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CARACTERISATION DE VACCINS A BASE DE GLUCIDESVariables influençant l'immunogénicité et propriétés physico-chimiques des vaccins glycoconjuguésDe nombreux aspects peuvent influer sur l'immunogénicité des vaccins conjugués et les principales variables étudiées jusqu'ici sont la taille du fragment saccharide et la nature des glycosides: taux de protéine dans le conjugué purifié, la stratégie de conjugaison, nature de l’espaceur et la protéine porteuse.La taille de la partie saccharidique et le ratio de cette partie / protéine a été étudiée dans différents travaux de Seppälä et Mäkelä,.Dans l'une des premières études sur l'effet de la taille sur l'immunogénicité de la protéine conjuguées à des dextrans, il a été montré que des dextrans de faible poids moléculaire conjugué à l’albumine sérique de poluet induit des réponses anti-dextran fortes chez la souris. L’augmentation de la taille du dextran, a abouti à une réduction de l’immunogénicité. Peeters et al. a montré qu’un tétramère synthétique de Hib unité capsulaire polysaccharide, conjuguée à un support protéique, induit chez des souris adultes, des niveaux d'anticorps primates non humains comparables à un conjugué Hib commercial. Ces niveaux sont plus élevées que ceux induits par un trimère répété, ce qui indique que pour le Hib un minimum de huit sucres est nécessaire pour une bonne réponse immunologique. Laferrière et al. a trouvé peu d'influence de la longueur de la chaîne glucidique sur l'immunogénicité des vaccins antipneumococciques conjugués chez la souris. Pozsgay et al. a étudié chez la souris, l'immunogénicité de l’oligosaccharides du LPS de Shigella dysenteriae de type 1 conjugué à l'albumine sérique humaine (HSA). Les auteurs ont constaté que les octa-, dodéca-, et des fragments de hexadécasaccharides induit des niveaux élevés d'anticorps IgG après trois injections. Ces niveaux sont supérieurs à ceux obtenus avec un conjugué tétrasaccharidique. L'influence du ratio glucides / protéine est différente pour les trois conjugués. Le conjugué octasaccharide-HSA avec la plus forte densité provoque une bonne réponse immunitaire, tandis que dans le cas des conjugués dodéca- et hexadécasaccharides, la densité médiane est optimal. Ces études suggèrent que la longueur de la chaîne d'oligosaccharides et le chargement de l’haptène peuvent être liés entre eux pour déterminer l'immunogénicité des vaccins glycoconjugués.L'espaceur est une molécule linéaire courte qui est généralement liée à la chaîne polysaccharidique et la protéine ou de fragments. Il ya des évidences dans la littérature qui suggèrent que les espaceurs rigides, contraints comme le maléimide cyclohexyle, provoquent une importante quantité d'anticorps indésirables, avec le risque de conduire à une réponse immunitaire éloignée de l'épitope ciblé sur la haptène. L'utilisation d'un alkyle souple type maleimido a été rapporté comme un moyen de surmonter l'immunogénicité observée précédemment. Un certain nombre de transporteurs protéiques ont été utilisés jusqu'ici dans l'évaluation préclinique et clinique de vaccins conjugués. Des protéines telles que les anatoxines diphtériques et tétaniques, qui dérivent des toxines respectives, après la décontamination chimique avec le formaldéhyde, ont été initialement choisies comme transporteur en raison de inocuité (tétanos et la vaccination contre la diphtérie). CRM 197, un mutant non toxique de la toxine 61 de la diphtérie a été largement utilisé comme support pour Hib. Un complexe protéique de la membrane externe de méningocoque du sérogroupe B a été utilisé par Merck comme support pour leur vaccin conjugué Hib. GSK dans leur vaccin antipneumococcique, conjugué multivalent, introduit la protéine D Hib liée à la plupart des polysaccharides inclus dans le vaccin. L'équipe de John Robbins fait un large usage de la forme recombinante non toxique de l’exo-toxine de Pseudomonas aeruginosa comme support contre Staphylococcus aureus de type 5 et 8 ainsi que pour Salmonella. / CHARACTERIZATION OF CARBOHYDRATE BASED VACCINES Variables influencing the immunogenicity and physicochemical properties of glycoconjugate vaccinesMany aspects can influence the immunogenicity of conjugate vaccines and the main variables investigated so far are the size of the saccharide moiety, the saccharide:protein ratio in the purified conjugate, the conjugation strategy, the nature of the spacer and the protein carrier. The size of the saccharide moiety and saccharide/protein ratio were investigated in different works such as Seppälä and Mäkelä in one of the first studies on the effect of size and chemistry on the immunogenicity of dextrans-protein conjugates found that dextrans of low molecular weight conjugated to chicken serum albumin, induced strong anti-dextran responses in mice, while increasing the dextrans' size resulted in reduced immunogenicity.47 Peeters et al. showed that a synthetic tetramer of Hib capsular polysaccharide repeating unit, conjugated to a protein carrier, induced in adult mice and non-human primates antibody levels comparable to a commercial Hib conjugate and higher than those induced by a trimer, indicating that for Hib a minimum of eight sugars is needed for a proper immunological response.48 Laferriere et al. found little influence of the carbohydrate chain length on the immunogenicity of pneumococcal conjugate vaccines in mice.49 Pozsgay et al. studied the immunogenicity in mice of synthetic Shigella dysenteriae type 1 LPS oligosaccharides conjugated to human serum albumin (HSA). The authors found that octa-, dodeca-, and hexadecasaccharide fragments induced high levels of lipopolysaccharide binding IgG antibodies in mice after three injections and were superior to a tetrasaccharide conjugate. The influence of the carbohydrate/protein ratio was different for the three conjugates. The octasaccharide-HSA conjugate with the highest density evoked a good immune response, while in the case of dodeca- and hexadecasaccharide conjugates, the median density was optimal.50 These studies suggest that oligosaccharide chain length and hapten loading might be interconnected in determining the immunogenicity of glycoconjugate vaccines. The spacer is a short linear molecule that is generally linked to the polysaccharide chain or to the protein or to both moieties, depending on the chemistry, used to facilitate the coupling between the protein and sugar. There are evidences in the literature which suggest that rigid, constrained spacers like cyclohexyl maleimide, elicit a significant amount of undesirable antibodies, with the risk of driving the immune response away from the targeted epitope on the hapten.51 52 The use of a flexible alkyl type maleimido spacer has been reported as a way to overcome the previous observed immunogenicity of cyclic maleimide linkers.53 A number of protein carriers have been used so far in preclinical and clinical evaluation of conjugate vaccines. 54 55 56 57 58 59 60Proteins such as diphtheria and tetanus toxoids, which derive from the respective toxins after chemical detoxification with formaldehyde, were initially selected as carrier because of the safety track record accumulated with tetanus and diphtheria vaccination. CRM197, a non-toxic mutant of diphtheria toxin61 which instead does not need chemical detoxification, has been extensively used as carrier for licensed Hib, pneumococcal, meningococcal conjugate vaccines and for other vaccines being developed. An outer membrane protein complex of serogroup B meningococcus has been used by Merck as carrier for their Hib conjugate vaccine.62 GSK in their multivalent pneumococcal conjugate vaccine introduced the use of the Hib-related protein D as carrier for most of the polysaccharides included into the vaccine.63 64 The team of John Robbins made extensive use of the recombinant non toxic form of Pseudomonas aeruginosa exo-toxin as carrier for Staphylococcus aureus type 5 and 8 as well as for Salmonella

Extracellular stimuli may influence the M1/M2 phenotypic polarization of macrophages. We examined M1/M2 biomarkers, phagocytic activity, and tumoricidal activity in RAW 264.7 mouse macrophages. Macrophages were treated with conditioned media (CM) from 4T1 breast cancer cells, curcumin, 22-oxacalcitriol, LPS, or a combination of the previously listed. Arginase activity, a M2 phenotypic biomarker, was upregulated by the treatment of macrophages with conditioned media. Curcumin, 22- oxacalcitriol, and LPS partially inhibited RAW 264.7 arginase activity in the presence of 4T1 breast cancer media. 22-oxacalcitriol increased the phagocytic ability of RAW 264.7 macrophages in the presence of M2 polarizing substances produced by the 4T1 breast cancer cells. Also, LPS increased RAW 264.7 phagocytic ability in the presence of 4T1 breast cancer CM. This study looked at the potential substances that would possibly reverse the M2 tumor promoting macrophage phenotype seen in the breast cancer tumor environment. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

Sjèogren's syndrome (S) is a chronic autoimmune disease characterized by ocular and oral dryness and primarily affects post menopausal women. In the present study we investigated the time course of lymphocytic infiltration, apoptosis, caspase-3 activity and different cytokines levels in the lacrimal glands of both genetically predisposed and control mice to elucidate immunopathological mechanism leading to dry eye. The results of our experiments showed that ovariectomy accelerated pathological findings of SS by increasing lympocytic infiltration, cytokine production, lacrimal gland cell death and cleaved caspase-3 activity, and these effects were more pronounced and persistent in the genetically predisposed mouse model of SS. In addition, we observed that lymphocytic infiltration occurred earlier compared to apoptosis which may perpetuate immune mediated destruction of lacrimal epithelial cells. Furthermore, treatment with physioloigical doses of 17-B Estradiol (E2) or DIhydrotestosterone (DHT) prevented all these pathological events observed after ovariectomy. / by Safinaz Mostafa. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Ho, Wing-Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 115-131). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.v / 摘要 --- p.ix / Publications --- p.xii / Table of Contents --- p.xiv / Chapter 1. --- General Introduction / Chapter 1.1. --- Diabetes Mellitus (DM) and Diabetic Nephropathy --- p.1 / Chapter 1.1.1. --- "Prevalence, Diagnosis and Classification of DM" --- p.1 / Chapter 1.1.2. --- Type 2 DM and its Complications: Diabetic Nephropathy --- p.5 / Chapter 1.1.3. --- Diagnosis and Impacts of Diabetic Nephropathy --- p.7 / Chapter 1.1.4. --- Current Treatment of Type 2 DM and Diabetic Nephropathy --- p.8 / Chapter 1.2. --- Cytokines and Chemokines --- p.9 / Chapter 1.2.1. --- Types and Properties --- p.9 / Chapter 1.2.2. --- Cytokines and chemokines in Type 2 DM and Diabetic Nephropathy --- p.13 / Chapter 1.3. --- T Lymphocyte Costimulatory Molecules --- p.15 / Chapter 1.3.1. --- Types and Properties --- p.15 / Chapter 1.3.2. --- T Lymphocyte Costimulatory Molecules in Type 2 DM and Diabetic Nephropathy --- p.16 / Chapter 1.4. --- Adhesion Molecules --- p.18 / Chapter 1.4.1. --- Types and Properties --- p.18 / Chapter 1.4.2. --- Adhesion Molecules in Type 2 DM and Diabetic Nephropathy --- p.20 / Chapter 1.5. --- Intracellular Signaling Pathways --- p.21 / Chapter 1.5.1. --- Types and Properties --- p.21 / Chapter 1.5.2. --- Intracellular Signaling Pathways in Type 2 DM and Diabetic Nephropathy --- p.23 / Chapter 1.6. --- Objectives of Our Study --- p.24 / Chapter 2. --- Materials and Methods / Chapter 2.1. --- Materials --- p.26 / Chapter 2.1.1. --- "Patients, Control Subjects and Blood Samples" --- p.26 / Chapter 2.1.2. --- Cell Line --- p.27 / Chapter 2.1.3. --- "Cell Culture Media, Buffers and Other Reagents" --- p.28 / Chapter 2.1.4. --- "Recombinant Human Cytokines, Inhibitors and Other Stimulators" --- p.30 / Chapter 2.1.5. --- Reagents and Buffers for Flow Cytometric Analysis --- p.31 / Chapter 2.1.5.1. --- Cytometric Bead Array (CBA) of Cytokines and Chemokines --- p.33 / Chapter 2.1.5.2. --- Multiplex Fluorescent Bead Immunoassay (FBI) of Soluble Adhesion Molecules --- p.33 / Chapter 2.1.5.3. --- Phosphorylation State Analysis of Signaling Molecules --- p.34 / Chapter 2.1.5.4. --- Immunofluorescent Staining of Cell Surface Molecules --- p.36 / Chapter 2.1.6. --- Reagents and Buffers for Protein Array Analysis --- p.37 / Chapter 2.1.7. --- "Reagents and Buffers for 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenylytetrazolium Bromide (MTT) Assay" --- p.37 / Chapter 2.1.8. --- Reagents for Human Enzyme-Linked Immunosorbent Assay (ELISA) --- p.37 / Chapter 2.2. --- Methods --- p.38 / Chapter 2.2.1. --- Whole Blood Culture Experiments --- p.38 / Chapter 2.2.2. --- "Collection of Serum and Plasma, and Purification of PBMC from EDTA-Blood" --- p.39 / Chapter 2.2.3. --- HK-2 Cell Cultures --- p.39 / Chapter 2.2.4. --- HK-2 Cell Treatments --- p.40 / Chapter 2.2.5. --- Flow Cytometric Analysis --- p.41 / Chapter 2.2.5.1. --- CBA of Cytokines and Chemokines --- p.41 / Chapter 2.2.5.2. --- Multiplex FBI of Soluble Adhesion Molecules --- p.41 / Chapter 2.2.5.3. --- Phosphorylation State Analysis of Signaling Molecules --- p.42 / Chapter 2.2.5.4. --- Immunofluorescent Staining of Cell Surface Molecules --- p.43 / Chapter 2.2.6. --- Protein Array Analysis --- p.44 / Chapter 2.2.7. --- MTT Assay --- p.44 / Chapter 2.2.8. --- ELISA --- p.45 / Chapter 2.2.9. --- Statistical Analysis --- p.46 / Chapter 3. --- "Clinical Study on the Expressions of Cytokines, Chemokines, Co-stimulatory Molecules, Phosphorylated Signaling Molecules in Patients with Diabetic Nephropathy" / Chapter 3.1. --- Introduction --- p.47 / Chapter 3.2. --- Results --- p.49 / Chapter 3.2.1. --- Demographic Data of Participants --- p.49 / Chapter 3.2.2. --- Expression Profile in Plasma of Patients --- p.49 / Chapter 3.2.2.1. --- Cytokines and Chemokines --- p.49 / Chapter 3.2.2.2. --- Soluble Costimulatory Molecules --- p.55 / Chapter 3.2.2.3. --- Soluble Adhesion Molecules --- p.55 / Chapter 3.2.2.4. --- "Correlations between Plasma Concentrations of Cytokines, Chemokines, soluble Costimulatory Molecules and soluble Adhesion Molecules and UACR in Patients" --- p.60 / Chapter 3.2.3. --- Effects ofTNF-α and IL-18 on the ex vivo Production from Whole Blood of Patients --- p.65 / Chapter 3.2.3.1. --- Ex vivo Production of Cytokines and Chemokines --- p.65 / Chapter 3.2.3.2. --- Ex vivo Production of Soluble Costimulatory Molecules --- p.70 / Chapter 3.2.4. --- "Expression of Phosphorylated p38 MAPK, JNK and ERK in PBMC of Patients" --- p.73 / Chapter 3.3. --- Discussion --- p.77 / Chapter 3.3.1. --- "Cytokines, Chemokines and Diabetic Nephropathy" --- p.77 / Chapter 3.3.2. --- Soluble Costimulatory Molecules and Diabetic Nephropathy --- p.80 / Chapter 3.3.3. --- Soluble Adhesion Molecules and Diabetic Nephropathy --- p.83 / Chapter 3.3.4. --- Intracellular Signaling and Diabetic Nephropathy --- p.87 / Chapter 4. --- In vitro Study on the Signal Transduction Mechanism Regulating the Expression of CCL2 and Cell Surface Adhesion Molecules in Tumour Necrosis Factor (TNF)-α-Stimulated HK-2 Cells / Chapter 4.1. --- Introduction --- p.90 / Chapter 4.2. --- Results --- p.93 / Chapter 4.2.1. --- Expression Profile of Cytokines and Chemokines of TNF-α-activated HK-2 Cells --- p.93 / Chapter 4.2.2. --- "TNF-α Upregulated CCL2, ICAM-1 and VCAM-1 Expression in HK-2 Cells" --- p.95 / Chapter 4.2.3. --- "TNF-α Activated the p38 MAPK, JNK and ERK Signaling Pathways in HK-2 Cells" --- p.96 / Chapter 4.2.4. --- Cytotoxicity of MAPK Inhibitors --- p.96 / Chapter 4.2.5. --- "Effects of p38 MAPK, JNK and ERK Inhibitors on TNF-α-induced Expressions of CCL2, ICAM-1 and VCAM-1" --- p.100 / Chapter 4.3. --- Discussion --- p.102 / Chapter 5. --- Conclusion and Future Prospects / Chapter 5.1. --- Conclusion --- p.107 / Chapter 5.2. --- Future Prospects --- p.111 / References --- p.115

Bibliography: leaves 119-136.

Antibacterial activities in the salivary glands of female mosquitoes Aedes aegypti were investigated in this study. The mean salivary bacteriolytic activity, during a period of 14-day of female mosquitoes exposed to five different concentrations of Gram-positive bacteria Micrococcus lysodeikticus in the sucrose meal, was detected by a lysoplate method. A logarithmic regression (R²=0.73) fits the different levels of bacteriolytic activity during the entire period. As the concentration of bacteria in the sugar meal increases, the level of mean salivary bacteriolytic activity increases. A maximum level of bacteriolytic factor may exist in the salivary gland when the concentration of M. lysodeikticus in the sucrose meal exceeds 0.6 g/100 ml. One way analysis of variance and multiple range analysis for the different levels of bacteriolytic activity further validate this finding. The mortality of mosquitoes in the different treatments was not affected by the quanti ties of this nonpathogenic bacteria in the sugar meal. Salivary bacteriostatic activity of female mosquitoes against Gram-negative bacteria Escherichia coli was not detectable in our study, despite positive preliminary results. / Graduation date: 1993

A foundation for the control of tumors is presented, based upon the formulation of a realistic, knowledge-based mathematical model of the interaction between tumor cells and the immune system. The parametric control variables relevant to the latest experimental data, e.g., the sigmoidal dose-response relationship and Michaelis-Menten dynamics, are also considered. The model consists of 12 states, each composed of first-order, nonlinear differential equations based on cellular kinetics and each of which can be modeled bilinearly. In recent years a great deal of clinical progress has been achieved in the use of optimal controls to improve cancer therapy patient care. For this study, a cancer immunotherapy problem is investigated in which the aim is to minimize the tumor burden at the end of the treatment period, while penalizing excessive administration of interleukin-2 as a limit of toxicity. The optimal solution developed for this investigation is a mixture of an initially large dose of interleukin-2, followed by a gradually decreased dosage and a continuing infusion to maintain the tumor cell population at its allowable limit. Sensitivity analysis is applied to an investigation of the influences of system parameters. It has been found that the immune system is influenced greatly by several parameters such as macrophage level, tumor killing rate, tumor growth rate, and IL-2 level. The simulation results suggest that parametric control variables are important in the destruction of tumors and that the application of exacerbation theory is a good method of tumor control. / Graduation date: 1991

published_or_final_version / Medicine / Master / Doctor of Medicine