Les neutrophiles constituent une première ligne de défense contre les agents infectieux. En revanche, leur activation incontrôlée peut exacerber certaines pathologies inflammatoires telles que les allergies cutanées. Notre équipe a montré précédemment que le facteur de transcription Nrf2 connu pour son rôle anti-oxydant, régulait l’inflammation cutanée dans l’hypersensibilité de contact (HSC). Ainsi ce travail a été mené pour évaluer in vitro l’implication de la voie Nrf2 dans les fonctions des neutrophiles et pour identifier son rôle dans le recrutement et l’activation des neutrophiles dans l’HSC.In vitro, nous montrons que la protéine Nrf2 est fortement exprimée dans les neutrophiles de la moelle osseuse. Nrf2 est fonctionnelle dans les neutrophiles stimulés : il active la transcription de gènes cibles cytoprotecteurs et diminue celle des gènes de l’inflammation. Ainsi, le prétraitement des neutrophiles avec un activateur de Nrf2 tel que le sulforaphane, réduit la production des formes réactives de l’oxygène (FRO)en réponse à une stimulation. En parallèle, l’absence de Nrf2 ne semble pas affecter la phagocytose et la nétose, deux fonctions clés du neutrophile. Enfin, Nrf2 est indispensable pour une migration optimale des neutrophiles en réponse aux chimiokines.Au cours de l’HSC induite par le dinitrochlorobenzène (DNCB), Nrf2 régule indirectement le recrutement des neutrophiles, en contrôlant le stress oxydant cutané et les voies inflammatoires impliquées dans la production de chimiokines, notamment CCL2, CCL4 et CCL11. En outre, Nrf2 induit l’augmentation d’expression du scavenger CD36 dans les macrophages et augmente ainsi leur capacité à éliminer les neutrophiles apoptotiques pour initier la résolution de l’inflammation.En conclusion, l’activation de Nrf2 dans les neutrophiles participe au contrôle de la production des FRO et la migration. En outre, Nrf2 émerge comme un effecteur clé dans le contrôle du recrutement et de la clairance des neutrophiles au cours de la réponse inflammatoire cutanée aux molécules allergisantes. La mise en évidence de ces mécanismes protecteurs de Nrf2 nous permet de proposer cette protéine comme nouvelle cible thérapeutique dans le contrôle d’inflammations cutanées chroniques. / Neutrophils form the first line of defense against infectious agents. However, their uncontrolled activation may exacerbate certain inflammatory conditions such as cutaneous allergies. Our team has previously shown that Nrf2 transcription factor known for its antioxidant role, regulates skin inflammation in contact hypersensitivity (CHS). Thus, our work was carried out to evaluate in vitro the involvement of Nrf2 pathway in neutrophil functions and to identify Nrf2 role in neutrophil recruitment and activation in CHS.In vitro, we showed that the protein Nrf2 was highly expressed in bone marrow neutrophils. Nrf2 is functional in stimulated neutrophils: it activates the transcription of cytoprotective genes and downregulates that of inflammatory genes. Thus, pretreatment of neutrophils with an Nrf2 activator such as sulforaphane reduces the production of reactive oxygen species (ROS) in response to stimulation. In parallel, Nrf2 does not affect two key functions of neutrophil, phagocytosis and netosis.Finally, Nrf2 is essential for optimal migration of neutrophils toward chemokines. In CHS induced by the dinitrochlorobenzene (DNCB), Nrf2 indirectly regulates the recruitment of neutrophils, through regulation of skin oxidant stress and inflammatory pathways that are involved in chemokines production, including CCL2, CCL4 and CCL11. In addition, Nrf2 induces the up-regulation of scavenger CD36 in macrophages and thus increases their ability to eliminate apoptotic neutrophils leading to the resolution of inflammation.In conclusion, Nrf2 activation in neutrophils participates in the control of ROS production and migration. In addition, Nrf2 emerges as an important effector in the control of neutrophil recruitment and clearance during the skin inflammatory response to allergenic molecules. The demonstration of Nrf2 protective mechanisms leads us to suggest this protein as a new therapeutic target in the control of chronic skin inflammations.

Indiana University-Purdue University Indianapolis (IUPUI)

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13557号 / 論医博第2286号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 伊藤 能永, 教授 森信 暁雄 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Abstract : Adverse drug reactions (ADRs) are undesirable effects caused after administration of a single dose or prolonged administration of drug or result from the combination of two or more drugs. Idiosyncratic drug reaction (IDR) is an adverse reaction that does not occur in most patients treated with a drug and does not involve the therapeutic effect of the drug. IDRs are unpredictable and often life-threatening. Idiosyncratic reaction is dependent on drug chemical characteristics or individual immunological response. IDRs are a major problem for drug development because they are usually not detected during clinical trials. In this study we focused on IDRs of Nevirapine (NVP), which is a non-nucleoside reverse transcriptase inhibitor used for the treatment of Human Immunodeficiency Virus (HIV) infections. The use of NVP is limited by a relatively high incidence of skin rash. NVP also causes a rash in female Brown Norway (BN) rats, which we use as animal model for this study. Our hypothesis is that idiosyncratic skin reactions associated with NVP treatment are due to post-translational modifications of proteins (e.g., glutathionylation) detectable by MS. The main objective of this study was to identify the proteins that are targeted by a reactive metabolite of Nevirapine in the skin. The specific objectives derived from the general objective were as follow: 1) To implement the click chemistry approach to detect proteins modified by a reactive NVP-Alkyne (NVP-ALK) metabolite. The purpose of using NVP-ALK was to couple it with Biotin using cycloaddition Click Chemistry reaction. 2) To detect protein modification using Western blotting and Mass Spectrometry techniques, which is important to understand the mechanism of NVP induced toxicity. 3) To identify the proteins using MASCOT search engine for protein identification, by comparing obtained spectrum from Mass Spectrometry with theoretical spectrum to find a matching peptide sequence. 4) To test if the drug or drug metabolites can cause harmful effects, as the induction of oxidative stress in cells (via protein glutathionylation). Oxidative stress causes cell damage that mediates signals, which likely induces the immune response. The results showed that Nevirapine is metabolized to a reactive metabolite, which causes protein modification. The extracted protein from the treated BN rats matched 10% of keratin, which implies that keratin was the protein targeted by the NVP-ALK. / Résumé : Les effets indésirables (EI) sont les effets indésirables causés après l'administration d'une dose unique ou une administration prolongée du médicament ou le résultat de la combinaison de deux médicaments ou plus. La Réaction idiosyncratique (IDR) est une réaction indésirable qui ne se produit pas dans la plupart des patients traités avec un médicament et qui ne comporte pas l'effet thérapeutique du médicament. IDR sont imprévisibles et peuvent mettre la vie du malade en danger. Cette réaction dépend des caractéristiques chimiques du médicaments et/ou de la réponse immunitaire individuelle du patient. IDR est un problème majeur pour le développement de médicaments car ils ne sont généralement pas détectés au cours des essais cliniques. Dans cette étude, nous nous sommes concentrés sur la Réaction idiosyncratique de névirapine (NVP) qui est un inhibiteur de transcriptase inverse non nucléosidique utilisé pour le traitement du virus d'immunodéficience humaine (VIH). L'utilisation de NVP est limitée par une incidence relativement élevée d'éruption cutanée. NVP provoque également une éruption cutanée chez les rats femelles de souche Brown Norway. Notre étude vise à mieux comprendre les IDRs induites par l'administration de NVP chez l'animal. La présente étude vise à vérifier l'hypothèse que les problèmes cutanés associés à la prise de NVP soient attribuables à la modification post-traductionnelle de protéines détactable par spectrométrie de masse. Les principaux objectifs de ce projet étaient : 1) Déterminer si la Nevirapine alcynes (NVP-ALK), un analogue de la NVP peut développer la même éruption cutanée que la NVP. La NVP-ALK a été couplé avec de la biotine en utilisant la réaction chimique (click chemistry). 2) Détecter les modifications post-traductionelles des proteines par Western blot et des techniques de spectrométrie de masse, pour comprendre le mécanisme de la toxicité induite par la NVP. 3) Identifier les protéines modifiées en utilisant le moteur de recherche MASCOT pour l'identification des protéines, en comparant le les spectres de masse obtenus avec les spectres théoriques pour trouver une séquence correspondante de peptide. 4) Tester si la NVP et ses métabolites peuvent provoquer des effets nocifs, comme l'induction d'un stress oxydatif dans les cellules (par la mesure de la glutathionylation des protéines). Les résultats ont montré que la névirapine est métabolisé en métabolite réactif ce qui provoque une modification de la kératine. Ainsi nos résultats suggèrent que la kératine est la cible des métabolites de la NVP-ALK.

BACKGROUND: Centres in Phases I and III of the International Study of Asthma and Allergies in Childhood (ISAAC) programme used the method of consent (passive or active) required by local ethics committees. METHODS: Retrospectively, relationships between achieved response rates and method of consent for 13-14 and 6-7-year-olds (adolescents and children, respectively), were examined between phases and between English and non-English language centres. RESULTS: Information was obtained for 113 of 115 centres for adolescents and 72/72 centres for children. Both age groups: most centres using passive consent achieved high response rates (>80% adolescents and >70% children). English language centres using active consent showed a larger decrease in response rate. Adolescents: seven centres changed from passive consent in Phase I to active consent in Phase III (median decrease of 13%), with five centres showing lower response rates (as low as 34%). Children: no centre changed consent method between phases. Centres using active consent had lower median response rates (lowest response rate 45%). CONCLUSION: The requirement for active consent for population school-based questionnaire studies can impact negatively on response rates, particularly English language centres, thus adversely affecting the validity of the data. Ethics committees need to consider this issue carefully. / Revisión por pares

Os antibióticos beta-lactâmicos, principalmente as aminopenicilinas (ampicilina e amoxicilina) são causa freqüente de exantema maculo papular e urticária tardia em pacientes tratados com estes medicamentos. Entretanto, a falta de métodos para diagnóstico destas reações constitui uma lacuna na abordagem destes indivíduos. Objetivos: Os objetivos deste estudo foram determinara a freqüência e significância de testes cutâneos positivos em pacientes com história de exantema maculo-papular e urticária tardias induzidos por antibióticos beta-lactâmicos e determinar a sensibilidade e especificade dos testes diagnósticos in vitro para o diagnóstico de alergia a drogas: proliferação linfocitária e dosagem de citocinas no sobrenadante da cultura linfocitária.. Métodos: Os pacientes foram submetidos à dosagem de IgE específica para os seguintes determinantes: benzilpeniciloil, amoxiciloil e ampiciloil. Testes cutâneos de puntura e intradérmico com benzilpeniciloil polilisina, mistura de determinantes menores da penicilina, benzilpenicilina (10.000 UI/ml), amoxicilina (2 mg/ml) e ampicilina (2mg/ml) foram realizados em todos os pacientes com leituras após 20 minutos, 48 horas e 72 horas. Foi realizado teste de contato com os mesmos medicamentos (amoxicilina 5%, ampicilina 5% e penicilina G 5000UI/g de vaselina sólida). Todos os testes foram feitos em duplicata com controles positivo e negativo. Amostras de sangue foram coletadas para a proliferação linfocitária (incorporação de timidina triciada) e dosagem de citocinas no sobrenadante da cultura (IL-5, IFN-gama e TNF- 17 alfa) através de ELISA. Resultados: Foram estudados 20 pacientes e 15 controles. Nenhum dos pacientes ou controles apresentou teste cutâneo de leitura imediata positivo. Três pacientes reagiram positivamente aos testes cutâneos de leitura tardia nas leituras de 48 e 72 horas. Não houve nenhum resultado positivo dos testes cutâneos no grupo controle. A proliferação linfocitária mostrou-se positiva em somente 2 controles (13%) e 10 pacientes (50%). A sensibilidade dos experimentos de proliferações linfocitária variou entre 25% com penicilina na concentração de 250 ?g/ml até 55% com ampicilina 1000 ?g/ml. Sua especificidade foi de 80% a 100%. Conclusão: os testes cutâneos de leitura tardia não constituíram métodos diagnósticos eficazes para as reações tardias aos antibióticos beta-lactâmicos. A proliferação linfocitária apresentou baixa sensibilidade, porém, alta especificidade para o diagnóstico das mesmas reações. A detecção de IFN-gama e IL-5 evidenciaram alta especificidade e baixa sensibilidade, enquanto a dosagem de TNF-alfa mostrou-se altamente sensível (95%), porém, pouco específica (40%) / The beta-lactam antibiotic, mainly aminopenicillins: ampicillin (AMP) and amoxicillin (AMX), are frequently cited as causes of morbiliforme eruptions and late onset urticaria. A major problem with existing data on beta-lactam reactions of the delayed type includes a lack of standardized testing methods. Objectives: The goal of this study is to establish the frequency and significance of positive skin testing in patients with history of beta-lactam induced maculo-papular exanthema and late onset urticaria and the sensibility/specificity of in vitro tests for drug allergy investigation: lymphocyte proliferation assay and cytokine detection. Methods: Patients were submitted to complete penicillin (PEN) allergy test: specific detection for serum IgE antibody to benzyl penicilloyl polylysine, amoxicilloyl polylysine and ampicilloyl polylysine; puncture and intradermal skin test with benzyl penicilloyl polylysine and minor determinants mixture, benzylpenicillin (10.000 UI/ml), amoxicillin (2mg/ml), ampicillin (2mg/ml) were performed and read at 20 minutes, 48hours and 72 hours. All patients had epicutaneous patch testing done with the same drugs (5% amoxicillin, 5% ampicillin and 5000 IU/g penicillin G all in petrolatum). All tests were performed in duplicate with positive and negative controls. All subjects had blood collected for lymphocyte proliferation assay (H3-thymidine incorporation assay) and IL-5, TNF-alpha and IFN-gamma profile were analyzed in culture supernatant by ELISA. Results: None of the patients or controls had positive immediated skin test. Only three patients reacted positively to the 48 and 72 hours 15 reading of intradermal skin test and patch test with the same reagents. There was no positive skin test result among the control group. The lymphocyte proliferation test (LTT) was positive in only two control subjects, while in the study group, 10 patients had positive proliferation tests (50%). The LTT sensitivity varied from 25% to 55% depending on the drug and concentration tested. The specificity ranged from 80% to 100%. IFN-gamma and IL-5 detection had a high specificity, but low sensitivity, while TNF-alpha showed high sensitivity (95%) and low specificity (40%)

O objetivo deste estudo foi o de avaliar os efeitos da irradiação laser de Er,Cr:YSGG em superfícies dentinárias no tratamento da hipersensibilidade dentinária cervical, em busca de um protocolo para ser aplicado clinicamente. Para o teste de permeabilidade, foram preparados 144 pré-molares, as coroas seccionadas e as raízes impermeabilizadas. Duas áreas de irradiação antagônicas foram delimitadas e livres da impermeabilização (controle e experimental). A seguir, os dentes foram subdivididos em dois sub-grupos, diferindo o condicionamento da superfície irradiada (EDTA 24% e ácido fosfórico 35%) e em grupos diferindo o protocolo:G1)Er:YAG, 60mJ/2Hz; G2)0,25W; G3)0,5W; G4)0,75W; G5)1W, G6)1,25W; G7)1,50W; G8)1,75W G9)2W. Após as irradiações, os espécimes foram imersos em azul de metileno 2% por 4 horas, e incluídos em resina epóxica para a realização dos cortes longitudinais. Estes tiveram suas imagens trabalhadas em computador para mensuração do nível de infiltração. Os dados foram submetidos à análise estatística. O Sub-grupo 1, condicionado com EDTA, apresentou diferenças entre os grupos sendo que as amostras irradiadas com Er:YAG demonstraram menor infiltração, diferindo estatisticamente dos grupos 3, 6 e 9. Os resultados do sub-grupo 2 mostraram que as médias das amostras irradiadas com Er:YAG tenderam a zero entretanto, não houve diferença entre os grupos. Na Fase B, fragmentos de dentina foram obtidos da região cervical de dentes humanos, preparados para análise em microscopia eletrônica de varredura. Morfologicamente, observou-se oclusão parcial dos túbulos dentinários após irradiação com Er:YAG e Er,Cr:YSGG nas condições de 0,25W e 0,50W. A partir de 0.75W, foi observado abertura dos túbulos e à medida que as energias aumentaram, foram observadas carbonização e fendas. De posse dos resultados dos trabalhos in vitro, foram selecionados os parâmetros para a aplicação e o acompanhamento clínico de 1 mês: G1)controle (placebo); G2)Er:YAG; G3)Er,Cr:YSGG 0,25W e G4)Er,Cr:YSGG 0,75W. Após a triagem, foram selecionados voluntários de acordo com critérios de exclusão e inclusão. O nível de sensibilidade de cada voluntário foi avaliado através da escala visual analógica de dor (VAS) com auxílio do ar da seringa tríplice 1 semana antes (Pré-1), 5 minutos anterior ao tratamento (Pré-2), após 5 minutos (Pós-1), 1 semana (Pós-2) e 1 mês após o tratamento (Pós-3). Os dados foram coletados e submetidos à análise estatística separadamente para os dois estímulos realizados: spray de ar e sonda exploradora. Tanto para o estímulo ar quanto para o estímulo sonda, não foram observadas diferenças estatisticamente significantes nas escalas Pré-1 e Pré-2, concluindo que os pacientes estavam padronizados e não houve efeito da profilaxia nos níveis de dor. Ao estímulo do spray de ar, nota-se uma redução nos níveis de dor na escala Pós-1, porém há uma estabilidade dos valores das escalas seguintes. O grupo 2 (Er:YAG) apresentou o menor nível de dor quando comparado aos outros grupos. Ao estímulo mecânico foi observado que o grupo 4 (Er,Cr:YSGG 0,50W) demonstrou o decréscimo de dor mais acentuado imediatamente após o tratamento, porém ao final do estudo, apresentou os maiores níveis de dor. Os grupos 1, 2 e 3 apresentam redução de dor, porém estatisticamente diferentes do grupo 4. Com base nos resultados apresentados e dentro dos limites e parâmetros estudados, pode-se concluir que nenhuma condição foi capaz de eliminar completamente a microinfiltração e diminuir a permeabilidade dentinária; porém o laser de Er:YAG e o laser de Er,Cr:YSGG 0,25W apresentaram resultados satisfatórios nos três estudos realizados. / The aim of the present study was to determine the correct parameters of the Er,Cr:YSGG laser in the treatment of cervical dentin hypersensitivity. This work was presented in 3 different stages. In the first one, it was evaluated the decrease in dentin permeability of dentinal tubules after Er,Cr:YSGG laser irradiation in radicular surfaces. Ninety premolars were prepared with its crows sectioned, and the roots completely impermeabilized. Two antagonist areas of irradiation were determined as control and experimental. Each surface was conditioned with EDTA 24% (sub-group1) and phosphoric acid 35% (sub-group2) and the selected parameters were: 1)Er:YAG, 60mJ, 2Hz, 4 irradiations of 20sec each, defocused mode; and groups 2 to 9 received irradiation with Er,Cr:YSGG laser, 20Hz, Z6 tip, 600µm diameter, defocus mode, 0% of air and water: 2)Er,Cr:YSGG 0,25W; 3) 0,5W; 4) 0,75W; 5) 1,0W; 6) 1,25W, 7) 1,50W, 8) 2W; 9) 2 W. After irradiation, samples were immersed in a solution of methylene blue for 4 hours, washed for 5 min and included in epoxy resin to allow longitudinal cuts. The images were digitalized and computed worked. Data was statistically analyzed. Although the samples irradiated with Er:YAG showed less microleakage, the sub-group 1 showed differences between the groups, statistically different from groups 3, 6 e 9. The results of the sub-group 2 showed that the mean values of the Er:YAG samples had a tendency to be negative, however, no differences were detected between the groups. In the second stage, cuts from the cervical area were obtained and prepared for scanning electron microscopy to evaluate the superficial morphology. It was observed the partial closure of dentinal tubules after the irradiation with Er:YAG and Er,Cr:YSGG laser in the 0.25 and 0.50W protocols. From the 0.75W until 2W, it was observed the dentinal tubules opened as the energy densities raised. With the results of the in vitro studies, the parameters were selected: G1)control; G2)Er:YAG; G3)Er,Cr:YSGG 0,25W e G4)Er,Cr:YSGG 0,75W. Thirty subjects, who met the entry criteria, were admitted into the study. The assessment method used to quantify sensitivity was the cold air syringe, recorded by the visual analogue scale (VAS), prior to treatment as baseline (Pre-1), immediately before (Pre-2) and immediately after (Post-1) the topical treatment, after 1 week (Post-2) and after 1 month (Post-3). Teeth were assigned to the 4 groups. After the follow-up, data was collected and submitted to statistical analysis for both kinds of stimulus: evaporative (air) and mechanical (probe). For both air and probe it was not observed differences among the Pre-1 and Pre-2 moments, for what it can be concluded that patients were standardized. Analyzing the evaporative stimulus, it can be observed the reduction in the pain level immediately after the treatment; however there was stability in the next values. Group 2 (Er:YAG) showed the least level of pain. Throughout the mechanical stimulus, it was observed that group 4 (Er,Cr:YSGG 0,50W) showed the most pronounced decrease of pain immediately after treatment, however, at the end of the study, the levels of pain increased. Groups 1, 2 e 3 showed reduction of pain, statistically different from the group 4. Based on the results and within the limits of this study, it can be concluded that none of the parameters and lasers studied were capable of eliminate the microleakage and decrease the dentin permeability, however, the Er:YAG and Er,Cr:YSGG 0,25W lasers showed satisfactory results in the three studies performed.

Les réactions d'hypersensibilité immédiate survenant au cours de l'anesthésie demeurent un sujet de préoccupation majeure pour les anesthésistes. Le médicament le plus fréquemment responsable d'une anaphylaxie sont les curares. Le but de ce travail est de préciser l'épidémiologie actuelle et d'identifier certains facteurs environnementaux susceptibles d'expliquer la fréquence élevée des réactions allergiques aux curares qui peuvent survenir en l'absence de toute exposition préalable. Dans la première partie de la thèse, une enquête nationale décrivant l'épidémiologie des réactions anaphylactiques peranesthésiques entre 2005 et 2007 en France. Elle a confirmé l'intérêt du dosage d'IgE spécifique anti-curare. La survenue de réactions d'hypersensibilité immédiate allergique à l'induction anesthésique lors d'une première exposition aux curares a conduit à évoquer l'existence d'une sensibilisation croisée avec des substances présentes dans l'environnement et possédant un ammonium quaternaire. Différentes hypothèses ont été proposées, notamment une exposition aux produits cosmétiques et/ou désinfectants, une exposition à des antigènes de type phosphorylcholine rencontrés dans les levures, ou certains parasites. Dans la deuxième partie de la thèse, deux populations professionnelles, des apprentis coiffeurs exposés à des agents chimiques contenant des ions ammoniums, et des boulanger-pâtissiers exposés à des allergènes de levures et de parasites, ont été comparées avec la population générale à la recherche de différences concernant la prévalence des anticorps IgE spécifique anti-curare. Au terme de ce travail, l'exposition à des produits cosmétiques chez les coiffeurs apparaît comme un facteur de risque de développer des anticorps IgE spécifique anti-curare, démontrant pour la première fois l'hypothèse du lien entre l'exposition répétée aux produits cosmétiques contenant l'ammonium quaternaire et la sensibilisation croisée aux curares / Immediate hypersensitivity reactions occurring during anesthesia remains a major concern for anesthesiologists. The drugs most frequently responsible for anaphylaxis are neuromuscular blocking agents (NMBAs). The purpose of this study is to determine the current epidemiology and to identify environmental factors that may explain the high frequency of allergic reactions to neuromuscular blocking agents that can occur without any prior exposure. In the first part of the thesis, a national survey describing the epidemiology of anaphylaxis during anesthesia between 2005 and 2007 in France was carried out. The value of testing specific IgE against NMBA was confirmed.The occurrence of immediate allergic hypersensitivity reaction when first exposure to an NMBA during induction led to evoke the existence of cross-sensitization with substances present in environment and having a quaternary ammonium compound. Different hypotheses have been proposed including exposure to cosmetics and / or disinfectants, exposure to antigens encountered in phosphorylcholine yeasts, or parasites, or exposure to a derivative opiate, pholcodine. In the second part of the thesis, two occupational populations, hairdressers apprentice exposed to chemical agents containing ammonium ions, baker and pastry maker exposed to allergens yeast and parasites were compared with the general population to search for differences regarding the prevalence of specific IgE antibody against NMBA. At the end of this work, hairdressers exposed to cosmetics agents appears to be a risk factor for developing specific IgE antibodies against NMBA and we demonstrated for the first time the hypothesis of the ross-sensitization between repeated exposures to cosmetics products containing quaternary ammonium and NMBAs

Les mécanismes physiopathologiques des hypersensibilités (HS) médicamenteuses ne sont que partiellement connus. Un terrain génétique favorisant est connu de longue date, mais peu de facteurs de risques sont formellement identifiés. A l’aide d’une approche par gènes candidats, nous avons évalué l’association entre des polymorphismes des gènes NOD1 et 2 et l’HS aux bétalactamines ; l’association entre plusieurs polymorphismes cytokiniques et différentes HS médicamenteuses. Enfin, nous avons utilisé une approche pangénomique à la recherche de gènes candidats au cours d’une HS spécifique, le DRESS. Parmi 368 cas et autant de contrôles italiens ainsi que 387 cas et 326 contrôles espagnols, nous avons mis en évidence une association entre l’un des polymorphismes de NOD2 et un faible risque d’HS immédiate aux bétalactamines chez les patients italiens, tandis qu’un autre polymorphisme de NOD2 était associé à un risque augmenté d’HS immédiate aux bétalactamines chez les patients espagnols. Aucune association avec le polymorphisme de NOD1 n’était identifiée. Parmi 118 patients et 236 contrôles, nous avons identifiés l’association entre les polymorphismes de l’IL1 (IL1-RN-A2 et IL1-[bêta] -511) et de l’IL10 (-592A) avec le risque de DRESS. Enfin, parmi 18 DRESS, l’analyse de puces CNV pangénomique nous a permis d’identifier des variations comportant les gènes KLRC2 et CESP1.Au total, nous avons pu démontrer l’implication de gènes modulant l’inflammation, la réponse antivirale ou le métabolisme des médicaments dans différentes HS médicamenteuses. Une confirmation à l’étage fonctionnelle de ces résultats est nécessaire / The physiopathology of drug hypersensitivity (HS) are only partially known. A genetic background for such drug allergy is still demonstrated but only few genes are identified. Using a candidate gene approach, we tested the association of NOD1 and 2 genes with betalactam HS and the association of several cytokines genes with some drug HS. Using a whole genome approach, we tried to discover new candidate gene for DRESS. Among 368 italian cases and controls and 387 spanish cases and 326 controls, we identified one polymorphism of NOD2 gene associated with a protective effect for italians and another polymorphism associated with higher risk of druh HS for spanians. No association with NOD1 polymorphims was identified. Among 118 cases and 236 controls, we noticed that IL1 polymorphisms (IL1-RN-A2 and IL1-? -511) and IL10 polymorphism (-592A) were associated with DRESS.Ending, among 18 DRESS, a whole-genome array let us identify variations containing KLRC2 and CESP1 genes. These studies demonstrate the implication of several genes involved in inflammation, antivirus response or drug metabolism in different drug HS.Fonctionnal studies are needed to confirm these results

O nim (Azadirachta indica) é conhecido na Ásia devido a várias propriedades biológicas conhecidas desde a antigüidade. Os estudos referentes à ação inseticida dessa planta restringem-se a análise de seus mecanismos de ação sobre insetos e também de seus efeitos sobre trabalhadores rurais que fazem uso de produtos a base de nim; não havendo, na literatura pesquisada, trabalhos relativos aos impactos causados sobre o sistema vegetal. As plantas, assim como outros organismos, possuem a capacidade de se defenderem contra ataque de patógenos. Uma das respostas desencadeadas pelo reconhecimento do patógeno pelas células vegetais é a reação de hipersensibilidade (RH), que envolve a morte imediata das células do sítio primário de infecção, oferecendo resistência ao crescimento do patógeno. A RH é caracterizada pela necrose dos tecidos onde primeiro se manifestou a infecção, e este processo de morte celular programada envolve uma série de sinais que ainda não estão completamente elucidados. Neste trabalho, foram estabelecidas as condições do meio de cultura de células de Rubus fruticosus para os estudos com extrato de sementes de nim, avaliado o efeito elicitor deste sobre a cultura. Foram obtidos extratos hidroalcoólicos E1 e E2 e suas respectivas frações lioflizadas, L1 e L2. Estes extratos apresentaram maior teor de açúcares e lipídeos em sua composição e revelaram potencial antioxidante. Detectou-se a presença de AZA-A em L1 e L2, por meio de CLAE, cujos teores foram de 5,03 e 1,1 mg/mL, respectivamente, com tempo de retenção em torno de 9,5 minutos, confirmado por meio de análises via espectrometria de massas. O extrato L2 foi fracionado nas frações L2 inicial e AZA2. O extrato L2, nas concentrações de 0,1; 0,5; 1 e 5 mg/mL, e destas frações AZA2 e L2 inicial nas proporções do extrato L2 nestas concentrações, elicitaram células de Rubus fruticosus. O extrato L2, nas concentrações de 0,1; 0,5; 1 e 5 mg/mL, e suas frações AZA2 e L2 inicial nas proporções do extrato L2 nestas concentrações, elicitaram células de Rubus fruticosus. As células de Rubus fruticosus (1,8g) foram incubadas em tampão citrato de sódio contendo o extrato L2 e as frações L2 inicial e AZA2, separadamente, até a concentração de 5 mg/mL, por 1h, em temperatura ambiente. Após este período, os compostos fenólicos, proteínas e açúcares redutores foram determinados no meio extracelular e intracelular por métodos colorimétricos. O efeito destas frações e do extrato L2, na produção de EROs em células intactas de Rubus fruticosus, foi analisado usando a sonda diacetato 2,7-diclorofluoresceína (LEE et al., 1999; MURATA et al., 2001). Os resultados obtidos indicam que AZA isolada não teve efeito sobre respostas de defesa. A fração L2 inicial teve aumento de fenólicos intracelulares, de açúcares redutores extracelulares e diminuição de EROs, com o aumento da concentração do elicitor, indicando potencial antioxidante e mecanismo de defesa. O extrato L2 também demonstrou potencial antioxidante e protetor das células com o aumento da concentração do elicitor, além de possuir ação inseticida. / The neem (Azadirachta indica) is known in Asia due to their several biological properties. The studies on the insecticide action of neem extracts have only been restrict to the insect mechanisms and their effects on rural workers; studies on the impact in the vegetable system are not available. The plants, like the other organisms, have the ability to self-defend against attack of patogens. The hypersensitive response (RH), a type of programmed cell death (PCD) in plants, is triggered by plant cells when they recognize the patogen, and is characterized by necrosis of tissues in the local region surrounding the infection; the signals involved are still not completely elucidated. The present study evaluated the effects of neem extracts in Rubus fruticosus cell. The powdered seeds were submitted to two consecutive extractions with ethanol:water (1:1, v/v) at room temperature for 10 minutes, yielding E1 and E2 fractions. The solvent was evaporated and the aqueous extracts were concentrated and lyophilized, resulting in two samples, L1 and L2. They are used for analyses by high performance liquid chromatograph (HPLC) in C-18 column (4.6 x 250 mm), with acetonitrile-water (4:6 v/v) as mobile phase, flow rate 1 mL/min, monitored at 214 nm. The principal compound of this fraction was azadirachtin (5.03 and 1.1 mg/mL, respectively), the retention time was 9.5 min; it was confirmed using mass spectrophotometry. The L2 extract was partially fractionated by high performance liquid chromatograph (HPLC) in semi preparative C-18 column. The main fractions, analyzed by colorimetric methods, ESI-MS, were L2 initial and AZA2. The Rubus fruticosus cells (18-21 days; 1.8 g) were incubated in sodium citrate buffer containing L2, L2 initial ans AZA2 at concentrations up to 5 mg/mL, for 1h, at room temperature. After this period, the phenolic compounds, proteins and reducing sugar were determined in the extracellular and intracellular medium by colorimetric methods. Also, the effects of these fractions over the production of reactive oxygen species (ROS) in intact cell of Rubus fruticosus, was analyzed using 2,7-dichloro-fluorescein diacetate. AZA2 had no effect on the defense response. The initial L2 fraction increased the phenolic compounds in the intracellular medium and the reducing sugars in the extracellular medium. The same fraction showed an inhibitory effect on ROS and also increased the concentration of the elicitor. These results indicate the antioxidant potential and protector effect of the L2 initial. The L2 extract also demonstrated antioxidant and protective potential of cells with the increase of the elicitor concentration. Therefore, in parallel with its insecticide action, the neem extract contributes to the self-defense ability of the plants.