La myélopoïèse est finement régulée au niveau métabolique. Les cellules myéloïdes (monocytes et macrophages) sont au centre de nombreuses pathologies. Ma thèse étudie le rôle du métabolisme des cellules myéloïdes dans les pathologies inflammatoires. Je me suis intéressée au métabolisme du cholestérol dans la prolifération anarchique des monocytes (cancérisation) et j’ai étudié des mutations du transporteur ABCA1 (ATP-Binding Cassette A1) impliqué dans l’efflux de cholestérol, qui sont à l’origine d’une prolifération accrue des monocytes, soulignant l’impact du métabolisme lipidique dans la régulation de la prolifération cellulaire. Le métabolisme du glucose est lui aussi impliqué dans la régulation de la myélopoïèse. Nous avons étudié le rôle du transporteur au glucose, Glut-1, dans l’athérosclérose, qui est une pathologie inflammatoire chronique. Dans ce contexte, les CSH et les progéniteurs myéloïdes sur-expriment le transporteur Glut-1, induisant une monocytose, permettant l’accumulation de macrophages dans les lésions. La déficience en Glut-1 prévient la monocytose et le développement des plaques d’athérosclérose. Je me suis intéressée au rôle de la lipase acide lysosomale (LIPA) dans la phagocytose des cellules apoptotiques (efferocytose) par les macrophages, où une grande quantité de cholestérol ingérée doit être dégradée. LIPA en est un acteur central. L’inhibition de cette enzyme provoque un stress oxydatif mitochondrial, et active l’inflammasome NLRP3, contribuant à une inflammation chronique. Cela réduit aussi l’activation des Liver-X-Receptor et induit un défaut d’efferocytose des macrophages, ce qui participe à l’apparition d’une inflammatoire chronique. / Myeloid cells are produced by hematopoiesis, from hematopoietic stem cells (HSCs), a metabolically fine-tuned process. In chronic inflammatory diseases, an increased amount of monocytes is observed (monocytosis). My thesis focuses on the role of myeloid cells metabolism in chronic inflammatory diseases. We focused on the impact of cholesterol metabolism alterations into the anarchic proliferation of monocytes (carcinogenesis). Novel somatic mutations in the cholesterol efflux transporter ATP-Binding Cassette A1 induce carcinogenesis of monocytes, highlighting the impact of cholesterol efflux pathway in monocyte proliferation. I studied glycolysis in atherosclerosis, a chronic inflammatory disease. HSCs and myeloid progenitors exhibited higher Glut-1 expression in a murine model of atherosclerosis, with an enhanced accumulation of macrophages into lesions. A partial deletion of Glut-1 reduced HSCs and progenitors proliferation, limiting monocytosis and atherosclerotic plaques development. I studied the role of lysosomal acid lipase (LIPA) in the phagocytosis of apoptotic cells (efferocytosis). When a macrophage phagocytized an apoptotic cell, an important amount of cholesterol has to be degraded. LIPA is a key player in this process. When LIPA is inhibited, we observed a reduced production of 25- and 27-hydroxycholesterol, leading to an increased mitochondrial oxidative stress, which activated NLRP3 inflammasome activation and a reduced LXR activation. LIPA inhibition leads to a defective efferocytosis in vitro and in vivo. LIPA enzyme is essential to prevent metabolic inflammation by maintaining effective efferocytosis.

Heme oxygenase (HO) is an enzyme that facilitates the oxidative breakdown of free heme into equi-molar concentrations of carbon monoxide (CO), the bile pigment biliverdin IX and free iron. These products have immuno-modulatory and antioxidative properties, which may be useful in the treatment of diseases characterised by low-grade inflammation and oxidative stress, such as insulin resistance and hyperglycaemia in type 2 diabetes. In fact, HO-1 protein levels and carbon monoxide generation are down-regulated in murine models of obesity and type 2 diabetes. Two independent research teams have reported that pharmacological induction of HO activity by protoporphyrin-based compounds, such as hemin and cobalt (III) protoporphyrin IX chloride (CoPP), exerts anti-diabetic effects, including protection from weight gain, systemic inflammation and peripheral insulin resistance, in various experimental models of type 2 diabetes. However, the relative insolubility and instability of hemin in solution and the multiple side-effects of CoPP, including weight loss, preclude their use for the treatment of patients in clinic. Heme arginate (HA) is a stable and soluble composition of hemin and L-arginine (LA) in a solution containing propylene glycol, ethanol and water. Furthermore, HA is licensed for the treatment of acute porphyria in several European countries. Therefore, HA may potentially be used in clinical trials. The current PhD thesis tests the hypothesis that the heme component of HA ameliorates hyperglycaemia via induction of HO activity in the leptin receptor deficient db/db (db/db) mouse model of type 2 diabetes. A preliminary in vivo study demonstrates that the heme but not the LA component of HA exerts an anti-hyperglycaemic effect in db/db mice. In a separate in vivo study, concomitant treatment of HA with stannous (IV) mesoporphyrin IX dichloride (SM), an inhibitor of HO activity, further improves the glycaemic control despite complete abrogation of the HA-mediated increase in HO activity in db/db mice. This result is in contrast to the above stated hypothesis, and demonstrates that the antihyperglycaemic effect of HA is due to a HO activity independent mechanism. Furthermore, the ameliorative effect of HA and HA+SM treatment on hyperglycaemia in db/db mice coincides with a gain in body and visceral fat weight, a reduction in islet β-cell inflammation and the preservation of islet β-cell function. Subsequent in vitro experiments demonstrate that HA exerts anti-inflammatory effects by a HO activity independent mechanism in pro-inflammatory in vitro models such as in cytokine mix-stimulated MIN6 β-cells and in classically activated bone marrow derived macrophages (BMDMs). In conclusion, the current thesis demonstrates the novel finding that the heme component of HA can exert anti-inflammatory and anti-diabetic effects via a HO activity independent mechanism. Future work should focus on studies to test the hypothesis that the interaction of heme with the nuclear receptor Rev-erb-α is responsible for the anti-inflammatory and anti-diabetic effects of HA.

No description available.

L’immunothérapie est une avancée majeure dans le traitement des cancers, même s’il reste fondamental d’identifier de nouvelles cibles pour optimiser la réponse anti-tumorale. Des enzymes impliquées dans le métabolisme d'acides aminés ont été décrites comme pouvant inhiber ces réponses immunitaires et ainsi favoriser la progression tumorale. Elles sont donc devenues des cibles thérapeutiques de premier choix. L’IL-4 induced gene 1 (IL4I1) est une phénylalanine oxydase qui, au cours de cette dernière décennie, s’est dévoilée dans la littérature comme un véritable outil de la tolérance périphérique. Ses fonctions restent cependant largement inexplorées. Au cours de ma thèse, j’ai étudié les propriétés immunorégulatrices de l’IL4I1 en contexte tumoral dans un modèle de mélanome spontané (souris Ret), mais également dans la physiologie des lymphocytes B (LB). Dans un premier temps, mes résultats mettent en avant que l’activité enzymatique de l’IL4I1 dans les rates des souris Ret est corrélée avec la sévérité de la maladie. De façon intéressante, l’inactivation génétique de l’IL4I1 dans ce modèle (RetIL4I1KO) retarde le développement de la tumeur primaire, mais aussi la dissémination métastatique. Nous démontrons que l’IL4I1, principalement exprimée par les cellules myéloïdes, a la capacité de façonner la composante immunitaire du microenvironnement tumoral, et ce, par le recrutement préférentiel de cellules myéloïdes au détriment d’effecteurs lymphocytaires T. Par ailleurs, mes données chez les souris déficientes pour l’IL4I1 (IL4I1KO) ont mis en évidence un rôle crucial de l’IL4I1, régulant de nombreuses étapes de la biologie des LB. Les souris IL4I1KO présentent une sortie accélérée des LB de la moelle osseuse, entraînant l'accumulation de LB folliculaires en périphérie. Ces souris ont un taux sérique élevé d'immunoglobulines naturelles et des anticorps auto-réactifs. Nous démontrons aussi que l’IL4I1 contrôle la réaction du centre germinatif, la différenciation des plasmocytes et la production d'anticorps spécifiques en réponse à une immunisation par un antigène T-dépendant. In vitro, l’absence de l’IL4I1 dans les LB augmente l’activation des voies de signalisation en aval du récepteur à l’antigène des LB (BCR), ainsi que leur prolifération. Par conséquent, nos résultats révèlent une propriété clé de l’IL4I1, contrôlant négativement l'activation dépendante du BCR. Compte tenu de l’effet majeur de l’IL4I1 sur la biologie des LB, il reste important d’investiguer si l’hyperréactivité du BCR des LB déficients pour l’IL4I1 participe au meilleur contrôle de la progression tumorale. / Immunotherapy is one of the most promising advance in cancer treatments. It remains essential to identify new targets to maximize the anti-tumor immune response. Enzymes involved in amino acid metabolism have been described to impede these responses and thus promote tumor progression. They became good therapeutic targets. IL-4-induced gene 1 (IL4I1) is a phenylalanine oxidase which, over the last decade, was unveiled as a real tool for peripheral tolerance. However its functions remain largely unexplored. During my PhD thesis, I studied the immunoregulatory properties of IL4I1 in a tumoral context involving a spontaneous melanoma model (Ret mice), but also in the B cell physiology. First, my results highlight that the IL4I1 enzymatic activity is positively correlated with disease aggressiveness in the spleen of Ret mice. Interestingly, genetic inactivation of IL4I1 in this model (RetIL4I1KO) delayed the development of both primary tumor and metastatic dissemination. We demonstrate that IL4I1, mainly expressed by myeloid cells, has the ability to shape the immune compartment within the tumor microenvironment, through recruitment of myeloid cells instead of activated T cells. Furthermore, my data in mice deficient for IL4I1 (IL4I1KO) have emphasized a crucial role of IL4I1 in regulating many steps of B cell biology. Indeed, IL4I1KO mice exhibit an accelerated B cell egress from the bone marrow, resulting in the accumulation of peripheral follicular B cells. These mice have a high serum level of natural immunoglobulins and auto-reactive antibodies. We also demonstrate that IL4I1 controls the germinal center reaction, plasma cell differentiation and specific antibody production in response to a T-dependent antigen immunization. In vitro, the absence of IL4I1 in B cells increases their proliferation and the activation of signaling pathways upon B cell receptor (BCR) engagement. Thus, our results reveal a key role of IL4I1, which negatively controls the BCR-dependent activation. Regarding these effects of IL4I1 on B cell biology, it remains important to evaluate whether the BCR-dependent hyperreactivity in IL4I1KO mice contributes in the tumor control.

Glioblastoma Multiforme (GBM) is an extremely aggressive grade IV brain tumour that is highly infiltrative and can spread to other parts of the brain quickly. It is the most common primary brain tumour where patients have a median survival of 14.6 months. Symptoms vary depending upon the location of the tumour and include seizures, progressive headaches and focal neurological deficit. The poor prognosis is characterised by deregulation of many key signalling pathways involving survival, growth, apoptosis and evasion of immune surveillance. In this study, we investigated whether complement factor H related protein 5 (CFHR5) from primary GBM cells direct from patients exhibited functional activity similar to factor H. The presence of CFHR5 was validated by western blot and ELISA technique from B30, B31 and B33 primary GBM cells. The functional capacity of CFHR5 was examined through the alternative pathway, co-factor, and decay acceleration assay. We demonstrated that CFHR5 was able to inhibit the alternative pathway through the same mechanism as factor H. Emerging evidence had shown that the innate immune protein surfactant protein D (SP-D) and recombinant human SP-D (rhSP-D) were able to induce apoptosis in eosinophilic leukaemic cells. We studied the ability of rhSP-D to induce apoptosis in U87 GBM cells through apoptotic and viability assays. rhSP-D was unable to mediate cell death and instead increased cell viability. This led us to investigate the expression of SP-D in U87 and B30 GBM cells through western blot, ELISA and immuno-fluorescence detection. We demonstrated novel information about the production of SP-D by GBM cells. To extend our study, we investigated the interaction of THP-1 macrophage with rhSP-D bound U87 cells. We carried out live cell imaging, RT-qPCR, and cell viability assays, to study the changes in cytokine expression and viability of cells. THP-1 did not engulf U87 cells; however, it did reduce the number of cells and decrease the expression of pro-tumourigenic cytokines. This study highlights the ability of primary GBM cells to evade innate immune detection by the secretion of functionally active CFHR5. It also demonstrated the ability of U87 to evade destruction by rhSP-D and THP-1 highlighting the extremely aggressive behaviour of the tumour and lack of new treatment to improve prognosis in over a decade.

Abstract At present, diagnostic reagent is the most fruitful and important realm in internal biotech product commercialization. Compared to other biotech medicine with the industrial characteristics of complications such as: high investment, high risk, and long-term research and development; diagnostic reagent, relatively, acquires a lower threshold for technique, a short-term research and development, less investment and a short time for feedback. In addition, people gradually acquire the notions of prophylaxis and health care, as a result, the potentiality of diagnostic reagent comes to the market¡¦s notice. The process of diagnostic reagent development can be divided by the mainstream techniques into four phases including: the technology of chemical test, the technology of enzyme test, the technology of immunoassay test, the technology of nuclease molecule test and biochip. From this, we know that the technology of immunity test is still the mainstream in the market, but there is a trend toward the development of the technology of nuclease molecule immunoassay. General Biologicals Corp. is the first built diagnostic reagent manufacture in our country, and, so far, it is the only diagnostic reagent manufacture that meets with the Department of Health¡¦s CGMP standard. The General Biologicals Corp. is the only internal manufacture that produces ELISA and EIA microplate. However, the General Biologicals Corps has less than 1% of six hundred million, and foreign manufactures have it all. The General Biologicals Corps went public in recent years, and the company has a microplate immunoassay diagnostic reagent factory for over twenty years. Those doctors, examiners and sales clerks who used the immunoassay reagent produced by the General Biologicals Corps are all in high positions in this industry. They know this company and also their reagent kits which have a low sensitivity. More important is that the reagent kits are not improved for all these years. The bad impression of the company makes the customers and collaborators have doubts about the quality, the attitude of the personnel and the sincerity of the company. It results in a trust crisis. In light of the five competitiveness analyses in the executive strategies: dynamic competition of the company in enterprise; S.W.O.T competitiveness analysis; smiling curve and bitter smiling curve; red ocean and blue ocean strategy and, finally, the administration of registering examined medical instrument, to see the integral competitiveness of the company in the industrial competitive environment. Can the General Biologicals Corp. seek league and collaboration in the competitors to seize the internal market, and then the overseas market. By these analyses and advises, to reengeneering the General Biologicals Corp. has a new management on the visible foundation. Key Word¡GImmunity Test, Nuclear Molecular Immuno assay. Five Force, Reengeneering

No description available.

Le diagnostic précoce de l'hypothyroïdie permet d'initier le traitement au plus tôt et ainsi de préserver la santé du patient. Le bénéfice du traitement de l'hypothyroïdie franche a été depuis longtemps établi, mais les critères de prise en charge des patients en hypothyroïdie fruste sont encore difficiles à définir. En effet, les symptômes ne sont pas toujours présents et leur appréciation est subjective. Afin d'établir le diagnostic et la prise en charge, le médecin s'appuie sur le dosage de la thyréostimuline (TSH) dans le sang, qui peut éventuellement être complété par le dosage des hormones thyroïdiennes. Le dosage de la TSH, très sensible, peut présenter sur un même échantillon sanguin d'importantes variations qui rendent d'autant plus difficiles la décision du médecin et le suivi du patient. Le polymorphisme naturel de la TSH peut expliquer en partie ces variations. La TSH appartient en effet à la famille des hormones glycoprotéiques et sa glycosylation peut constituer jusqu'à 30% de son poids. Dans le cas de l'hypothyroïdie en particulier, ces glycanes sont modifiés et présentent une plus grande quantité d'acides sialiques terminaux. Ainsi, certaines variations entre les dosages de la TSH, qui freinent actuellement leur harmonisation, peuvent être dues à des différences de reconnaissance de glycoformes par les anticorps utilisés dans les dosages. Dans ce contexte, l'objectif de de ces travaux était de contribuer à la construction de dosages plus performants que ceux actuellement utilisés dans le diagnostic de l'hypothyroïdie. Un nouveau calibrateur recombinant sialylé plus proche de la TSH circulante dans l'hypothyroïdie a alors été produit. De nouvelles associations d'anticorps monoclonaux ont été utilisées pour construire des dosages. Les nouveaux dosages sélectionnés ont ensuite été calibrés avec la TSH sialylée produite et le calibrateur de référence international. Ils ont alors servi à doser plusieurs séries de sérums de patients. Ces travaux ont donc validé l'utilisation d'un nouveau calibrateur d'origine recombinante pour les dosages de la TSH, ce qui devrait à l'harmonisation des dosages existants. / If iodine deficiency is the first cause of low thyroid hormone levels in the world, there are also other etiologies to thyroid disorders. Diagnosis of those allow an early treatment to preserve patient's health. Although there is a general agreement concerning treatment of overt hypothyroidism, treatment of subclinical hypothyroidism is still under debate. In these cases, symptoms are, by definition, not always present. In order to establish diagnosis, the clinicians rely on the measurement of circulating thyroid stimulating hormone (TSH, potentially completed with thyroid hormones measurement). TSH assays are now very sensitive, but can present important between assays variations. The diagnosis and follow up of the patient are consequently complicated. Natural polymorphism of TSH can explain a part of this variability. TSH belongs to the glycoprotein hormones family and its glycans can count for more than 30% of its weight. In hypothyroidism, these glycans are subject of modulation and present higher levels of terminal sialylation. Variation in immuno-assays can be explained by these modifications of sialylation if recognition by antibodies used in immuno-assays is glycosylation dependent. In this context, the aim of this work was to contribute to the construction of new immuno-assays, more reliable in the early diagnosis of subclinical hypothyroidism. During this thesis a new recombinant standard closer to circulating TSH was produced. The total level of sialylation was higher and better mimic the circulating forms in hypothyroidism. In order to select the best antibodies associations in immuno-assays, new antibodies were obtained and associated with commercially available antibodies. New immuno assays improvement is based on the following two approaches: the first one is the use of a new standard which presents glycoformes closer to the circulating TSH and the second one consists in an appropriate selection of antibodies involved in the assays. The new assays were used to measure TSH concentration in blood samples. These studies associated with validation steps allow us to select four assays and constitute a proof of concept for the use of a new sialylated recombinant standard for TSH assays. This can contribute to the needed harmonization of TSH assays.

Semiconductor nanocrystals, otherwise known as Quantum dots (Q dots), are a new type of fluorophore that demonstrates many advantages over conventional organic fluorophores. These advantages offer the opportunity to improve known immunofluorescent methods and immunofluorescent biosensors for rapid and portable bacterial detection in water. The detection of the micro organism Escherichia coli O157:H7 by attenuation of a fluorophore’s signal in water was evaluated alone and in the presence of another bacterial species. A sandwich immunoassay with antibodies adhered to SU-8 as a conventional comparison to our novel attenuation detection was also conducted. The assays were tested for concentration determination as well as investigation for cross reactivity and interference from other bacteria and from partial target cells. In order to immobilize the capture antibodies on SU-8, an existing immobilization self-assembly monolayer (SAM) for glass was modified. The SAM was composed of a silane ((3-Mercaptopropyl) trimethoxysilane (MTS)) and hetero-bifunctional cross linker (N-γ-maleimidobutyryloxy succinimide ester (GMBS)) was utilized in this procedure. The SU-8 surface was activated using various acids baths and oxygenated plasma, and it was determined that the oxygenated plasma yielded the best surface attachment of antibodies. The use of direct fluorophore signal attenuation for detection of the target E. coli resulted in the lowest detectable population of 1x10¹ cfu/mL. It was not specific enough for quantitative assessment of target concentration, but could accurately differentiate between targeted and non-targeted species. The sandwich immunofluorescent detection on SU-8 attained the lowest detectable population of 1x10⁴ cfu/ml. The presence of Klebsiella pneumoniae in solution caused some interference with detection either from cross reactivity or binding site blocking. Partial target cells also caused interference with the detection of the target species, mainly by blocking binding sites so that differences in concentration were not discernable. The signal attenuation not only had a better lowest detectable population but also had less measurement error than the sandwich immunoassay on SU-8 which suffered from non-uniformed surface coverage by the antibodies.

Le diagnostic précoce de l'hypothyroïdie permet d'initier le traitement au plus tôt et ainsi de préserver la santé du patient. Le bénéfice du traitement de l'hypothyroïdie franche a été depuis longtemps établi, mais les critères de prise en charge des patients en hypothyroïdie fruste sont encore difficiles à définir. En effet, les symptômes ne sont pas toujours présents et leur appréciation est subjective. Afin d'établir le diagnostic et la prise en charge, le médecin s'appuie sur le dosage de la thyréostimuline (TSH) dans le sang, qui peut éventuellement être complété par le dosage des hormones thyroïdiennes. Le dosage de la TSH, très sensible, peut présenter sur un même échantillon sanguin d'importantes variations qui rendent d'autant plus difficiles la décision du médecin et le suivi du patient. Le polymorphisme naturel de la TSH peut expliquer en partie ces variations. La TSH appartient en effet à la famille des hormones glycoprotéiques et sa glycosylation peut constituer jusqu'à 30% de son poids. Dans le cas de l'hypothyroïdie en particulier, ces glycanes sont modifiés et présentent une plus grande quantité d'acides sialiques terminaux. Ainsi, certaines variations entre les dosages de la TSH, qui freinent actuellement leur harmonisation, peuvent être dues à des différences de reconnaissance de glycoformes par les anticorps utilisés dans les dosages. Dans ce contexte, l'objectif de de ces travaux était de contribuer à la construction de dosages plus performants que ceux actuellement utilisés dans le diagnostic de l'hypothyroïdie. Un nouveau calibrateur recombinant sialylé plus proche de la TSH circulante dans l'hypothyroïdie a alors été produit. De nouvelles associations d'anticorps monoclonaux ont été utilisées pour construire des dosages. Les nouveaux dosages sélectionnés ont ensuite été calibrés avec la TSH sialylée produite et le calibrateur de référence international. Ils ont alors servi à doser plusieurs séries de sérums de patients. Ces travaux ont donc validé l'utilisation d'un nouveau calibrateur d'origine recombinante pour les dosages de la TSH, ce qui devrait à l'harmonisation des dosages existants. / If iodine deficiency is the first cause of low thyroid hormone levels in the world, there are also other etiologies to thyroid disorders. Diagnosis of those allow an early treatment to preserve patient's health. Although there is a general agreement concerning treatment of overt hypothyroidism, treatment of subclinical hypothyroidism is still under debate. In these cases, symptoms are, by definition, not always present. In order to establish diagnosis, the clinicians rely on the measurement of circulating thyroid stimulating hormone (TSH, potentially completed with thyroid hormones measurement). TSH assays are now very sensitive, but can present important between assays variations. The diagnosis and follow up of the patient are consequently complicated. Natural polymorphism of TSH can explain a part of this variability. TSH belongs to the glycoprotein hormones family and its glycans can count for more than 30% of its weight. In hypothyroidism, these glycans are subject of modulation and present higher levels of terminal sialylation. Variation in immuno-assays can be explained by these modifications of sialylation if recognition by antibodies used in immuno-assays is glycosylation dependent. In this context, the aim of this work was to contribute to the construction of new immuno-assays, more reliable in the early diagnosis of subclinical hypothyroidism. During this thesis a new recombinant standard closer to circulating TSH was produced. The total level of sialylation was higher and better mimic the circulating forms in hypothyroidism. In order to select the best antibodies associations in immuno-assays, new antibodies were obtained and associated with commercially available antibodies. New immuno assays improvement is based on the following two approaches: the first one is the use of a new standard which presents glycoformes closer to the circulating TSH and the second one consists in an appropriate selection of antibodies involved in the assays. The new assays were used to measure TSH concentration in blood samples. These studies associated with validation steps allow us to select four assays and constitute a proof of concept for the use of a new sialylated recombinant standard for TSH assays. This can contribute to the needed harmonization of TSH assays.