and quantitatively on all detectable polypeptide spots as well as on food allergen spots. Results showed that the major source of variability was the number of gel replicates. Other sources were minor. This has a direct practical impact on the laboratory work as this supports the utilization of three or four gel replicates to get robust results. Furthermore, this implies that the study can be run over several days, and be performed by several trained operators, without impacting its reproducibility. Furthermore, 2-DE could detect a 2-fold change between two samples with an acceptable rate of false positives (below 7%). This level of sensitivity is acceptable in the context of safety assessment of GM soybean as the biological variability of proteins in soybean is higher than the technical variability shown in this study. Overall, the 2-DE technique is suitable for investigating endogenous food allergen variability between several soybean seeds, including GM and non-GM counterpart

Supplementary data on the characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, which confers herbicide tolerance, and on the compositional assessment of field grown MST-FGØ72-2 soybean expressing HPPD W336

Supplementary data are provided which are supportive to the research article entitled “Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean” (Dreesen et al., 2018) [1]. The conducted supplementary analyses include the characterization of additional Escherichia coli-produced HPPD W336 protein batches used as a surrogate in HPPD W336 safety studies, the assessment of potential glycosylation and monitoring of stability in simulated intestinal fluid and during heating of the HPPD W336 protein. Furthermore, data are provided on conducted field trials and subsequent compositional analysis in MST-FGØ72-2 soybean grain of compounds related to the tyrosine degradation pathway and the metabolism of homogentisate

Protease resistance of food proteins : a mixed picture for predicting allergenicity but a useful tool for assessing exposure

Background: Susceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios. Aim: To evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability. Methods: Four pairs of established allergens and their related non/weakly-allergenic counterparts (seed albumins, muscle tropomyosins, plant lipid transfer proteins [LTP] and collagens) plus fish parvalbumin, were subjected to nine combinations of pH (1.2-2.5-4.0) and pepsin-to-protein ratio (PPR: 10-1-0.1 U/µg) for pepsin digestion, followed by pancreatin digestion in the presence of bile salts. Digestion was monitored by SDS-PAGE in conjunction with Coomassie staining and immunoblotting using rabbit antisera and human IgE. Results: At pH 4.0 and at PPR 0.1 most proteins, both allergen and non-allergen, were highly resistant to pepsin. Under conditions known to favor pepsin proteolysis, the established major allergens Ara h 2, Pru p 3 and Pen a 1 were highly resistant to proteolysis, while the allergen Cyp c 1 was not. However, this resistance to pepsin digestion only made Ara h 2 and to a lesser extent Pen a 1 and Pru p 3 stand out compared to their non-allergenic counterparts. Largely irrespective of preceding pepsin digestion conditions, pancreatin digestion was very effective for all tested proteins, allergens and non-allergens, except for Cyp c 1 and bovine collagen. Conclusions: Sub-optimal pH, low pepsin-to protein ratio, and sequential pepsin and pancreatin digestion protocols do not improve the predictive value in distinguish allergens from non-allergens. Digestion conditions facilitating such distinction differ per protein pair