Made available in DSpace on 2014-08-20T13:33:00Z (GMT). No. of bitstreams: 1 dissertacao_raquel_rosa_costa.pdf: 808388 bytes, checksum: 18bdb94a8d12b8f7a746b7ee2613012a (MD5) Previous issue date: 2011-06-27 / he main objective of this work was to introduce the gene bvlI with confirmed potential antifungal of lectin from Bauhinia variegata, Apple Tree (Malus domestica Borkh) cv. Gala, to establish resistance to fungus Coletotrichum gloeosporioides, causing Leaf Spot. Multiplication experiments were performed in vitro regeneration of leaf explant culture and two protocols via Agrobacterium tumefaciens transformation aimed at inserting the gene bvll. The multiplication of shoots the medium that has best results was MS supplemented with 0.1 mg L-1 ANA and 8.3 mg L-1 2iP and, for the regeneration of the best responses were obtained with using MS, supplemented with 30 g L-1 sorbitol, 0.5 mg L-1 ANA and 5 mg L-1 TDZ, reaching a rate of 94.5% of regenerations. As regards the processing protocols, which showed best results was the P1 Protocol, which through analysis of PCR were obtained 15% shoots containing the gene bv/l. However, the expression of BVLI protein was not detected by Dot Blot, indicating the need for further procedure, such as refinement of protein extraction protocol, as well as utilization other techniques of analyses, such as Real Time. / O objetivo principal do presente trabalho foi introduzir o gene bvlI com potencial antifúngíco confirmado da lectina de Bauhinia variegata, em macieira (Malus domestica Borkh), cv. Gala, para estabelecer resistência ao fungo Coletotrichum gloeosporioides, causador da Mancha Foliar. Foram realizados experimentos de multiplicação in vitro, regeneração de explantes foliares e, dois protocolos de transformação via Agrobacterium tumefaciens visando a inserção do gene bvll. Na multiplicação de brotações o meio que apresentou melhores resultados foi o MS suplementado com 0,1 mg L-1 ANA e 8,3 mg L-1 de 2iP e, para a regeneração as melhores respostas foram obtidas com meio MS, suplementado com 30 g L-1 de sorbitol, 0,5 mg L-1 ANA e 5 mg L1 de TDZ, atingindo um percentual de 94,5% de regenerações. Quanto aos protocolos de transformação, o que apresentou melhores resultados foi o protocolo P1, no qual através da análise de PCR foram obtidas 15% de brotações contendo o gene bv/l. No entanto, a expressão da proteína BVLI não foi detectada através de Dot Blot, indicando a necessidade de novos procedimentos, como refinamento do protocolo de extração de proteínas, bem como utulização de outras técnicas de análises, como Real Time.

La maladie de Crohn (MC) est une maladie inflammatoire chronique de l’intestin qui peut être expliquée par une dysbiose et une dérégulation de la réponse immunitaire. La mannose-binding lectin (MBL), récepteur lectinique et les Toll-like récepteurs jouent un rôle crucial dans la défense contre les pathogènes et dans le développement de la réponse inflammatoire. Ils peuvent reconnaitre de nombreux micro-organismes dont Candida albicans. Cette levure commensale est un immunogène des anticorps anti-Saccharomyces cerevisiae (ASCA), anticorps utilisés dans le diagnostic de la MC. Une association entre un déficit en MBL et des taux élevés d’ASCA a été montrée chez les patients atteints de MC. Ce déficit en MBL est fréquemment associé à un phénotype sévère de la maladie. Le récepteur TLR2, associé à TLR1 ou à TLR6, est également impliqué dans la reconnaissance de C. albicans et le maintien de la barrière intestinale. L’objectif de ce projet a été d’étudier le rôle de la MBL et des récepteurs TLR2/TLR1/TLR6 dans l’homéostasie intestinale et l’élimination de C. albicans du tube digestif. Pour cela, nous avons exploré, les effets de la MBL et des TLR sur la colonisation intestinale à C. albicans et l’inflammation intestinale. Par ailleurs, nous avons déterminé, chez l’homme, l’influence des polymorphismes du gène MBL2 sur la modulation de l’activité et des taux de MBL au cours de la MC.A l’aide d’un modèle murin, nous avons mis en évidence l’expression de MBL-A et de MBL-C par les cellules épithéliales intestinales et leur implication dans l’homéostasie intestinale. Un déficit en MBL favorise la colonisation par C. albicans et une dissémination de la levure, en présence d’une colite chimio-induite. Les récepteurs TLR1 et TLR2 participent également dans la défense contre la colonisation par C. albicans, sans pour autant que leurs déficits n’entrainent de dissémination de la levure. A l’inverse, le récepteur TLR6 favorise la colonisation par C. albicans. La MBL et les récepteurs TLR1/TLR2/TLR6 régulent aussi l’expression des cytokines pro-inflammatoires impliquées dans les réponses Th1 et Th17. Ces résultats expérimentaux ont incité à envisager la poursuite de notre travail en approfondissant l’étude clinique sur des variations quantitatives et qualitatives de la MBL chez les patients atteints de MC et leur lien éventuel avec la persistance de l’inflammation intestinale. L’étude sur la cohorte de patients MC a montré que le gène MBL2 était associé à un déficit quantitatif en MBL et un déficit qualitatif du complexe MBL-MASP chez les sujets sains et les patients. Ce polymorphisme est également associé à des taux élevés d’ASCA chez les patients MC (p<0.05) de la maladie et il est fréquemment associé aux formes sévères de la maladie. De plus, le variant rs2066844 du gène NOD2, variant associé à la MC, est significativement corrélé à une diminution de l’activité fonctionnelle du complexe MBL-MASP, sans diminution quantitative des taux de MBL.Ces données montrent, pour la première fois, la production intestinale de MBL, qui est modulée par la colonisation par C. albicans. Elles confirment le rôle de la MBL et des récepteurs TLR dans l’homéostasie intestinale et dans la défense contre C. albicans. De plus, l’étude clinique sur la cohorte de patients MC révèle un défaut d’activité fonctionnelle du complexe MBL-MASP chez les patients MC ayant des polymorphismes des gènes MBL2 et NOD2, pouvant mener à un phénotype sévère de la maladie. Ces études expérimentales et cliniques apportent des nouvelles données sur le lien entre les récepteurs de l’immunité innée et la maladie de Crohn ainsi que la colonisation/infection fongique et ouvrent des nouvelles perspectives sur le lien entre la persistance de la colonisation fongique et le déficit quantitatif/qualitatif de la MBL chez les patients MC. / Crohn’s disease (CD) is an inflammatory bowel disease characterized by a dysregulation of the inflammatory response caused by dysbiosis and immune system disorders. Mannose Binding Lectin (MBL) and Toll like receptors (TLR) are involved in recognition of microorganisms and inflammatory response. They can recognize different patterns on the surface of the pathogens including Candida albicans. This pathogenic yeast is an immunogen for anti-Saccharomyces cerevisiae antibody (ASCA), diagnostic marker of CD. An association between MBL-deficiency and ASCA is observed in CD, and this MBL deficiency is frequently associated with a severe clinical phenotype of CD. In addition to MBL, TLR2, which forms heterodimers with either TLR1 or TLR6, have a major role in the innate immune defense against C. albicans and promotes intestinal homeostasis. In this project, we studied the role of MBL, TLR1, TLR2, and TLR6 in intestinal homeostasis and elimination of C. albicans in the intestinal tract. In the first part this study; we assessed the effect of either MBL or TLR deficiencies on C. albicans colonization and intestinal inflammation in a murine model. In the second part of this study, we assessed the role of MBL polymorphisms in the modulation of MBL level and activity in CD patients.In murin model, we showed that MBL is locally produced by the epithelial cells in response to C. albicans sensing and to intestinal inflammation and this lectin is required for the intestinal homeostasis. MBL-deficient mice had a higher level of colonization than wild-type mice. DSS-induced colitis promoted a high C. albicans colonization and dissemination to the kidneys and lungs of MBL-deficient mice. MBL-deficient mice exhibited reduced expression of Il-1β and IL-6 and elevated expression of IL-17, IL-23, dectin-1 and TLR-4. In terms of mice deficient in TLRs, DSS treatment and C. albicans oral challenge induced greater weight loss, worse clinical signs of inflammation, higher histopathologic scores, and increased mortality rates in TLR1-/- and TLR2-/- mice when compared to TLR6-/- and wild-type mice. Cytokine expression (TNF, IL-1β, IL-10, and IL-17A) was significantly increased in TLR1-/- and TLR2-/- mice, while they were decreased in TLR6-/- mice. In addition to the experimental studies, we observed in the clinical cohort of CD patients that MBL2 variant rs5030737 (codon 52) was associated with a low level of MBL that leads to impaired MBL-MASP functional activities in both CD patients and healthy subjects. Furthermore, this variant was also associated with a higher level of ASCA in CD patients (p<0.05). Increased ASCA levels were found in CD patients with stricture formation and penetrating disease complications, 42% and 21% respectively. Besides, we observed that NOD2 variant rs2066844, associated with susceptibility to CD, is significantly correlated with the impairment of the functional activity of MBL-MASP complex.Overall, this study emphasizes the role of MBL and TLR in intestinal homeostasis and host defense against C. albicans and shows for the first time that MBL could be produced locally in the intestinal epithelial cells in response to C. albicans sensing. In terms of the clinical study, we observed that CD patients with a severe clinical phenotype have an impairment in MBL-MASP functional activity, and that this defect is associated with MBL2 and NOD2 polymorphisms These experimental studies contribute to understand the link between innate immunity receptors, Crohn’s disease and fungal colonization/infection. Finally, this study leads to new objectives which are to study the link between intestinal microbiota and MBL variation in Crohn’s disease patients.

A galectina-1 (Gal-1) é uma proteína que reconhece ?-galactosídeos e participa de vários processos biológicos, incluindo a modulação da resposta imunológica. Vários relatos da literatura reportam o potencial uso terapêutico da Gal-1 para doenças auto-imunes, inflamatórias e infecciosas. Entretanto, são escassos os relatos sobre o envolvimento da Gal-1 na infecção causada por Tripanosoma cruzi. Neste trabalho foi avaliada a participação da Gal-1 endógena e a administração de Gal-1 exógena na evolução da infecção aguda induzida experimentalmente por T. cruzi. Foram realizados experimentos in vivo e/ou in vitro com o uso de Gal-1 recombinante humana, camundongos C57BL/6 deficientes do gene da Gal-1 (Gal-1 KO, knock-out) ou do gene Toll Like Receptor 4 (TLR-4 KO, knock-out) e selvagens (WT, wild type), além de seus macrófagos. A forma de T. cruzi utilizada foi a tripomastigota da cepa Y. Os parâmetros analisados na caracterização do processo de infecção foram: i) parasitemia e sobrevivência de animais; ii) histopatologia do tecido cardíaco; iii) imunofenotipagem de leucócitos; iv) dosagem de citocinas; v) determinação de taxas de invasão e liberação de parasitas a partir de células infectadas; vi) produção de óxido nítrico por macrófagos. A Gal-1 e/ou anticorpos anti-glicopeptídeos miméticos de mucina de T. cruzi ligam-se à glicanas da superfície deste parasita e impedem sua invasão em fibroblastos e sua captura por macrófagos. O tratamento de macrófagos infectados com Gal-1 reduz a liberação de parasitas e aumenta a produção de óxido nítrico (NO) por um mecanismo, aparentemente, independente da sinalização via TLR-4. Os camundongos KO Gal-1 e os WT tratados com Gal-1 exógena apresentaram as menores taxas de parasitemia, sendo que os primeiros são mais resistentes à infecção aguda por T. cruzi. A ausência da Gal-1 endógena nos animais infectados provocou vários efeitos como a redução no infiltrado inflamatório e carga parasitária no tecido cardíaco, níveis séricos elevados de citocinas (IL-2, IL-4, IL-6, IL-10 e IL-17A), menor porcentagem de linfócitos T CD4+ e maior de T CD8+ no coração dos animais, aumento do influxo de neutrófilos na cavidade peritoneal e no coração. Com base nesse conjunto de resultados sugerimos que a ausência de Gal-1 endógena ou o tratamento de animais com Gal- 1 exógena promoveram perfis imunológicos (resposta inata e adaptativa) favorecedores da resolução da infecção experimental aguda por T. cruzi / Galectin-1 (Gal-1) is a protein that recognizes ?-galactosides and participates in many biological processes, including the modulation of the immune response. Several reports in the literature show the potential therapeutic use of Gal-1 to autoimmune, inflammatory and infectious diseases. However, there are few reports on the involvement of Gal-1 on infection caused by Trypanosoma cruzi. Here, we evaluated the involvement of endogenous Gal-1 and Gal-1 administration of exogenous in the development of acute experimental infection by T. cruzi. Recombinant human Gal-1, C57BL/6 mice deficient for Gal-1 gene (Gal-1 KO, knockout) or for Toll like receptor 4 gene (TLR-4 KO, knock-out) or C57BL/6 wild type mice (WT), and macrophages from these animals were used in experiments in vivo and / or in vitro. The form of T. cruzi used in this work was trypomastigotes from Y strain. The analyzed parameters characterizing the process of infection were: i) parasitemia and survival of animals; ii) histopathology of cardiac tissue; iii) leucocyte immunophenotyping; iv) cytokine assay; v) determination of invasion and release rates of parasites from infected cells; vi) nitric oxide production by macrophages. The Gal-1 and / or antibodies anti- glycopeptides that mimics T. cruzi mucin bind to glycans on the surface of this parasite and prevent invasion of the parasite in fibroblasts and its capture by macrophages. Treatment of infected macrophages with Gal-1 promotes a lower release of parasites and increased production of nitric oxide (NO) by these phagocytes, and this production of NO is independent of TLR-4 signaling pathway. The Gal-1 KO mice and WT mice treated with exogenous Gal-1 had the lowest rates of parasitemia and the first group is more resistant to acute infection with T. cruzi. The absence of endogenous Gal-1 in infected animals caused various effects such as a reduction in the inflammatory infiltrate and the parasite load in the cardiac tissue, elevated serum levels of cytokines (IL-2, IL-4, IL-6, IL-10 and IL- 17A), a lower percentage of T CD4+ and increased T CD8+ the hearts of animals, increased influx of neutrophils into the peritoneal cavity and heart tissue. Based on this set of results we suggest that the absence of endogenous Gal-1 or treatment of animals with exogenous Gal-1 promoted immunological profiles (innate and adaptive response) favoring the resolution of acute experimental T. cruzi infection.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23072号 / 医博第4699号 / 新制||医||1049(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 川口 義弥, 教授 浅野 雅秀, 教授 福田 和彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L'inflammation du tissu adipeux, le stress oxydatif ou encore les modifications du microbiote intestinal semblent constituer des facteurs essentiels dans l'initiation, l'aggravation, voire même la "chronicisation" du syndrome métabolique et de l'insulinorésistance. La lectine Reg3a (également appelée HIP/PAP) est un composant de l'immunité innée qui déploie des activités anti-bactérienne, anti-oxydante et anti-inflammatoire. Dans ces conditions, Reg3a pourrait jouer un rôle clé en tant que modulateur du dialogue moléculaire entre le foie, le tissu adipeux et le tube digestif. Via son expression dans le foie de souris transgéniques, ou bien son administration par voie sous-cutanée, nous montrons que la protéine humaine Reg3a présente des effets bénéfiques sur le phénotype des animaux, en contribuant à leur capacité à mieux gérer les troubles du métabolisme d’origine à la fois glucidique et lipidique liés au vieillissement, à un régime alimentaire hyperlipidique ou une obésité et une insulinorésistance génétiquement acquises. Elle permet une diminution de la proportion de masse grasse et une meilleure régulation de la glycémie. Les mécanismes moléculaires sous-jacents restent à déterminer mais pourraient d’une part impliquer le régulateur énergétique AMPK, d’autre part être en lien avec le phénomène de brunissement du tissu adipeux. / Inflammation of adipose tissue, oxidative stress or modifications of intestinal microbiota seem to stand among crucial factors contributing to the initiation, worsening or even "chronification" of metabolic syndrome and insulin resistance. The lectin Reg3a (also called HIP/PAP) is a component of innate immunity with antibacterial, antioxidant and anti-inflammatory activities. In these conditions, Reg3a could play a key role as a modulator of the molecular dialogue between the liver, the adipose tissue and the gut. Through its expression in the liver of transgenic mice or its subcutaneous administration, we show that the human protein Reg3a has a positive impact on animals’ phenotype and contribute to their capacity to better manage both glucose and lipid metabolic disorders related to ageing, a high fat diet or genetically acquired obesity and insulin resistance. Notably it leads to a decrease in the proportion of fat and allows a better control of glycaemia. The associated molecular mechanisms remain to be determined but they could involve the energy sensor AMPK and the phenomenon of adipose tissue browning.

Pseudomonas aeruginosa and Burkholderia cepacia are opportunistic human pathogens. Hazards pose to immunocompromited patient groups, most of whom are patients suffering from cystic fibrosis, for which is the infection caused by Pseudomonas aeruginosa or Burkholderia cepacia, often lethal. Bacteria exhibit resistance to most antibiotics and the immunization of patients, paradoxically, worsens the patients' condition. Hen antibodies, unlike mammalian do not activate the complement cascade and do not cause the inflammatory response, therefore, seem to be a suitable tool to protect high risk populations as a means of passive immunization. In this work were prepared specific chicken antibodies against significant virulent factor - bacterial lectins PAIIL and BClA responsible for adhesion. Antibody specificity was demonstrated by ELISA and Western blot. Antibodies were affinity purified and cleaved to fragments. Pneumocytes type II from the lungs of patients with Cystic Fibrosis were isolated and cultured. Tests of the adhesion of bacteria cells were performed on them using ELISA. As an alternative model rat pneumocytes and tumor cell line A549 has been used. Prepared antibodies specifically detect bacterial lectins on the surface of bacteria. Antibody has been shown to reduce adhesion of bacteria to...

β-Glucans are structural components of fungal cell walls which are involved in the immune recognition of fungal pathogens and possess beneficial immunomodulatory activities in isolated form. Here we have developed a soluble chimeric form of the major mammalian β-glucan receptor, Dectin-1, and demonstrate its application for the detection and characterisation of soluble and insoluble β-glucans, including fungal particles, using ELISA, flow cytometric and fluorescence-based microscopy assays.

Les lectines de type C (CLRs) sont des récepteurs impliqués dans la reconnaissance d’oligosaccharides et principalement exprimés à la surface des cellules présentatrices d’antigène (APCs) et notamment des cellules dendritiques (DCs), véritable sentinelle de notre système immunitaire. Elles sont impliquées dans la reconnaissance de motifs spécifiques exprimés à la surface d’agents pathogènes et sont capables de stimuler le système immunitaire afin de déclencher une réponse adaptée. Ce rôle crucial joué par les CLRs dans l’équilibre de la réponse immunitaire confère aux interactions CLR/glycane des perspectives d’applications pharmaceutiques. L’objectif à long-terme du projet de recherche dans lequel cette thèse s’intègre consiste à utiliser ces CLRs pour modeler les réponses du système immunitaire. A cette fin, des néoglycoconjugués spécifiques de chaque CLR doivent être développés. Au cours de cette thèse, 9 CLRs ont été produits BDCA2, DC-SIGN, DC-SIGNR, dectin-1, dectin-2, langerin, LSECtin, MCL and Mincle. Différentes stratégies de production ont été testées en parallèle, incluant des techniques d’adressage au périplasme en vue d’obtenir des protéines solubles et fonctionnelles et de l’expression cytoplasmique, sous forme de corps d’inclusion suivie d’étapes de renaturation qui s’est révélé la plus efficace au final. Une stratégie permettant de construire des tétramères artificiels de CLRs, appelés TETRALEC, a été mise au point. Cet outil permettant le criblage et la caractérisation des lectines a été obtenu avec DC-SIGNR par un marquage spécifique de la lectine. Le complexe TETRALEC a été caractérisé au niveau structural et des tests fonctionnels ont été menés sur des puces à glycanes et des cellules pathogènes. La série de CLRs que nous avons produites a été utilisée pour cribler des puces à glycanes et à glycomimétiques. Ces études nous ont permis de mettre en évidence des interactions dépendantes de l’environnement du glycane et d’identifier de nouveaux glycanes ou glycomimétiques spécifiques de certains CLRs. En effet, de manière étonnante, plusieurs des CLRs testés sont capables, pour un glycane donné, de discriminer des isomères de position ouvrant ainsi de nouveaux questionnements sur la signification biologique de cette sélectivité. De plus des glycomimétiques reconnaissant préférentiellement dectin-2 par rapport à DC-SIGN, DCSIGNR et langerin ont été identifiés. Le choix des glycomimétiques et l’évaluation des étapes de leur optimisation ont été permis par diverses études biophysiques qui ont quantifié la force et la spécificité des interactions. Ceci a permis le développement d’un ligand optimisé sélectif de DC-SIGN. La co-cristallisation de la protéine avec ce ligand a révélé un intéressant mode de liaison qui amène également de nouvelles questions. Simultanément à l’optimisation de ligands monovalents, un premier pas a été réalisé vers la conception d’une molécule pour permettre une vaccination contre le cancer médiée par les CLRs. Les résultats de SPR ont identifié des candidats potentiellement intéressants et des tests biologiques préliminaires ont été réalisés. / C-type Lectin Receptors (CLRs) are carbohydrate-binding proteins mainly expressed on Antigen Presenting Cells (APCs), including dendritic Cells (DCs), the sentinel of the innate immune system. They recognize pathogens or damaged cells by interacting with glycan features and the encounter between the CLR and its ligand constitutes a necessary step for the activation of the adaptive immune system. This crucial role played by CLRs in the balance of immune responses offers to CLR-glycan interactions pharmaceutical applications. The long-term objective of the research project in which this PhD is included is to use these CLRs as modulators in order to tailor the immune system responses. To do so, neoglyco-conjugates selective to each individual CLR have to be developed.Nine different CLRs were produced in this work: BDCA2, DC-SIGN, DC-SIGNR, dectin-1, dectin-2, langerin, LSECtin, MCL and Mincle.Several approaches have been explored in parallel for CLR production, ranking from bacterial periplasmic targeting, aiming to express soluble and functional protein, to inclusion bodies production into the bacterial cytoplasm, with subsequent protein refolding. Our collection of CLRs were used to screen glycan and glycomimetic arrays, highlighting context-dependent binding and identifying natural ligands or glycomimetics selective to each CLRs. Thus, several CLRs were surprisingly able to differentiate between positional isomers of a given N-Glycan, which opens new questions regarding the biological significance. Moreover, glycomimetics with a selectivity towards dectin-2 over DC-SIGN, DC-SIGNR and langerin CLRs have been identified.To guide the choice of the glycomimetics and estimate their optimisation, diverse biophysical studies were performed to evaluate the strength and specificity of the interaction. This enabled the development of an ultimate ligand selective towards DC-SIGN. A co-crystallised structure of the protein with this ligand revealed an interesting binding mode that also opens new questions.Simultaneously to monovalent ligand optimization, a first step towards the design of a highly defined molecule for cancer vaccination by CLR targeting was made. SPR results revealed potential candidates to exploit and preliminary biological assays were performed. Finally, a strategy for tetrameric lectin engineering as been explored, termed TETRALEC. This tool for screening and lectin characterization, has been obtained with one the lectin of the study, DC-SIGNR, by a site-specific labelling of the lectin. The TETRALEC complex was structurally characterised and functional assays were performed on glycan array and pathogen cells.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20442号 / 農博第2227号 / 新制||農||1050(附属図書館) / 学位論文||H29||N5063(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 澤山 茂樹, 教授 左子 芳彦, 准教授 中川 聡 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Rationale: CGD is characterized by recurrent life-threatening infections with bacterial and/or fungal pathogens and granuloma formation. This is caused by defects in the phagocyte NADPH oxidase systems. Deficiency of MBL caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. Combined deficiencies of MBL and NADPH oxidase have not been described commonly, and we report one such case. Methods: The patient records were reviewed, and laboratory data collected. Genetic mutation analysis for MBL2 gene was done at IBT laboratory while CGD mutation analysis is pending. Results: A 2 y/o Caucasian male presents with an intermittent fever for several weeks with an enlarging blister in the right anterolateral thigh. The patient had a history of recurrent pneumonias. Chest CT demonstrated prominent interstitial markings with pulmonary nodules. Lung Biopsy revealed multifocal nodular necrotizing granulomas. Neutrophil Oxidative Burst Test was consistent with X-linked CGD (Patient 14.3/Control 1375.5). The serum level of MBL was 25ng/mL (N=>500). MBL genetic analysis showed LXPA/LYPB (the latter being the defective haplotype). Elevated CRP and polyclonal hyperglobulinemia were consistent with previous report in CGD. The patient was treated with itraconazole, bactrim and interferon gamma with consistent improvement. Conclusions: The role of MBL deficiency in this patient with severe necrotizing granulomatous disease of the lung is unclear. However it seems likely that MBL defects will contribute to worse infections in patients with phagocyte malfunction. The true prevalence of MBL defect in CGD needs to be studied.