Background: Hemophilia A is X-linked recessive congenital bleeding disorder. Exogenous infusion of FVIII is the treatment of choice in hemophilia A patients. However, inhibitor development remains the major problem in management of Hemophilia A. It has been showed that IVIG has immunomodulatory effects and it has been being used in the treatment of several autoimmune and inflammatory disorders. Here, we investigated the effect of co-administration of FVIII with IVIG on the development of inhibitor in naive and previously immunized hemophilia A mice. Methods: Initially, hemophiliac mice were immunized by weekly intraperitoneal injection of human recombinant FVIII (rFVIII). The mice then were treated, either by rFVIII/IVIG co-injection or rFVIII alone. In the other experimental group, naive hemophiliac mice were treated with rFVIII/IVIG co-injection for four weeks followed by injection of either rFVIII or rFVIII/IVIG. Plasma's anti-FVIII Ab titer was measured using ELISA. Results: Weekly injection of rFVIII led to the development of anti-FVIII Ab in all previously untreated mice. Treatment of those immunized mice with rFVIII/IVIG co-injection did not reduce the level of pre-existing Ab. On the other hand, naive mice treated with rFVIII/IVIG co-injection showed significantly less Ab titer compared to the mice received rFVIII alone after 4 weeks (mean Ab titre of 1 compared to 39, in rFVIII/IVIG co-injection and rFVIII groups respectively). Although the rFVIII/IVIG-treated mice developed immune response following the injection of rFVIII alone, Ab titer in those that kept receiving rFVIII/IVIG co-injection remained lower compared to other groups during the whole twelve weeks of the experiment. Conclusions: Co-injection of rFVIII with IVIG decreased the anti-FVIII immune response in previously untreated hemophilia A mice. These findings suggest that IVIG co-administration can be effective in management of hemophilia A patients at risk of inhibitor development. / Thesis / Master of Applied Science (MASc)

The infusion of intravenous immunoglobulin therapy in the home setting requires a critical nursing assessment and interventions aimed at managing and preventing the escalation of adverse events. Some patients experience side effects that necessitate a rapid response by field nurses, requiring standing orders for nursing administration and the availability of essential medications to alleviate symptoms in the patient's home. The clinical practice issue was that the home health agency did not have a uniform clinical practice nursing guideline to assist field nurses in providing rapid responses for managing infusion-related reactions. The purpose of this project was to develop an evidence-based clinical practice guideline using standing orders for the comprehensive management of immunoglobulin side effects in the patient's home. The practice-focused question centered on whether the use of a nursing practice guideline based on interprofessional collaboration could manage the side effects of patients in the home by decreasing the use of emergent care and improved quality of care for those patients susceptible to significant side effects. An interdisciplinary expert panel experience in IVIG l used Newman's system theory and the reach, effectiveness, adoption, implementation, maintenance framework for interprofessional collaboration in developing a clinical nursing guideline with a standing order for rating side effects. Panelists used the appraisal of guidelines, research, and evaluation II tool to appraise the evidence for the guideline. The use of clinical guideline with standing orders to address the needs of patients in the home setting may lead to positive social change by enabling more rapid management of symptoms, more effective care in the home, and improved patient outcomes

La maladie du greffon contre l’hôte (GvHD) est une complication majeure des greffes de cellules souches hématopoïétiques (HSCT) qui survient dans 30 à 70% des cas et peut causer la mort, malgré un traitement prophylactique bien conduit. Il existe donc une réelle demande clinique pour améliorer ces traitements prophylactiques. Parce que ces traitements prophylactiques reposent en général sur des agents immunosuppresseurs, ceux-ci contribuent à diminuer la reconstitution immunitaire du patient, ce qui a un impact défavorable sur les infections et les taux de rechute d’hémopathie maligne, et donc limite leur utilisation. Les immunoglobulines (IVIG) pourraient représenter une alternative intéressante puisqu’elles ont des propriétés immunomodulatrices et qu’elles sont de plus couramment utilisées en clinique pour traiter des patients ayant un déficit immunitaire. Leur capacité à réduire l’apparition et la sévérité de la GvHD, sans toutefois inhiber ou nuire à la reconstitution immunitaire chez le patient n’a néanmoins jamais été clairement démontrée. Les objectifs de ce projet sont donc d’évaluer l’efficacité des IVIG à réduire l’incidence et la sévérité de la GvHD dans un modèle murin humanisé de GvHD, ainsi que de déterminer le mécanisme d’action des IVIG. Ce modèle consiste à injecter des huPBMCs à des souris immunodéprimées ne pouvant les rejeter. Les résultats obtenus suggèrent que les IVIG possèdent un effet immunomodulateur permettant de réduire les signes cliniques et de retarder l’apparition de la GvHD, tout en permettant l’apparition de cellules NK. Les IVIG agiraient de façon indirecte sur les huPBMCs afin d’induire l’apparition des cellules NK. / Graft-versus-host disease (GvHD) is a major complication following hematopoietic stem cell transplantation (HSCT), occuring in 30 to 70% of HSCT despite proper prophylactic treatment, and can result in death. It is therefore primordial to improve the prophylactic treatments in order to reduce the frequency and severity of GvHD. Since these prophylactic treatments are based on the use of immunosuppressive agents, they inhibit the immune reconstitution and thus increase the risk of infections and relapse. Because of their immunomodulating properties, immunoglobulins (IVIG) represent a promissing alternative. Furthermore, IVIG are already widely used in patients with immune deficits. Since no study has yet clearly shown that IVIG can reduce GvHD without inhibiting the immune reconstitution in the patient, the scientific community is still debating their usefullness in GvHD prevention. Should their efficacy to reduce the incidence and severity of GvHD without impairment to the immune reconstitution be demonstrated, the use of IVIG could change the clinical outcome for HSCT patients, improving their remission and quality of life. The objectives of this project are to evaluate IVIG efficacy in GvHD prevention and to define the mechanism of action of IVIG. To fulfill these objectives, we will use a GvHD humanized mouse model, which consists of injecting huPBMCs in immunodeficient mice who can not reject them. Our results demonstrate an immunomodulatory effect of IVIG, allowing for reduced clinical signs and a slighlty increased survival in GvHD. A population of NK cells also appeared upon IVIG treatment and is believed to be indirectly induced by IVIG.

Therapeutische Erfolge von IVIG sind gut dokumentiert, aber die zu Grunde liegenden molekularen Mechanismen sind noch nicht vollständig erforscht. Molekulare Analysen unseres Labors über die Interaktion von IVIG mit Fabs von Patienten, die an einer autoimmunen Thrombozytopenie (ITP) leiden zeigten, dass die am häufigsten selektierten Fab von den V3-23 und V3-30 VH-Keimbahngenen abstammten. Eine weitere Studie mit IgG und IgM Phagen-Display Bibliotheken von einem gesunden Spender zeigten ebenfalls eine bevorzugte Reaktivierung von IVIG mit Fabs vom Ursprung der V3-23 und V3-30 Gene. Es konnte gefolgert werden, dass diese Interaktion von IVIG mit Fabs von diesen zwei VH-Genen weder alleine auf den Gesundheitsstatus des Spenders zurückzuführen war, noch auf eine zuvor erfolgte Behandlung mit IVIG. Diese Dissertation wurde unter Verwendung der Phagen-Display Technologie unternommen, um die molekulare Interaktion von IVIG mit Antikörpern zu erforschen, die von einem Patienten kloniert wurden, der an einem systemischen Lupus erythematodes und rheumatischem Fieber leidet. Die Resultate waren mit den früheren Studien zu vergleichen, insbesondere mit den Daten eines Patienten, der zu der ITP einen Lupus entwickelte. 23 Fabs, welche 7 unabhängige Klone repräsentierten, wurden isoliert. Im Gegensatz zu von Patienten mit ITP abstammenden Klonen reagierte keines von den in dieser Studie selektierten Fabs mit Thrombozyten. Die über IVIG gebundene Fab-Phagen stammten hierbei ausschließlich von den V3-23 und V3-30 VH-Genen ab. Darüber hinaus wurde beobachtet, dass von diesen Fabs verschiedene CDR3 Regionen einschließlich verschiedenen D- und JH-Gensegmenten benutzt wurden. Die Ergebnisse zeigten weiterhing, dass die Bindung von IVIG an die Fabs unabhängig von der Leichten Kette war. Ihrem Keimbahngen-Ursprung entsprechend hatten die Fabs Aminosäuren an Positionen in den FR1, FR3 und im 3'-Ende von CDR2, die dafür bekannt sind, dass sie für die Bindung des B-Zell-Superantigens Staphylococcus Protein A (SpA) essentiell sind. Es wurde gezeigt, dass sich zwar einige von den Fabs stark an SpA banden, aber keine Korrelation in der Intensität zur Bindung mit IVIG vorlag. Einige Fabs zeigten eine schwache Bindung an HIV gp120, einem anderen B-Zell-Superantigen. Zusammenfassend lässt sich aus der vorliegenden Studie und den vorherigen Ergebnissen schließen, dass ein Anteil von IVIG wie ein B-Zellen Superantigen funktionieren könnte, das für die Bildung und Regulation des normalen B-Zellen Repertoires wichtig ist. Der Bindungsmechanismus scheint ähnlich, aber nicht identisch mit dem der anderen getesteten B-Zellen-Superantigene zu sein. / The beneficial therapeutic effects of IVIG are well documented, but the underlying molecular mechanisms are not fully understood. Recent investigations from our laboratory into the molecular analysis of Fabs bound by IVIG from patients suffering from autoimmune thrombocytopenia revealed that the most frequently selected Fabs originated from the V3-23 and V3-30 VH germline genes. A subsequent study with IgG and IgM phage display libraries from a healthy donor also demonstrated a preferential reactivity of IVIG to Fabs of V3-23 and V3-30 origin. That study revealed that the unique reactivity of IVIG to Fabs of these two VH gene loci was not restricted to the autoimmune nature of the donors, neither to previous treatment with IVIG. One of the thrombocytopenia patients developed lupus. This study was undertaken to study the molecular interaction of IVIG with antibodies selected from a patient suffering from systemic lupus erythematosus and rheumatic fever using phage display technology, and to compare the results with the previous studies. Twenty-three Fabs representing seven independent clones were isolated. In contrast to ITP-derived clones, none of the Fabs selected in this study reacted with platelets. The Fab phages bound by IVIG were sequenced in order to determine their VH gene usage and clonal relatedness. V3-23 and V3-30 VH genes were found to be exclusively utilized by the Fab phages bound by IVIG. Moreover, different CDR3 regions including different D and JH gene segments were observed to be used by these Fabs. The results further showed that the binding of IVIG to the Fabs was independent of the light chain since different light chains were observed to be associated with the VH3 immunoglobulins. Detailed sequence analysis of the Fabs revealed the presence of amino acid residues at positions within FR1, FR3, and the 3' end of CDR2 that are known to be contacted by the B cell superantigen Staphylococcus protein A (SpA). Some of the Fabs were shown to bind strongly to SpA, but there was no correlation with the binding-intensity to IVIG. Some bound very weakly to HIV gp120, another B cell superantigen. This study, together with previous results, suggests that a subset of IVIG may function as a B cell superantigen that may significantly shape the B cell repertoire. The binding mechanism appears to be similar but not identical to the other tested B cell superantigens.

La maladie du greffon contre l’hôte (GvHD) est une complication majeure des greffes de cellules souches hématopoïétiques (HSCT) qui survient dans 30 à 70% des cas et peut causer la mort, malgré un traitement prophylactique bien conduit. Il existe donc une réelle demande clinique pour améliorer ces traitements prophylactiques. Parce que ces traitements prophylactiques reposent en général sur des agents immunosuppresseurs, ceux-ci contribuent à diminuer la reconstitution immunitaire du patient, ce qui a un impact défavorable sur les infections et les taux de rechute d’hémopathie maligne, et donc limite leur utilisation. Les immunoglobulines (IVIG) pourraient représenter une alternative intéressante puisqu’elles ont des propriétés immunomodulatrices et qu’elles sont de plus couramment utilisées en clinique pour traiter des patients ayant un déficit immunitaire. Leur capacité à réduire l’apparition et la sévérité de la GvHD, sans toutefois inhiber ou nuire à la reconstitution immunitaire chez le patient n’a néanmoins jamais été clairement démontrée. Les objectifs de ce projet sont donc d’évaluer l’efficacité des IVIG à réduire l’incidence et la sévérité de la GvHD dans un modèle murin humanisé de GvHD, ainsi que de déterminer le mécanisme d’action des IVIG. Ce modèle consiste à injecter des huPBMCs à des souris immunodéprimées ne pouvant les rejeter. Les résultats obtenus suggèrent que les IVIG possèdent un effet immunomodulateur permettant de réduire les signes cliniques et de retarder l’apparition de la GvHD, tout en permettant l’apparition de cellules NK. Les IVIG agiraient de façon indirecte sur les huPBMCs afin d’induire l’apparition des cellules NK. / Graft-versus-host disease (GvHD) is a major complication following hematopoietic stem cell transplantation (HSCT), occuring in 30 to 70% of HSCT despite proper prophylactic treatment, and can result in death. It is therefore primordial to improve the prophylactic treatments in order to reduce the frequency and severity of GvHD. Since these prophylactic treatments are based on the use of immunosuppressive agents, they inhibit the immune reconstitution and thus increase the risk of infections and relapse. Because of their immunomodulating properties, immunoglobulins (IVIG) represent a promissing alternative. Furthermore, IVIG are already widely used in patients with immune deficits. Since no study has yet clearly shown that IVIG can reduce GvHD without inhibiting the immune reconstitution in the patient, the scientific community is still debating their usefullness in GvHD prevention. Should their efficacy to reduce the incidence and severity of GvHD without impairment to the immune reconstitution be demonstrated, the use of IVIG could change the clinical outcome for HSCT patients, improving their remission and quality of life. The objectives of this project are to evaluate IVIG efficacy in GvHD prevention and to define the mechanism of action of IVIG. To fulfill these objectives, we will use a GvHD humanized mouse model, which consists of injecting huPBMCs in immunodeficient mice who can not reject them. Our results demonstrate an immunomodulatory effect of IVIG, allowing for reduced clinical signs and a slighlty increased survival in GvHD. A population of NK cells also appeared upon IVIG treatment and is believed to be indirectly induced by IVIG.

Mucocutaneous lymph node syndrome or Kawasaki syndrome is very severe disease.The most common symptom includes a high fever which is probably due to inflammation of blood vessels so-called vasculitis. . Several months old babies to preschool children are the most often affected group of patients. Specific and typical symptoms of Kawasaki disease include long term fever, conjunctivitis, erythema, lymphadenopathy, mucosal changes as red swollen lips and strawberry tongue and multiple rashes.Qualitative and quantitative research was applied in the diploma thesis "Kawasaki disease in the contemporary society through nurse´s eyes". Three basic objectives and three research questions were established in this diploma thesis.

Mucocutaneous lymph node syndrome or Kawasaki syndrome is very severe disease. The most common symptom includes a high fever which is probably due to inflammation of blood vessels so-called vasculitis. The characteristic statement is that the etiology of disease remains unknown and the origin is not clarified. Several months old babies to preschool children are the most often affected group of patients. It is relatively rare, modern and mystery disease in contemporary and industrial developed society. The first appearance of this disorder is in 20th century. The disorder was first described by well-known Tokyo origin pediatrician Tomisaku Kawasaki in Japan, who studied this disease very thoroughly. This rare disease is considered autoimmune in origin triggering by an infectious agent especially in those who are genetically predisposed. Specific and typical symptoms of Kawasaki disease include long term fever, conjunctivitis, erythema,lymphadenopathy, mucosal changes as red swollen lips and strawberry tongue and multiple rashes. Related cardiovascular complications should be pointed out especially coronary or other major arteries aneurysms and their ruptures, pericardial effusion, heart inflammations, coronary thrombosis, pericardial exudates, arrhythmias, or mitral valve disease. There is no specific test for identification of Kawasaki disease despite the contemporary technological and diagnostic options. So, the easiest way to establish diagnosis is to recognize typical symptoms, blood/urine/spinal fluid testing and then performing X-ray, electrocardiogram and echocardiogram. This disease has very low mortality, the short-term prognosis is excellent and relapse of symptoms is rare. In total, 337 children were diagnosed with Kawasaki disease and admitted to hospital during the evaluation period (2007-2015) in the Czech Republic. This is significantly lower incidence comparing to the other countries in the world. Diploma thesis "Kawasaki syndrome in contemporary society from a nurse´s point of view" was designed as theoretical with a supplement of short case study. The goal of presence of this case study is to better comprehend presented topic. The scope of problem is described from theoretical point of view in partial chapters of this diploma thesis. All the information mentioned in this thesis quotes verified sources, publications written by the Czech and foreign specialists in this particular field. The goal of the thesis was to describe, based on available literature, the issue of Kawasaki disease in children focused on specifics of nursing. All the information was searched in bibliographical issued writings, in databases, or on the Internet. Based on the goal determined in advance, scientific methods such as explanation, analysis, synthesis and demonstration of data were chosen for composing this diploma thesis. The output of theoretical work is to present complex view on the issue of Kawasaki syndrome particularly for non-medical professionals. It came out that the profession of nurse has its own irreplaceable place in the context of Kawasaki disease. As well as competences of nurse are essential. These competences are provided for improving quality of life of children with this unfamiliar and life-threatening disease.

Οι ανοσολογικές διαταραχές στην οξεία θρομβοπενική πορφύρα (ΙΘΠ) της παιδικής ηλικίας εξακολουθούν να αποτελούν αντικείμενο μελέτης. Η έκφραση των Th1/Th2 κυτταροκινών έχει μελετηθεί σε αυτοάνοσα νοσήματα και φαίνεται ότι παίζει ανοσορυθμιστικό ρόλο. Έτσι, θεωρήθηκε σκόπιμη η μελέτη της έκφρασης Th1/Th2 κυτταροκινών καθώς και των επιπέδων του TGF-β1 στο πλάσμα σε 18 παιδιά (μέση ηλικία 6,4 χρόνια) με οξεία ΙΘΠ πριν και 24 ώρες μετά τη χορήγηση IVIG, καθώς και μετά από 0,5-5 χρόνια. Οι ασθενείς αυτοί παρουσίαζαν μειωμένο αριθμό CD4 κυττάρων και ανεστραμμένη αναλογία CD4/CD8. Μελετήθηκε η έκφραση των αποπτωτικών γονιδίων Fas και Bcl-2 και παρατηρήθηκε μειωμένη έκφραση του γονιδίου Fas στους ασθενείς, και φυσιολογική έκφραση του Bcl-2. Η θεραπεία με IVIG δε φάνηκε να επηρεάζει αυτές τις παραμέτρους. 12 από τα 18 παιδιά παρουσίαζαν χαμηλή έκφραση Th0/Th1 κυτταροκινών, με ταυτόχρονη έκφραση IL-10, ή δεν εξέφραζαν καμία κυτταροκίνη in vivo. 24 ώρες μετά τη χορήγηση IVIG, 5/12 παιδιά δεν εξέφραζαν καμμία κυτταροκίνη ενώ τα υπόλοιπα έδωσαν είτε ένα Th2, είτε ένα Th0/Th1 πρότυπο. Μελέτες που έγιναν κάποια χρόνια αργότερα έδειξαν ότι αυτά τα 12 παιδιά δεν παρουσίασαν δεύτερο επεισόδιο και in vivo δεν εξέφραζαν καμμία κυτταροκίνη ή μόνο IL-4. Σε 4/18 παιδιά παρατηρήθηκε ένα Th1 ή Th0/Th1 πρότυπο έκφρασης κυτταροκινών και έκφραση IL-10, το οποίο διατηρήθηκε και μετά τη θεραπεία με IVIG. 0,5-5 χρόνια αργότερα παρατηρήθηκε έκφραση Th1 κυτταροκινών και IL-10 και στα 4 παιδιά, τα οποία ανέπτυξαν μία υποτροπιάζουσα μορφή ΙΘΠ με σπάνια επεισόδια επαγόμενα από ιογενείς λοιμώξεις. 2/18 παιδιά παρουσίασαν κατά την εμφάνιση της νόσου ένα καθαρό Th1 πρότυπο, που χαρακτηριζόταν από αυξημένη έκφραση IFN-γ και το οποίο διατηρήθηκε τόσο μετά τη χορήγηση IVIG, όσο και αργότερα και σ’ αυτά η νόσος χαρακτηρίστηκε σαν χρόνια IΘΠ. Τα επίπεδα του TGF-β1 στο πλάσμα των ασθενών ήταν χαμηλά όταν ή νόσος ήταν σε έξαρση και αυξάνονταν όταν ή νόσος ήταν σε ύφεση. Γενικά, φαίνεται ότι μια σταθερή κατάσταση ύφεσης της νόσου σχετίζεται με απουσία έκφρασης κυτταροκινών ή με Τh2 πρότυπο. Έκφραση των μεταγραφικών παραγόντων AP-1, NFAT και NFκΒ υπήρχε στα Τ κύτταρα των ασθενών κατά την εμφάνιση της νόσου και όχι όταν η νόσος ήταν σε ύφεση, ενώ ο NFAT-S απουσίαζε. / Childhood ITP resolves usually after the rst episode, although it may recur, and in 10-20% of cases develops into a chronic disorder. Evidence of the immunoregulatory role of Th1/Th2 responses in autoimmune diseases, prompted us to perform a prospective study of Th1/Th2 gene expression proles and TGF-β plasma levels in 18 children (median age 6.4 years) with acute ITP, before and after IVIG infusion, and during a follow- up period (0.5-5 years). The patients had a decreased CD4 cell population and an inverted CD4/CD8 ratio. RT-PCR of their Fas and Bcl-2 genes revealed increased Fas activity but normal expression of Bcl-2. IVIG therapy had no effect on these parameters. 12 of 18 patients had either low Th0/Th1+IL-10 or no in vivo cytokine gene expression (0). 24h after IVIG infusion this pattern became 0 or Th2 or remained low Th0/Th1. During follow-up these patients did not relapse and maintained 0 or Th2 pattern without IL-10. Of the remaining 6 patients, 4 presented with a Th1 or Th0/Th1 pattern +IL-10 that persisted after IVIG treatment (though IFN-γ expression diminished) and stabilized to Th1+IL-10 at follow-up, which was marked by infrequent episodes of ITP. Two patients presenting with a strict Th1 pattern characterized by high expression of IFN-γ which remained unchanged after IVIG and at follow-up, can be characterized as chronic ITP. TGF-β plasma levels were low in patients with active disease and increased in remission. Overall, acute ITP presents with Th1, Th0/Th1 or 0 in vivo cytokine gene expression. Stable remission is associated with a 0 or Th2 pattern. A 0 or Th2 pattern after IVIG gave the best prognosis, whereas sustained high expression of IFN-γ and refractoriness to IVIG were the main indicators of poor prognosis. The transcription factors AP-1, NFAT and NFκΒ were studied in 2 patients in acute phase and in remission. They were found to be constitutive in the patients’ ex-vivo and stimulated T cells, but absent from the patients’ ex-vivo T cells in remission, while the NFAT transcriptional silencer was missing.

The IgG Fc domain mediates a range of antibody effector functions, including antibody dependent cell-mediated cytotoxicity (ADCC), complement activation, phagocytosis, and the recently emerged general anti-inflammatory effect of immunoglobulin therapy (IVIg). The conserved N-glycan attached to Fc N297 maintains the Fc structural integrity for the effector functions, while its glycoform is known to modulate the affinity for the Fc γ-receptors (FcγRs), complement, and the C-type lectin DC-SIGN. IgG Fc exhibits protein-directed glycosylation characterized by a series of biantennary complex type glycoforms, with a small population of sialylated species. The sialylated Fc has been proposed to bind DC-SIGN and initiate an anti-inflammatory signalling pathway. The restricted Fc glycan processing is partially attributed to the hydrophobic interaction between Fc glycan and the hydrophobic Fc protein backbone. Mutations within the hydrophobic Fc protein-glycan interface dramatically increases Fc glycan processing, while concomitantly decreases Fc affinity for the FcγRs. However, it is unclear whether this disrupted Fc-FcγR interaction was due to the increased terminal glycan processing, or the perturbed Fc protein-glycan interface. Here, the integrity of the Fc protein-glycan interface was demonstrated to be important in maintaining the productive Fc-FcγR interaction independently of glycoform. This glycoform-independent effect was exploited to generate novel inhibitory Fc variants. In addition, the interaction between sialylated IgG and the putative IVIg receptor DC-SIGN was re-evaluated. Analysis shows that IVIg binds DC-SIGN in a glycan-independent, Fab-mediated manner. Furthermore, the effect of IVIg sialylation on human antigen presenting cells was examined; evidence presented here indicate that IVIg deglycosylation, not desialylation, has an anti-inflammatory effect on human dendritic cells (DCs). These data suggest the need for a general re-evaluation of the current mechanistic model of anti-inflammatory IVIg.

La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu fréquente des IgG humaines. Pourtant, elle est l’objet de beaucoup d’attention depuis que deux articles fondateurs ont été publiés, qui montrent l’un que la sialylation des IgG diminue leur capacité à déclencher la cytotoxicité cellulaire dépendant de l’anticorps (ADCC), et l’autre que les IgG sialylées en α2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thérapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifère, connaissent depuis la fin des années 90 un succès et une croissance phénoménaux sur le marché pharmaceutique. La maîtrise de la N-glycosylation du Fc des IgG est une clé de l’efficacité des anticorps monoclonaux. Si les IgG sialylées sont des molécules peu fréquentes in vivo, elles sont très rares en culture cellulaire. Dans cette étude, nous avons développé une méthode de production d’IgG avec une sialylation de type humain en cellules CHO. Nous avons travaillé principalement sur la mise au point d’une stratégie de production d’IgG sialylées par co-expression transitoire d’une IgG1 avec la β1,4-galactosyltransférase I (β4GTI) et la β-galactoside-α2,6-sialyltransférase I (ST6GalI). Nous avons montré que cette méthode permettait d’enrichir l’IgG1 en glycane fucosylé di-galactosylé mono-α2,6-sialylé G2FS(6)1, qui est le glycane sialylé présent sur les IgG humaines. Nous avons ensuite adapté cette méthode à la production d’IgG présentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. L’analyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec l’IgG1 native ou une version mutante de l’IgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilisé les IgG1 produites avec différents profils de glycosylation afin d’évaluer l’impact de la sialylation α2,6 sur l’interaction de l’IgG avec le récepteur FcγRIIIa, principal récepteur impliqué dans la réponse ADCC. Nous avons montré que la sialylation α2,6 augmentait la stabilité du complexe formé par l’IgG avec le FcγRIIIa, mais que ce bénéfice n’était pas directement traduit par une augmentation de l’efficacité ADCC de l’anticorps. Enfin, nous avons débuté le développement d’une plateforme d’expression stable d’IgG sialylées compatible avec une production à l’échelle industrielle. Nous avons obtenu une lignée capable de produire des IgG enrichies en G2FS(6)1 à hauteur de 400 mg/L. Cette étude a contribué à une meilleure compréhension de l’impact de la sialylation sur les fonctions effectrices des IgG, et a permis d’augmenter la maîtrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire. / Only a fraction of the N-glycans present on the Fc fragment of the human IgGs is sialylated. However, a new interest for sialylation has risen since two major articles were published, one showing that sialylation reduces the capacity of the antibody to trigger antibody-dependent cell cytotoxicity (ADCC), whereas the other showed that the IgGs carrying α2,6-sialic acids on their Fc N-glycans were responsible for the anti-inflammatory activity of intravenous immunoglobulins (IVIGs) injected at high doses. Therapeutic monoclonal antibodies (mAbs) are in majority recombinant IgGs produced in mammalian cell culture. Since the end of the nineties, mAbs have become a major class of pharmaceutical products, and their success is still growing. The control of Fc N-glycosylation is a key parameter for the improvement of the therapeutic efficacy of mAbs. Sialylated IgGs are found only as traces in the classic CHO cell culture processes. In this study, we developed a method for the production of IgGs with a human-like sialylation in CHO cells. We focused on a production strategy relying on the transient co-expression of an IgG1 with the β1,4-galactosyltransferase I (β4GTI) and the β-galactoside-α2,6-sialyltransferase I (ST6GalI). We showed that this method allowed the enrichment of the IgG1 glycoprofile in the fucosylated di-galactosylated mono-α2,6-sialylated glycane G2FS(6)1, which is the main sialylated glycan found in human IgGs. We then adapted this method to the production of highly galactosylated or highly sialylated IgGs with and without core-fucosylation. The analysis of the glycosylation profiles obtained using the various enzyme combinations co-expressed with the native IgG1 or the mutant IgG1 F243A allowed us to discuss the influence of the under-galactosylation found in IgGs produced in CHO cells versus the Fc structural constraints on the limitation of IgG sialylation in CHO cells. We used the IgG1 glycovariants produced with our method to assess the impact of Fc α2,6-sialylation on the interaction of the IgG with the receptor FcγRIIIa, which is the main receptor mediating the ADCC response. We showed that the presence of α2,6-sialylation in the Fc increased the stability of the IgG-FcγRIIIa complex. This benefit however did not translate into an improved ADCC capacity. Finally, we initiated the development of a stable expression platform for the production of sialylated IgGs at yields relevant for the industry. We obtained a cell line capable of producing IgGs enriched in G2FS(6)1 at 400 mg/L. This may eventually represent a novel approach to manufacture a recombinant IVIG surrogate. With this work, we contributed to a better understanding of the impact of sialylation on the effector functions of IgGs. We also improved our understanding of the techniques allowing for the modification and control of the glycosylation profile of IgGs in cell culture.