Os vasos sanguíneos são formados, entre outros componentes, por células endoteliais as quais fazem parte da microvasculatura óssea e são capazes de regular o desenvolvimento ósseo tendo em vista de que os processos de osteogênese e angiogênese estão interligados. Os leucotrienos (LTs) são mediadores lipídicos envolvidos no recrutamento de leucócitos e na regulação da síntese de citocinas. O tratamento com o leucotrieno B4 (LTB4) induz a angiogênese pela superexpressão do fator de crescimento endotelial vascular (VEGF). Assim, o objetivo do trabalho foi investigar o efeito das células endoteliais reguladas pelo LTB4 na diferenciação osteogênica. Para isso, células endoteliais primárias de aorta foram isoladas e cultivadas por até 4 dias e, quando apropriado, foi realizado o tratamento das mesmas com o LTB4. O meio condicionado das células endoteliais foi armazenado para os experimentos com osteoblastos. Células osteoblásticas foram isoladas da calvária e cultivadas por até 21 dias, avaliando-se, portanto, os efeitos das células endoteliais reguladas ou não pelo LTB4 e a resposta dos estímulos exógenos LTB4, o inibidor da síntese de LTs MK 886 e o antagonista do receptor do LTB4, o U75302. Tais respostas foram observadas na fase de crescimento celular, por meio da viabilidade, proliferação e produção de marcadores osteogênicos e angiogênicos como o RANKL, OPG e o VEGF por meio da redução do MTT, citometria de fluxo e western blotting, respectivamente. A diferenciação foi avaliada por meio dos ensaios de fosfatase alcalina (ALP) e expressão gênica por meio de ensaio enzimático e qRT-PCR e mineralização por Vermelho de alizarina. Resultados mostraram que tanto as células endoteliais, mediadas ou não pelo LTB4, quanto os estímulos exógenos não foram capazes de modular a proliferação dos osteoblastos. Porém, durante a diferenciação, o LTB4 inibiu a atividade da ALP, a expressão gênica do BLT1, ALP, BGLAP (osteocalcina) e OPG (osteoprotegerina) foi aumentada, e os genes RANKL e VEGF tiveram a sua expressão diminuída pelo tratamento com o meio condicionado das células endoteliais, mediadas ou não pelo LTB4 (P<0,05). Além disso, a mineralização dos osteoblastos foi aumentada pelas células endoteliais e diminuída pelas células endoteliais mediadas pelo LTB4 (P<0,05). Assim, podemos concluir que os fatores angiogênicos das células endoteliais, mediadas ou não pelo LTB4, exercem um papel importante na regulação da diferenciação osteogênica e formação óssea contribuindo, portanto, para a compreensão de mecanismos que regulam a patofisiologia de doenças ósseas. / Endothelial cells make blood vessels and are involved in the regulation of tissue metabolism. Endothelial cells from bone microvasculature are capable of regulating bone development in view of the fact that the processes of osteogenesis and angiogenesis are interconnected. Leukotrienes (LTs) are lipid mediators involved in leukocyte recruitment and regulation of cytokine synthesis. It is known that the treatment with LTB4 induces angiogenesis by overexpression of vascular endothelial growth factor (VEGF). Thus, the aim of this study was to investigate the effect of LTB4-regulated endothelial cells on osteogenic differentiation. For this, primary endothelial cells from aorta were isolated and cultured for up to 4 days and, where appropriate, the treatment with LTB4 was done. The conditioned medium of these cells was stored for osteoblast experiments. Osteoblastic cells were isolated from calvaria and cultured for up to 21 days, assessing the effects of endothelial cells regulated or not by LTB4 and the response of exogenous LTB4 stimuli, the inhibitor of LTs synthesis MK 886 and the antagonist of LTB4 receptor, U75302. Such responses were observed in the cell growth phase through the viability, proliferation and production of osteogenic and angiogenic markers such as RANKL, OPG and VEGF by MTT assay, flow cytometry and western blotting, respectively. The cell differentiation was evaluated by alkaline phosphatase (ALP), gene expression and mineralization assay through ALP enzymatic assay, qRT-PCR and Alizarin Red staining. Results showed that both endothelial cells, mediated or not by LTB4 and exogenous stimuli were not able to modulate the osteoblasts proliferation. However, during the differentiation, LTB4 inhibited ALP activity, the gene expression of BLT1, ALP, BGLAP (osteocalcin) and OPG (osteoprotegerin) was increased, and the RANKL and VEGF genes had their expression decreased by the treatment with the endothelial cells conditioned medium, mediated or not by LTB4 (P <0.05). In addition, the osteoblasts mineralization was increased by endothelial cells conditioned medium (CM-EC) and decreased by LTB4-mediated endothelial cells conditioned medium (CM-EC-LTB4) (P <0.05). Thus, we can conclude that the angiogenic factors of the endothelial cells, mediated or not by LTB4, play an important role in the regulation of osteogenic differentiation and bone formation, thus contributing to the understanding of mechanisms that regulate the pathophysiology of bone diseases.

O Histoplasma capsulatum (H.capsulatum) é um fungo dimórfico patogênico e responsável por graves lesões pulmonares, as quais se caracterizam pelo acúmulo de leucócitos ao redor do fungo, resultando na formação de granulomas. A infecção ocorre principalmente pela inalação de conídios ou pequenos fragmentos de micélio que alcançam os alvéolos, onde se transformam em leveduras, que é a forma patogênica do fungo. Na resposta imune do hospedeiro, as integrinas participam nos mecanismos fagocíticos, essenciais na resposta à histoplasmose. As 2integrinas contêm uma cadeia 2, também conhecida como CD18, comum a várias moléculas de adesão, e uma cadeia variável. Até o momento foram identificadas quatro cadeias distintas: L, a qual forma o dímero L2, também conhecido como LFA-1 (do inglês leukocyte function antigen-1) ou CD11aCD18; m, formando m2, chamado Mac-1 (do inglês macrophage differentiation antigen 1) ou CR3 (do inglês complement receptor 3) ou CD11bCD18; x, formando x2, CD11cCD18, gp150, 95 ou CR4 (do inglês complement receptor 4) e a cadeia d, formando d2, CD11dCD18. Neste trabalho, investigamos o papel da molécula CD18 em macrófagos alveolares (MAs) e macrófagos peritoneais (MPs) nas funções efetoras contra H. capsulatum e a relação do leucotrieno B4 (LTB4) nestas respostas. Inicialmente confirmamos que MAs e MPs provenientes dos animais CD18low, expressam baixa porcentagem de CD11bCD18 (CR3). Demonstramos que, como esperado, MAs e MPs de ambos os grupos fagocitam mais leveduras opsonizadas com complemento do que não opsonizadas. Surpreendentemente, MAs de animais CD18low fagocitam 136% mais leveduras opsonizadas do que MAs de C57BL/6. Também, MPs destes animais fagocitam aproximadamente 240% mais leveduras quando infectados com H. capsulatum e opsonizados, quando comparados aos MPs de C57BL/6. A adição de LTB4 aumenta a atividade fagocítica em 520% por MAs de animais C57BL/6 e 200% por MAs de CD18low, enquanto que a adição de LTB4 aumentou a fagocitose dos MPs de animais C57BL/6 em 600% vezes quando comparado aos MPs de CD18low. Este fenômeno foi inibido pela pré-incubação destas células com antagonista específico do receptor BLT1 apenas em animais C57BL/6. A adição de LTB4 na cultura de MPs reduziu a porcentagem de morte das leveduras apenas nos animais C57BL/6. Os animais CD18low produzem espontaneamente mais LTB4 e apresentaram um grande aumento na produção de óxido nítrico quando comparados aos animais C57BL/6. Pacientes acometidos pela Doença Granulomatosa Crônica (DGC) possuem deficiência congênita da molécula CD18. Células fagocíticas isoladas do sangue periférico de pacientes com DGC foram incubadas com leveduras opsonizadas e assim como macrófagos de animais deficientes de CD18, fagocitam mais leveduras opsonizadas (900%) ou não (300%), quando comparado com células de indivíduos sadios. Sugerimos que a molécula CD18 tem importante participação nos mecanismos efetores da imunidade inata, por mecanismo dependente de mediadores lipídicos, como o LTB4, no controle dos mecanismos de defesa contra H. capsulatum. / Histoplasma capsulatum (H. capsulatum) is a pathogenic dimorphic fungus and its infection is characterized by accumulation of leukocytes and granuloma formation. Infection occurs mainly by fungal inhalation that reaches the alveoli, which became yeast (the pathogenic form). in the immune response of host, integrins participate in phagocytic mechanisms, fundamental in the response against histoplasmosis.,8^2-integrin has a 02 chain known as CD18, usual to many adhesion molecules, and a variable a chain. Until the moment, it was identified four variable a chains: aL, that constitutes the dimer aL,82, also known as LFA-1 (leukocyte function antigen) or CD11aCD18; am forming the a^m,B^2 or Mac-1 (macrophage differentiation antigen 1) and CR3 (complement receptor 3) and CD11bCD18; ax, constituting the dimer Able CD11cCD18, gp150, 95 or CR4 (complement receptor 4) and ad chain, that constitutes a^d,8^2, CD11dCD18. In the present study, we sought to investigate the effect of CD18 in alveolar (AMs) and peritoneal macrophages (PMs) effecter functions against H. capsulatum and the relation of LTB^4 in those responses. We confirm that AMs and PMs of CD18\'°^W mice have low expression of ,32-integrin compared to wild type mice (WT). We demonstrate that, as expected, AMs and PMs from WT and CD18\'°^W, phagocytosed more complement (C)-opsonized yeasts than the unopsonized yeasts. Surprisingly, AMs from CD18\'°^Wmice phagocytosed 136% more (C)-opsonized yeasts than AMs obtained from WT. Also, PMs of CD18^b°^W mice phagocytosed 240% more (C)-opsonized yeasts than PMs of WT. The addition of LTB^4, increases the phagocytic activity by AMs of WT mice in 520% and by AMs from CD18\'°^W mice in 200%, while the addition of LTB^4 only increased the phagocytosis of C-opsonized H. capsulatum by PMs of C57BL/6 mice in 600%, when compaired with PMs from CD18\'°^W mice. This phenomenon was inhibited by pretreatment of these cells with an especific BLT1 receptor antagonist only in PMs from C57BU6 mice. The addition of LTB^4 in the culture of MPs reduced the percentage of death of yeasts in animals C57BL/6. CD18\'°^W mice, spontaneously produce more LTB^4 and showed a large increase in the production of nitric oxide when compared to C57BU6. Patients affected by Chronic Granulomatous Disease (DGC) have congenital deficiency of the CD18 molecule. Phagocytic cells isolated from peripheral blood of patients with DGC were incubated with C-opsonized yeasts and as well as macrophages from CD18\'°^W, phagocytosed more C-opsonized yeasts (900%) or not (300%) when compared with cells from healthy individuals.Therefore, we suggest that the CD18 molecule has important participation in the effector mechanisms of innate immunity, a mechanism dependent on lipid mediators such as LTB^4, to control these mechanisms in defense against H. capsulatum.

O Histoplasma capsulatum (H.capsulatum) é um fungo dimórfico patogênico e responsável por graves lesões pulmonares, as quais se caracterizam pelo acúmulo de leucócitos ao redor do fungo, resultando na formação de granulomas. A infecção ocorre principalmente pela inalação de conídios ou pequenos fragmentos de micélio que alcançam os alvéolos, onde se transformam em leveduras, que é a forma patogênica do fungo. Na resposta imune do hospedeiro, as integrinas participam nos mecanismos fagocíticos, essenciais na resposta à histoplasmose. As 2integrinas contêm uma cadeia 2, também conhecida como CD18, comum a várias moléculas de adesão, e uma cadeia variável. Até o momento foram identificadas quatro cadeias distintas: L, a qual forma o dímero L2, também conhecido como LFA-1 (do inglês leukocyte function antigen-1) ou CD11aCD18; m, formando m2, chamado Mac-1 (do inglês macrophage differentiation antigen 1) ou CR3 (do inglês complement receptor 3) ou CD11bCD18; x, formando x2, CD11cCD18, gp150, 95 ou CR4 (do inglês complement receptor 4) e a cadeia d, formando d2, CD11dCD18. Neste trabalho, investigamos o papel da molécula CD18 em macrófagos alveolares (MAs) e macrófagos peritoneais (MPs) nas funções efetoras contra H. capsulatum e a relação do leucotrieno B4 (LTB4) nestas respostas. Inicialmente confirmamos que MAs e MPs provenientes dos animais CD18low, expressam baixa porcentagem de CD11bCD18 (CR3). Demonstramos que, como esperado, MAs e MPs de ambos os grupos fagocitam mais leveduras opsonizadas com complemento do que não opsonizadas. Surpreendentemente, MAs de animais CD18low fagocitam 136% mais leveduras opsonizadas do que MAs de C57BL/6. Também, MPs destes animais fagocitam aproximadamente 240% mais leveduras quando infectados com H. capsulatum e opsonizados, quando comparados aos MPs de C57BL/6. A adição de LTB4 aumenta a atividade fagocítica em 520% por MAs de animais C57BL/6 e 200% por MAs de CD18low, enquanto que a adição de LTB4 aumentou a fagocitose dos MPs de animais C57BL/6 em 600% vezes quando comparado aos MPs de CD18low. Este fenômeno foi inibido pela pré-incubação destas células com antagonista específico do receptor BLT1 apenas em animais C57BL/6. A adição de LTB4 na cultura de MPs reduziu a porcentagem de morte das leveduras apenas nos animais C57BL/6. Os animais CD18low produzem espontaneamente mais LTB4 e apresentaram um grande aumento na produção de óxido nítrico quando comparados aos animais C57BL/6. Pacientes acometidos pela Doença Granulomatosa Crônica (DGC) possuem deficiência congênita da molécula CD18. Células fagocíticas isoladas do sangue periférico de pacientes com DGC foram incubadas com leveduras opsonizadas e assim como macrófagos de animais deficientes de CD18, fagocitam mais leveduras opsonizadas (900%) ou não (300%), quando comparado com células de indivíduos sadios. Sugerimos que a molécula CD18 tem importante participação nos mecanismos efetores da imunidade inata, por mecanismo dependente de mediadores lipídicos, como o LTB4, no controle dos mecanismos de defesa contra H. capsulatum. / Histoplasma capsulatum (H. capsulatum) is a pathogenic dimorphic fungus and its infection is characterized by accumulation of leukocytes and granuloma formation. Infection occurs mainly by fungal inhalation that reaches the alveoli, which became yeast (the pathogenic form). in the immune response of host, integrins participate in phagocytic mechanisms, fundamental in the response against histoplasmosis.,8^2-integrin has a 02 chain known as CD18, usual to many adhesion molecules, and a variable a chain. Until the moment, it was identified four variable a chains: aL, that constitutes the dimer aL,82, also known as LFA-1 (leukocyte function antigen) or CD11aCD18; am forming the a^m,B^2 or Mac-1 (macrophage differentiation antigen 1) and CR3 (complement receptor 3) and CD11bCD18; ax, constituting the dimer Able CD11cCD18, gp150, 95 or CR4 (complement receptor 4) and ad chain, that constitutes a^d,8^2, CD11dCD18. In the present study, we sought to investigate the effect of CD18 in alveolar (AMs) and peritoneal macrophages (PMs) effecter functions against H. capsulatum and the relation of LTB^4 in those responses. We confirm that AMs and PMs of CD18\'°^W mice have low expression of ,32-integrin compared to wild type mice (WT). We demonstrate that, as expected, AMs and PMs from WT and CD18\'°^W, phagocytosed more complement (C)-opsonized yeasts than the unopsonized yeasts. Surprisingly, AMs from CD18\'°^Wmice phagocytosed 136% more (C)-opsonized yeasts than AMs obtained from WT. Also, PMs of CD18^b°^W mice phagocytosed 240% more (C)-opsonized yeasts than PMs of WT. The addition of LTB^4, increases the phagocytic activity by AMs of WT mice in 520% and by AMs from CD18\'°^W mice in 200%, while the addition of LTB^4 only increased the phagocytosis of C-opsonized H. capsulatum by PMs of C57BL/6 mice in 600%, when compaired with PMs from CD18\'°^W mice. This phenomenon was inhibited by pretreatment of these cells with an especific BLT1 receptor antagonist only in PMs from C57BU6 mice. The addition of LTB^4 in the culture of MPs reduced the percentage of death of yeasts in animals C57BL/6. CD18\'°^W mice, spontaneously produce more LTB^4 and showed a large increase in the production of nitric oxide when compared to C57BU6. Patients affected by Chronic Granulomatous Disease (DGC) have congenital deficiency of the CD18 molecule. Phagocytic cells isolated from peripheral blood of patients with DGC were incubated with C-opsonized yeasts and as well as macrophages from CD18\'°^W, phagocytosed more C-opsonized yeasts (900%) or not (300%) when compared with cells from healthy individuals.Therefore, we suggest that the CD18 molecule has important participation in the effector mechanisms of innate immunity, a mechanism dependent on lipid mediators such as LTB^4, to control these mechanisms in defense against H. capsulatum.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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.