Chronic myeloid leukemia (CML) is a disease characterized by the expansion of granulocytic cells. The BCR-ABL tyrosine kinase inhibitor imatinib, the frontline treatment for Ph+ leukemias, can induce complete hematologic and cytogenetic response in most chronic phase CML patients. Despite the remarkable initial clinic effects, it is now recognized that imatinib will unlikely cure patients because a small cell population containing leukemic stem cells (LSCs) with self-renewal capacity is insensitive to tyrosine kinase inhibitors. In Chapter I, I briefly review the BCR-ABL kinase and its related signaling pathways. BCR-ABL kinase activates several signaling pathways including MAPK, STAT, and JNK/SAPK. BCR-ABL also mediates kinase-independent pathways through SRC family kinases. I will also discuss pathways involving β-catenin, hedgehog, FoxO and Alox5 are critical to the regulation of self-renewal and differentiation in LSC of CML. As detailed in Chapter II, I describe our work evaluating the effects of omacetaxine, a novel CML drug inducing cell apoptosis by inhibition of protein synthesis, on self-renewal and differentiation of LSCs and BCR-ABL-induced CML and acute lymphoblastic leukemia (B-ALL) in mice. We found that treatment with omacetaxine decreased the number of LSCs and prolonged the survival of mice with CML or B-ALL. In chapter III, I describe that Alox5 is an essential gene in the function of LSCs and CML development. We show evidence that Alox5 affects differentiation, cell division, and survival of long-term LSCs. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly and prolonged survival. In chapter IV, I present evidence of our work showing a further dissection the Alox5 pathway by comparing the gene expression profiles of wild type and Alox5-/- LSCs. We show that Msr1 deletion causes acceleration of CML development. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and β-catenin. Taken together, these results demonstrate that some pathways including Alox5 and Msr1 play an important role in regulating the self-renewal and differentiation of LSC. More efforts should be put into developing the novel strategies that may effectively target LSCs and thus cure CML.

Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.

Fatty acid amide hydrolase (FAAH) is a widely conserved amidase in eukaryotes, best known for inactivating the signal of N-acylethanolamine (NAE) lipid mediators. In the plant Arabidopsis thaliana, FAAH-mediated hydrolysis of NAEs has been associated with numerous biological processes. Recently, the phylogenetic distribution of FAAH into two major branches (group I and II FAAHs) across angiosperms outside of Arabidopsis (and in other Brassicaceae), suggests a previously unrecognized complexity of this enzyme. Although A. thaliana has long been used to assess biological questions for plants, in this case it will fall short in understanding the significance of multiple FAAHs in other plant systems. Thus, in this study, I examined the role (s) of six FAAH isoforms in upland cotton (Gossypium hirsutum L.) and two FAAHs in the legume Medicago truncatula.

L’arthrose (OA) est une maladie dégénérative et multifactorielle caractérisée par une destruction de cartilage, une formation d’ostéophytes et une inflammation au niveau de la membrane synoviale. Le 4-hydroxynonénal (HNE), un produit final de la peroxydation lipidique, a été identifié récemment comme un facteur catabolique et un médiateur inflammatoire dans le cartilage arthrosique humain. Notre projet vise à étudier l’effet du HNE sur la régulation de la prostaglandine E2 synthase-1 microsomale (mPGES-1) et de la protéine activante 5-lipoxygénase (FLAP)/5-lipoxygénase (5-LOX) dans les chondrocytes arthrosiques humains. Lorsque les cellules sont traitées une seule fois avec 10 µM HNE, les résultats de Western blot et de PCR en temps réel montrent que l’expression de la cyclooxygénase-2 (COX-2) et de la mPGES-1 augmente de manière significative et atteint respectivement le maximum après 8 et 16 heures d’incubation puis diminue graduellement. Cependant, lorsque les cellules sont traitées plusieurs fois avec 10 µM HNE à 2 heures d’intervalle, l’expression de la COX-2 et de la mPGES-1 augmente en fonction du temps sans subir une baisse après 24 heures d’incubation. Le HNE induit l’activité du promoteur de la mPGES-1 via l’activation du facteur de transcription Egr-1. L’investigation de la 2ème voie du métabolisme de l’acide arachidonique, à savoir 5-LOX/FLAP, montre que le HNE induit l’expression de FLAP après 24 heures de stimulation et celle de 5-LOX seulement après 48 heures. Ceci semble survenir à l’étape de transcription au cours de laquelle HNE induit l’expression de l’ARNm et l’activité du promoteur du gène 5-LOX. Nous avons démontré aussi que le niveau de leukotriène B4 (LTB4) augmente et suit le même profil que celui de la 5-LOX. L’étude des mécanismes moléculaires susceptibles d’être impliqués dans la régulation de la 5-LOX/FLAP par le HNE montre que ce dernier stimule leur expression via l’action de prostaglandine E2 (PGE2) et du facteur de croissance transformant-beta 1 (TGF-β1). En conclusion, notre étude démontre que le HNE induit à court-terme d’incubation la voie de COX-2/mPGES-1 puis par la suite stimule celle de FLAP/5-LOX à long-terme d’incubation dans les chondrocytes arthrosiques humains. Ces résultats suggèrent que la mPGES-1 et 5-LOX/FLAP sont des potentielles cibles thérapeutiques intéressantes pour contrôler la production de PGE2 et LTB4 dans OA. / 4-hydroxynonenal (HNE), a lipid peroxidation end-product, is produced abundantly in osteoarthritic (OA) articular tissues. Recently, we reported that HNE-induced cyclooxygenase-2 (COX-2) decreased gradually in human OA chondrocytes after 8 h of incubation. This study aimed to investigate whether COX-2 down-regulation is attributed to HNE depletion and is responsible for the switch from COX-2 to 5-lipoxygenase-activating protein (FLAP)/5-lipoxygenase (5-LOX). Treatment of chondrocytes with 10 µM HNE induced prostaglandin E2 (PGE2) release as well as COX-2 and microsomal prostaglandin E2 synthase-1 (mPGES-1) expression at the protein and mRNA levels, with a plateau reached at 8-16 h of incubation, followed by a subsequent decline. However, 8 repeated treatments with 10 µM HNE prevented the reduction of COX-2 and mPGES-1 expression. We demonstrated that HNE induced mPGES-1 promoter activity mainly through transcription factor Egr-1 activation. On the other hand, when COX-2 expression decreased, leukotriene B4 (LTB4) level rose after a long period of stimulation (48 and 72 h). At the mRNA level, HNE induced FLAP and 5-LOX expression after 24 and 48 h of stimulation, respectively. The addition of a nonspecific COX-2 inhibitor (naproxen) to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that 10 µM HNE significantly induced transforming growth factor-beta 1 (TGF-β1) production. The addition of anti-TGF-β antibody to culture medium reduced HNE-induced 5-LOX/FLAP expression by 40%, indicating the involvement of a TGF-β1-dependent mechanism. Our data demonstrate that the shunt to the FLAP/5-LOX pathway in HNE-induced human OA chondrocytes is attributed to COX-2 inhibition, probably due to HNE depletion. PGE2 and TGF-β1 are suggested to be involved in this regulation. Further experiments are in progress to determine other molecular mechanisms underlying this switch in OA chondrocytes.

L’arthrose (OA) est une maladie dégénérative et multifactorielle caractérisée par une destruction de cartilage, une formation d’ostéophytes et une inflammation au niveau de la membrane synoviale. Le 4-hydroxynonénal (HNE), un produit final de la peroxydation lipidique, a été identifié récemment comme un facteur catabolique et un médiateur inflammatoire dans le cartilage arthrosique humain. Notre projet vise à étudier l’effet du HNE sur la régulation de la prostaglandine E2 synthase-1 microsomale (mPGES-1) et de la protéine activante 5-lipoxygénase (FLAP)/5-lipoxygénase (5-LOX) dans les chondrocytes arthrosiques humains. Lorsque les cellules sont traitées une seule fois avec 10 µM HNE, les résultats de Western blot et de PCR en temps réel montrent que l’expression de la cyclooxygénase-2 (COX-2) et de la mPGES-1 augmente de manière significative et atteint respectivement le maximum après 8 et 16 heures d’incubation puis diminue graduellement. Cependant, lorsque les cellules sont traitées plusieurs fois avec 10 µM HNE à 2 heures d’intervalle, l’expression de la COX-2 et de la mPGES-1 augmente en fonction du temps sans subir une baisse après 24 heures d’incubation. Le HNE induit l’activité du promoteur de la mPGES-1 via l’activation du facteur de transcription Egr-1. L’investigation de la 2ème voie du métabolisme de l’acide arachidonique, à savoir 5-LOX/FLAP, montre que le HNE induit l’expression de FLAP après 24 heures de stimulation et celle de 5-LOX seulement après 48 heures. Ceci semble survenir à l’étape de transcription au cours de laquelle HNE induit l’expression de l’ARNm et l’activité du promoteur du gène 5-LOX. Nous avons démontré aussi que le niveau de leukotriène B4 (LTB4) augmente et suit le même profil que celui de la 5-LOX. L’étude des mécanismes moléculaires susceptibles d’être impliqués dans la régulation de la 5-LOX/FLAP par le HNE montre que ce dernier stimule leur expression via l’action de prostaglandine E2 (PGE2) et du facteur de croissance transformant-beta 1 (TGF-β1). En conclusion, notre étude démontre que le HNE induit à court-terme d’incubation la voie de COX-2/mPGES-1 puis par la suite stimule celle de FLAP/5-LOX à long-terme d’incubation dans les chondrocytes arthrosiques humains. Ces résultats suggèrent que la mPGES-1 et 5-LOX/FLAP sont des potentielles cibles thérapeutiques intéressantes pour contrôler la production de PGE2 et LTB4 dans OA. / 4-hydroxynonenal (HNE), a lipid peroxidation end-product, is produced abundantly in osteoarthritic (OA) articular tissues. Recently, we reported that HNE-induced cyclooxygenase-2 (COX-2) decreased gradually in human OA chondrocytes after 8 h of incubation. This study aimed to investigate whether COX-2 down-regulation is attributed to HNE depletion and is responsible for the switch from COX-2 to 5-lipoxygenase-activating protein (FLAP)/5-lipoxygenase (5-LOX). Treatment of chondrocytes with 10 µM HNE induced prostaglandin E2 (PGE2) release as well as COX-2 and microsomal prostaglandin E2 synthase-1 (mPGES-1) expression at the protein and mRNA levels, with a plateau reached at 8-16 h of incubation, followed by a subsequent decline. However, 8 repeated treatments with 10 µM HNE prevented the reduction of COX-2 and mPGES-1 expression. We demonstrated that HNE induced mPGES-1 promoter activity mainly through transcription factor Egr-1 activation. On the other hand, when COX-2 expression decreased, leukotriene B4 (LTB4) level rose after a long period of stimulation (48 and 72 h). At the mRNA level, HNE induced FLAP and 5-LOX expression after 24 and 48 h of stimulation, respectively. The addition of a nonspecific COX-2 inhibitor (naproxen) to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that 10 µM HNE significantly induced transforming growth factor-beta 1 (TGF-β1) production. The addition of anti-TGF-β antibody to culture medium reduced HNE-induced 5-LOX/FLAP expression by 40%, indicating the involvement of a TGF-β1-dependent mechanism. Our data demonstrate that the shunt to the FLAP/5-LOX pathway in HNE-induced human OA chondrocytes is attributed to COX-2 inhibition, probably due to HNE depletion. PGE2 and TGF-β1 are suggested to be involved in this regulation. Further experiments are in progress to determine other molecular mechanisms underlying this switch in OA chondrocytes.

As frutíferas nativas do Brasil são subexploradas comercialmente, sendo que os programas de melhoramento genético são bastante incipientes, devido a muitas limitações associadas à fisiologia e biologia dessas espécies, embora apresentem potencial econômico tanto para o consumo in natura como para o processamento industrial. No Brasil, o maracujá-doce (Passiflora alata) está conquistando novos clientes e mercados, e tem atingido preços até três vezes maiores do que os do maracujá-azedo, amplamente cultivado no país. Os frutos do maracujá-doce são muito apreciados, embora apenas a seleção empírica tenha sido praticada. Evidentemente, seria muito vantajoso obter uma variedade geneticamente superior em termos de qualidade e produção de frutos. Nesse sentido, nosso grupo de pesquisa tem focado esforços visando a caracterizar geneticamente uma população segregante de maracujá-doce. Aqui, foram avaliados 30 genótipos desta população, previamente selecionados como superiores. Estes foram plantados sob três condições experimentais: Anhumas (2014/2015 e 2015/2016) e Piracicaba (2015/2016). Foi utilizado o delineamento em blocos casualizados, sendo que as análises genético-estatísticas tiveram por base a abordagem de modelos mistos para a predição dos valores genéticos (BLUPS). Foram estimados parâmetros genéticos e fenotípicos, herdabilidade, correlações genéticas e resposta esperada à seleção relativamente a peso (PF), comprimento (CF) diâmetro (DF) do fruto, espessura (EC) e peso (PC) da casca, peso (PP) e rendimento de polpa (RP), teor de sólidos solúveis totais (SST) e Produtividade. Os resultados mostraram haver altas correlações genéticas entre os principais atributos do fruto, assim como valores elevados de herdabilidade (desde 0,45 para PF a 0,94 para EC), valores que favorecem a prática de seleção. Em seguida, foi aplicado índice multiplicativo para fins de seleção e seis genótipos foram indicados como superiores, quais sejam: 49, 21, 107, 125, 140 e 122. Nossos resultados devem auxiliar outros estudos e iniciativas de seleção de genótipos mais produtivos, evidenciando a grande utilidade de ferramentas estatísticas modernas para se obter maior resposta à seleção. Em paralelo, foi estudada a resposta do maracujá-doce à infecção causada por Xanthomonas axonopodis. Dois genes, previamente identificados como superexpressos no maracujá-azedo, e que codificam as enzimas lipoxigenase-2 e (+)-neomentol desidrogenase foram aqui analisados via qPCR. No maracujá-doce observou-se maior expressão da (+)-neomentol desidrogenase, enzima associada à resposta de hipersensibilidade. Os sintomas da doença sugerem que a resposta de hipersensibilidade é particular no maracujá-doce, já que ocorre a queda precoce das folhas, evitando a disseminação sistêmica da bactéria. / Brazilian native fruit species are under-exploited commercially, and breeding programs are quite incipient due to many limitations associated with the physiology and biology of these species, although having economic potential both for in natura consumption as well as for industrial processing. In Brazil, the sweet passion fruit (Passiflora alata) is conquering new customers and markets, and has riched up to triple the sour passion fruit prices, widely grown in the country. The fruits of the sweet passion fruit are much appreciated, although only empirical selections have been practiced. Evidently, it would be very beneficial to obtain a genetically superior variety in terms of fruit quality and production. In this scenario, our research group has focused efforts to genetically characterize a segregating population of sweet passion fruit. Here, we evaluated 30 genotypes previously selected from this population by field-cultivating them under three experimental conditions: Anhumas (2014/2015 and 2015/2016) and Piracicaba (2015/2016). A randomized block design was used, and the genetic-statistical analyzes were based on a mixed-effects model approach in order to predict the genetic values (BLUPS). We estimated genetic and phenotypic parameters, as heritability, genetic correlations and expected response to selection for weight (WF), length (LF) diameter (DF) of the fruit, thickness (TS) and weight (WS) of the shell, weight (WP) and pulp yield (PY), total soluble solids (SS) and fruit yield. We found high genetic correlations between the main fruit attributes, as well as high values of heritability (from 0.45 for WF up to 0.94 for TS), values that may favor selection practices. Then, we applied a multiplicative selection index and six superior genotypes were selected, which are: 49, 21, 107, 125, 140 and 122. Our results may assist other studies and initiatives of selection of more productive genotypes, showing the great usefulness of modern statistical tools to obtain greater responses to selection. In parallel, we studied the response of sweet passion fruit to infection caused by Xanthomonas axonopodis. Two genes, previously identified as overexpressed in sour passion fruit, and that encode the enzymes lipoxygenase-2 and (+)-neomentol dehydrogenase were herein analyzed via qPCR. In the sweet passion fruit an increased expression of (+)- neomentol dehydrogenase, an enzyme associated with hypersensitivity response, was observed. The disease symptoms suggest that the response of hypersensitivity is particular in sweet passion fruit, since it provokes the premature fall of the leaves, thereby preventing the systemic bacterial dissemination.

Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.

No description available.

Contexte - La prévalence de la maladie de Crohn (MC), une maladie inflammatoire chronique du tube digestif, chez les enfants canadiens se situe parmi les plus élevées au monde. Les interactions entre les réponses immunes innées et acquises aux microbes de l'hôte pourraient être à la base de la transition de l’inflammation physiologique à une inflammation pathologique. Le leucotriène B4 (LTB4) est un modulateur clé de l'inflammation et a été associé à la MC. Nous avons postulé que les principaux gènes impliqués dans la voie métabolique du LTB4 pourrait conférer une susceptibilité accrue à l'apparition précoce de la MC. Dans cette étude, nous avons exploré les associations potentielles entre les variantes de l'ADN des gènes ALOX5 et CYP4F2 et la survenue précoce de la MC. Nous avons également examiné si les gènes sélectionnés montraient des effets parent-d'origine, influençaient les phénotypes cliniques de la MC et s'il existait des interactions gène-gène qui modifieraient la susceptibilité à développer la MC chez l’enfant. Méthodes – Dans le cadre d’une étude de cas-parents et de cas-témoins, des cas confirmés, leurs parents et des contrôles ont été recrutés à partir de trois cliniques de gastro-entérologie à travers le Canada. Les associations entre les polymorphismes de remplacement d'un nucléotide simple (SNP) dans les gènes CYP4F2 et ALOX5 ont été examinées. Les associations allélique et génotypiques ont été examinées à partir d’une analyse du génotype conditionnel à la parenté (CPG) pour le résultats cas-parents et à l’aide de table de contingence et de régression logistique pour les données de cas-contrôles. Les interactions gène-gène ont été explorées à l'aide de méthodes de réduction multi-factorielles de dimensionnalité (MDR). Résultats – L’étude de cas-parents a été menée sur 160 trios. L’analyse CPG pour 14 tag-SNP (10 dans la CYP4F2 et 4 dans le gène ALOX5) a révélé la présence d’associations alléliques ou génotypique significatives entre 3 tag-SNP dans le gène CYP4F2 (rs1272, p = 0,04, rs3093158, p = 0.00003, et rs3093145, p = 0,02). Aucune association avec les SNPs de ALOX5 n’a pu être démontrée. L’analyse de l’haplotype de CYP4F2 a montré d'importantes associations avec la MC (test omnibus p = 0,035). Deux haplotypes (GAGTTCGTAA, p = 0,05; GGCCTCGTCG, p = 0,001) montraient des signes d'association avec la MC. Aucun effet parent-d'origine n’a été observé. Les tentatives de réplication pour trois SNPs du gene CYP4F2 dans l'étude cas-témoins comportant 225 cas de MC et 330 contrôles suggèrent l’association dans un de ceux-ci (rs3093158, valeur non-corrigée de p du test unilatéral = 0,03 ; valeur corrigée de p = 0.09). La combinaison des ces deux études a révélé des interactions significatives entre les gènes CYP4F2, ALOX et NOD2. Nous n’avons pu mettre en évidence aucune interaction gène-sexe, de même qu’aucun gène associé aux phénotypes cliniques de la MC n’a pu être identifié. Conclusions - Notre étude suggère que la CYP4F2, un membre clé de la voie métabolique LTB4 est un gène candidat potentiel pour MC. Nous avons également pu mettre en évidence que les interactions entre les gènes de l'immunité adaptative (CYP4F2 et ALOX5) et les gènes de l'immunité innée (NOD2) modifient les risques de MC chez les enfants. D'autres études sur des cohortes plus importantes sont nécessaires pour confirmer ces conclusions. / Background - The rates of Crohn’s disease (CD) a chronic inflammatory disease of the gastrointestinal tract, among Canadian children are the world’s highest. Interactions between the host microbial–innate-immune-responses are thought to underplay transition from physiological to pathological inflammation. Leukotriene B4 (LTB4) is a key modulator of inflammation and has been shown to be associated with CD. We postulated that key genes involved in the LTB4 metabolic pathway could confer susceptibility for early-onset CD. In this study we implemented a candidate gene approach to test for associations between DNA variants in the ALOX5 and CYP4F2 genes and early-onset of CD. We also explored whether the selected genes demonstrated parent-of-origin effects, influenced CD clinical phenotypes and whether there were gender-gene and gene-gene interactions that determined CD susceptibility. Methods – The study consisted of an exploratory phase (case-parent design) followed by a replication phase (case-control design). Confirmed cases, parents and controls were recruited from three tertiary gastroenterology clinics across Canada. Associations between tag-single nucleotide polymorphisms in the CYP4F2 and ALOX5 genes were examined. Allelic and/or genotype associations were examined using conditional on parental genotype (CPG) analysis for the case-parent data and contingency table and logistic regression for the case-control data. Gene-gene interactions were explored using multi-factor dimensionality reduction (MDR) methods. Results – The first phase of the study was based on 160 trios (case-parent design). CPG analysis for 14 tag-SNPs (i.e. 10 in the CYP4F2 and 4 in the ALOX5 gene, respectively) revealed significant allelic or genotypic associations between 3 tag-SNPs in the CYP4F2 gene (rs1272, p=0.04, rs3093158, p=0.00003, and rs3093145, p=0.02). No associations with ALOX5 tag-SNPs were evident. CYP4F2-haplotype analysis showed significant associations with CD (omnibus test p-value=0.035). Two specific haplotypes (GAGTTCGTAA, p=0.05; GGCCTCGTCG, p=0.001) showed evidence for association with CD. No parent-of-origin effects were observed. The second phase of the study retested the three CYP4F2 SNPs that showed association in the first stage and was based on 223 CD cases and 330 controls. Some indications of association with one SNP i.e. rs3093158 were present (genotypic uncorrected 1-sided p-value=0.03); however this genotype association did not withstand correction. Combining cases from the two phases of the study revealed significant interactions between the CYP4F2, ALOX and NOD2 genes. No gene-gender interactions were obvious nor were the study genes associated with specific clinical phenotypes of CD. Conclusions - Our study suggests that the CYP4F2, a key member of the LTB4 metabolic pathway is a potential candidate gene for CD. Furthermore there was evidence that interactions between adaptive immunity genes (CYP4F2 and ALOX5) and innate immunity genes (NOD2) genes modify risk for CD in children. Further studies on larger cohorts are required to confirm these findings.

Contexte - La prévalence de la maladie de Crohn (MC), une maladie inflammatoire chronique du tube digestif, chez les enfants canadiens se situe parmi les plus élevées au monde. Les interactions entre les réponses immunes innées et acquises aux microbes de l'hôte pourraient être à la base de la transition de l’inflammation physiologique à une inflammation pathologique. Le leucotriène B4 (LTB4) est un modulateur clé de l'inflammation et a été associé à la MC. Nous avons postulé que les principaux gènes impliqués dans la voie métabolique du LTB4 pourrait conférer une susceptibilité accrue à l'apparition précoce de la MC. Dans cette étude, nous avons exploré les associations potentielles entre les variantes de l'ADN des gènes ALOX5 et CYP4F2 et la survenue précoce de la MC. Nous avons également examiné si les gènes sélectionnés montraient des effets parent-d'origine, influençaient les phénotypes cliniques de la MC et s'il existait des interactions gène-gène qui modifieraient la susceptibilité à développer la MC chez l’enfant. Méthodes – Dans le cadre d’une étude de cas-parents et de cas-témoins, des cas confirmés, leurs parents et des contrôles ont été recrutés à partir de trois cliniques de gastro-entérologie à travers le Canada. Les associations entre les polymorphismes de remplacement d'un nucléotide simple (SNP) dans les gènes CYP4F2 et ALOX5 ont été examinées. Les associations allélique et génotypiques ont été examinées à partir d’une analyse du génotype conditionnel à la parenté (CPG) pour le résultats cas-parents et à l’aide de table de contingence et de régression logistique pour les données de cas-contrôles. Les interactions gène-gène ont été explorées à l'aide de méthodes de réduction multi-factorielles de dimensionnalité (MDR). Résultats – L’étude de cas-parents a été menée sur 160 trios. L’analyse CPG pour 14 tag-SNP (10 dans la CYP4F2 et 4 dans le gène ALOX5) a révélé la présence d’associations alléliques ou génotypique significatives entre 3 tag-SNP dans le gène CYP4F2 (rs1272, p = 0,04, rs3093158, p = 0.00003, et rs3093145, p = 0,02). Aucune association avec les SNPs de ALOX5 n’a pu être démontrée. L’analyse de l’haplotype de CYP4F2 a montré d'importantes associations avec la MC (test omnibus p = 0,035). Deux haplotypes (GAGTTCGTAA, p = 0,05; GGCCTCGTCG, p = 0,001) montraient des signes d'association avec la MC. Aucun effet parent-d'origine n’a été observé. Les tentatives de réplication pour trois SNPs du gene CYP4F2 dans l'étude cas-témoins comportant 225 cas de MC et 330 contrôles suggèrent l’association dans un de ceux-ci (rs3093158, valeur non-corrigée de p du test unilatéral = 0,03 ; valeur corrigée de p = 0.09). La combinaison des ces deux études a révélé des interactions significatives entre les gènes CYP4F2, ALOX et NOD2. Nous n’avons pu mettre en évidence aucune interaction gène-sexe, de même qu’aucun gène associé aux phénotypes cliniques de la MC n’a pu être identifié. Conclusions - Notre étude suggère que la CYP4F2, un membre clé de la voie métabolique LTB4 est un gène candidat potentiel pour MC. Nous avons également pu mettre en évidence que les interactions entre les gènes de l'immunité adaptative (CYP4F2 et ALOX5) et les gènes de l'immunité innée (NOD2) modifient les risques de MC chez les enfants. D'autres études sur des cohortes plus importantes sont nécessaires pour confirmer ces conclusions. / Background - The rates of Crohn’s disease (CD) a chronic inflammatory disease of the gastrointestinal tract, among Canadian children are the world’s highest. Interactions between the host microbial–innate-immune-responses are thought to underplay transition from physiological to pathological inflammation. Leukotriene B4 (LTB4) is a key modulator of inflammation and has been shown to be associated with CD. We postulated that key genes involved in the LTB4 metabolic pathway could confer susceptibility for early-onset CD. In this study we implemented a candidate gene approach to test for associations between DNA variants in the ALOX5 and CYP4F2 genes and early-onset of CD. We also explored whether the selected genes demonstrated parent-of-origin effects, influenced CD clinical phenotypes and whether there were gender-gene and gene-gene interactions that determined CD susceptibility. Methods – The study consisted of an exploratory phase (case-parent design) followed by a replication phase (case-control design). Confirmed cases, parents and controls were recruited from three tertiary gastroenterology clinics across Canada. Associations between tag-single nucleotide polymorphisms in the CYP4F2 and ALOX5 genes were examined. Allelic and/or genotype associations were examined using conditional on parental genotype (CPG) analysis for the case-parent data and contingency table and logistic regression for the case-control data. Gene-gene interactions were explored using multi-factor dimensionality reduction (MDR) methods. Results – The first phase of the study was based on 160 trios (case-parent design). CPG analysis for 14 tag-SNPs (i.e. 10 in the CYP4F2 and 4 in the ALOX5 gene, respectively) revealed significant allelic or genotypic associations between 3 tag-SNPs in the CYP4F2 gene (rs1272, p=0.04, rs3093158, p=0.00003, and rs3093145, p=0.02). No associations with ALOX5 tag-SNPs were evident. CYP4F2-haplotype analysis showed significant associations with CD (omnibus test p-value=0.035). Two specific haplotypes (GAGTTCGTAA, p=0.05; GGCCTCGTCG, p=0.001) showed evidence for association with CD. No parent-of-origin effects were observed. The second phase of the study retested the three CYP4F2 SNPs that showed association in the first stage and was based on 223 CD cases and 330 controls. Some indications of association with one SNP i.e. rs3093158 were present (genotypic uncorrected 1-sided p-value=0.03); however this genotype association did not withstand correction. Combining cases from the two phases of the study revealed significant interactions between the CYP4F2, ALOX and NOD2 genes. No gene-gender interactions were obvious nor were the study genes associated with specific clinical phenotypes of CD. Conclusions - Our study suggests that the CYP4F2, a key member of the LTB4 metabolic pathway is a potential candidate gene for CD. Furthermore there was evidence that interactions between adaptive immunity genes (CYP4F2 and ALOX5) and innate immunity genes (NOD2) genes modify risk for CD in children. Further studies on larger cohorts are required to confirm these findings.