This literature review will discuss Zika virus and the salient research on antibody dependent enhancement and how this mechanism may lead to congenital defects. Specific objectives include: the mechanism of antibody dependent enhancement, Zika and dengue virus pathogenesis, placenta pathophysiology, and how changes in viral virulence may play a role the pathogenesis of neurologic congenital defects seen in infants infected with Zika virus in utero. While some cohort studies have examined the relationship between prior dengue immunity, Zika virus infection in pregnancy, and effects on neonatal outcomes further prospective studies using large cohorts and more detailed lab testing and imaging is essential to better understand this relationship. A proposed study enrolling a large cohort of women in the 6th- 8th week of pregnancy from Northeastern Brazil will seek to further describe what additional risk dengue immunity may pose in the context of Zika virus. This risk is essential to understand, as Zika and Dengue viruses co-circulate in many regions of the world. Furthermore, participants in the proposed study will undergo bi-weekly screening for Zika virus through laboratory and ultrasound testing until their delivery. Infants will then have full neurologic testing and MRI scanning for the following year after birth to characterize any congenital defects. Neonates born to mothers with prior dengue immunity who contract Zika virus during pregnancy will be compared to neonates not exposed to Zika virus in utero. This experiment will illuminate the associated risk and evidence of ADE occurring with prior dengue immunity and Zika virus infection during pregnancy. Results from this study will not only help define risks of congenital defects with Zika virus, it will inform vaccine research and elucidate challenges in the administration of the current tetravalent dengue vaccine.

Introdução: Anemias hemolíticas induzidas por fármacos (AHIF) são caracterizadas por uma anemia hemolítica imune após exposição a um fármaco. Testes sorológicos têm o potencial de confirmar a presença de anticorpo contra fármacos, mas a realização destes testes ainda é um desafio. Os anticorpos IgM e IgG envolvidos na AHIF são principalmente de dois tipos: fármaco-dependentes e fámaco-independentes. Os anticorpos fármaco-independentes reagem com os eritrócitos \"in vitro\" sem a presença do fármaco, e os resultados imunohematológicos são idênticos aos encontrados nas anemias hemolíticas autoimunes idiopáticas. Anticorpos fármaco-dependentes resultam em teste da antiglobulina direto (TAD) positivo e eluato negativo, os anticorpos só reagem \"in vitro\" com os eritrócitos na presença do fármaco. Indivíduos saudáveis podem desenvolver anticorpos contra fármacos, mesmo na ausência de anemia hemolítica. Os indivíduos podem ser sensibilizados durante exposição prévia a fármacos, exposição a antibióticos usados em ração animal, tanques de piscicultura, berçários de criação de suínos ou tratamento de mastite em bovinos. Estes indivíduos apresentam um maior risco de desenvolvimento de AHIF grave na ocasião de uma segunda exposição ao fármaco. Objetivos: Padronizar testes imunohematológicos para a detecção de anticorpos dirigidos contra fármaco-dependentes. Materiais e métodos: Amostras de 162 doadores de sangue foram submetidas a pesquisa de anticorpos fármaco-dependentes anti-cefalexina, anti-rifampicina e anti-diclofenaco de sódio. Amostra de um doador de plaquetas colhidas por aférese com TAD positivo foi investigada para detecção de anticorpos fármaco-dependentes anti-valsartana. Amostras de 8 pacientes com suspeita diagnóstica de AHIF foram investigadas para a presença de anticorpos contra os fármacos que foram prescritos ao paciente no período de duas semanas prévias ou até o momento em que a hemólise aguda foi constatada. Anticorpos fármaco-dependentes que reagem na presença do fármaco ligado covalentemente à membrana da hemácia, ou na presença do fármaco em solução, foram investigados em doadores de sangue (técnicas em gel) e em pacientes (técnicas em tubo e em gel). Resultados: Os testes laboratoriais detectaram a presença de anticorpo anti-valsartana no eluato de um doador de plaquetas colhidas por aférese com TAD positivo e a presença de anticorpo anti-ceftazidima no soro de um paciente com suspeita clínica de AHIF. A investigação de anticorpo anti-cefalexina e anti-rifampicina foi negativa em amostras de doadores de sangue. O teste laboratorial para investigação de anticorpo anti-diclofenaco de sódio não foi finalizado devido a presença de hemólise em hemácias sensibilizadas com o fármaco e após a adição do fármaco na solução de hemácias. Conclusão: A detecção de anticorpo anti-valsartana em um doador de plaquetas colhidas por aférese evidenciou a presença de anticorpo anti-fármaco na ausência de hemólise. A detecção de anticorpo anti-ceftazidima em amostra de um paciente com anemia hemolítica confirmou a suspeita clínica de AHIF. Reconhecer os sinais clínicos da AHIF precocemente e a disponibilidade de testes sorológicos é essencial para confirmação diagnóstica, orientação terapêutica e prevenção de uma hemólise potencialmente fatal. / Introduction: Drug-induced immune hemolytic anemia (DIHA) is characterized by immune hemolytic anemia following exposure to a drug. Serological testing has the potential to confirm the presence of anti-drug antibodies, but the testing is still a challenge. The IgM and IgG antibodies involved in DIHA comprise mainly two types: drug-dependent and drug-independent. Drug-independent antibodies react with erythrocytes in vitro without the presence of the drug, and the immunohematologic results are identical to those found in idiopathic autoimmune hemolytic anemias. Drugdependent antibodies induce positive direct antiglobulin tests (DAT) and negative eluates, and in vitro antibody reactions with erythrocytes are only detected in presence of the drug. Healthy individuals can develop antibodies against drugs even in the absence of hemolytic anemia. Individuals may be sensitized during previous exposure to drugs, exposure to antibiotics used in animal feed, fish culture ponds, swine nurseries or mastitis treatment in bovines. These individuals present higher risk of developing severe DIHA on the second exposition. Aims: To standardize immunohematological tests for detection of anti-drug dependent antibodies. Materials and methods: Samples from 162 blood donors were tested for anti-cephalexin, anti-rifampicin and antidiclofenac antibodies. Sample from one platelet apheresis donor with positive DAT was investigated for anti-valsartan drug-dependent antibody. Samples from 8 patients with suspected DIHA were investigated for the presence of antibodies directed against the drugs that were prescribed to the patient in the period of two weeks prior to or until the time when acute hemolysis was detected. Drug-dependent antibodies that react in presence of the drug covalently attached to the erythrocyte membrane, or in presence of the drug in solution were investigated in samples from blood donors (gel techniques) and from patients (tube and gel techniques). Results: Laboratory tests detected antivalsartan antibody in the eluate of an platelet apheresis donor with positive TAD and anti-ceftazidime antibody in the serum of a patient with clinical suspicion of DIHA. Investigation of anti-cephalexin and anti-rifampicin antibody was negative in blood donor samples. The laboratory test for anti-diclofenac antibody was not possible due to the presence of hemolysis in erythrocytes sensitized with the drug and when the drug in solution was added to the erythrocyte. Conclusion: The detection of anti-valsartan antibody in an platelet apheresis donor revealed the presence of an anti-drug antibody in the absence of hemolysis. Detection of anti-ceftazidime antibody in a patient with hemolytic anemia confirmed the clinical suspicion of DIHA. It is essential to recognizing early clinical signs of DIHA and have serological tests available for diagnostic confirmation, therapeutic guidance and prevention of potentially fatal hemolysis.

O óxido nítrico (NO) é um mensageiro biológico que tem importância vital em muitos processos fisiológicos e apresenta uma multiplicidade de funções de regulação no corpo humano, tais como neurotrasmissão, vasodilatação, participa nas respostas imunes e em vários processos associados com o desenvolvimento dos tumores. Vários estudos fornecem evidências sobre as propriedades tumoricidas do óxido nítrico e doadores de NO que podem ser usados para o tratamento de tumores malignos e são objetos de interesse na atualidade. Baseado nisto o objetivo deste trabalho foi de sintetizar e descrever aspectos estruturais dos compostos [Ru(bpy)2(dcbpy)] (dcbpy = ácido 4,4\'-dicarboxi-2,2\'-bipiridina, bpy = 2,2\'-bipyridina) [Ru(bpy)(dcbpy)2], cis-[RuCl2(dcbpy)2], cis-[Ru(dcbpy)2NO(L)]n+ (L = cloreto), [Ru(TERPY)(dcbpy)Cl] (TERPY = 2,2\': 6\'\', 2\'\' - terpiridina) and [Ru(TERPY)(dcbpy)NO], além de ensaios de citotoxicidade in vitro para alguns dos complexos sintetizados. Ensaios de citotoxicidade com solução aquosa de complexos rutênio-nitrosilo em linhagem de células metastáticas B16-F10 mostrou resultados relativamente baixos. Medidas de viabilidade celular mostrou que estes decrescem cerca de 10 % em relação ao controle. Este resultado foi interpretado como sendo devido a baixa interação entre o complexo e a célula. Bioconjugação de rutênio-nitrosilo com anticorpo policlonal IgG foi obtido por interação convalente e mostrou maior especificidade com um alvo na célula. Cromatografia de exclusão foi utilizada para separar o bioconjugado --IgG, que foi caracterizado por teste Western Blotting. Após a bioconjugação, o --IgG foi submetido a estudos de citotoxicidade com células metastáticas e a viablidade celular avaliada com ensaios MTT. Os resultados exibiram um incrível aumento na citotoxicidade de células B16-F10. Viabilidade celular foi determinada e valores de morte celular de cerca de 90 % obtida para uma das frações de --IgG. Nossos resultados demonstraram que o bioconjugado --IgG pode aumentar a resposta citotóxica e quiçá ser útil em terapia clínica. / Nitric oxide (NO) is a biological messenger that has vital importance in many physiological processes and shows a multitude of regulatory roles in the human body, such as neurotrasmission, vasodilatation, immune responses and also participates in various processes associated with cancer development. Several studies provide evidences of the tumoricidal properties of NO donors that can be used for the treatment of malignant tumors and nowadays are objects of interest. Based on this the aim of this work was synthesize compounds that in a controlled manner can deliver NO in a biological process. The synthesis, structural aspects, and in vitro cytotoxic properties of [Ru(bpy)2(dcbpy)] (dcbpy = 2,2\'-bipyridine-4,4\'-dicarboxylic acid), 2\'-bipyridine; bpy = 2,2\'-bipyridine) [Ru(bpy)(dcbpy)2], cis-[RuCl2(dcbpy)2], cis-[Ru(dcbpy)2NO(L)]n+ (L = chloride), [Ru(TERPY)(dcbpy)Cl] (TERPY = 2,2\': 6\'\', 2\'\' - terpiridine) and [Ru(TERPY)(dcbpy)NO] are described. Citotoxicity assays with aqueous nitrosyl ruthenium complex in metastatic B16-F10 cells displayed very little effect. Cell viability measurement showed decrease around 10 % in comparison to the control. It was associated due to the low interaction between nitrosyl ruthenium specie and the cell. Bioconjugation of nitrosyl ruthenium specie with polyclonal antibody IgG was achieved by covalent interaction and showed more specific interaction between bioconjugated and target cell. Exclusion chromatography was used to isolate --IgG conjugated, which was characterized by Western Blotting test. Following bioconjugation, the --IgG was submitted to cytotoxic studies with metastatic cells and the viability evaluated by MTT assay. The results displayed incredible increase of citotoxicity for B16F10 cells. Cell viability was achieved to decrease until to 90 % in comparison to the control when one fraction of --IgG was used. Taken together, the present findings demonstrate that the --IgG complex may elicit citotoxicity responses that may find useful applications in clinical therapy.

The aim of this thesis was to establish the therapeutic value of the IFITM1 monoclonal antibodies and to design and develop therapeutically valuable recombinant monoclonal antibodies so as to study the implication of these novel antibodies in cancer therapy. Cancer metastasis is one of the main interests that has given rise to the design and development of innovative strategies for cancer therapeutics. The Interferon Induced Transmembrane Protein 1(IFITM1), a notable member of the IFITM family of proteins has been identified as one of the most up-regulated trans-membrane proteins in metastatic breast cancer and cervical adenocarcinoma. This interferon-regulated protein is also involved in cell migration, invasion in glioma and squamous cancers. This PhD aimed to study IFITM1 as a pro-invasive cancer target by the use of IFITM1 monoclonal antibodies that were raised against the extracellular domain of the human IFITM1 gene. The epitope mapping of IFITM1 revealed the binding activity of the IFITM1 monoclonal antibody. This gave the opportunity to design and generate to new IFITM1-specific molecular tools, in the form of recombinant IFITM1 targeted murine scFv antibody, IFITM1-CPG2 yeast fusion protein antibody for potential application in ADEPT as well as a Mouse-Human Chimeric IFITM1 antibody secreting mammalian cell line. The immunohistochemical staining of IFITM1 in tissue micro array from breast, colon and oeosphegal cancer has revealed that the majority of these cancers produce this protein. However, IFITM1 is over produced in cervical cancer indicating it’s selective over expression in cervical cells. This PhD endeavored to investigate the expression of IFITM1 at a translational and transcriptional level and to study the clinical significance of IFITM1 in cervical cancer. The antibody dependent cell mediated cytotoxic activity of the chimeric IFITM1 antibody was found to be cytotoxic to SiHa cells in vitro. In the future these molecular tools could be used to regulate and further characterize the activity of this transmembrane protein antibody. In an effort to better understand the mechanisms that regulate the activity and the over production of the IFITM1 gene and its interacting proteins, a proteomic screen of cervical cancer cells was carried out using data-independent SWATH-MS on an AB SCIEX TripleTOF™ mass spectrometer. This Mass Spec analysis provided us with a host of IFITM1 biomarkers and revealed that the IFITM1 gene and its binding proteins also cross link with the IRF1 pathway. The data presented in this thesis, demonstrates that the IFITM1 gene can be targeted to either stimulate or inhibit IFITIM1 signaling to engage IFITM1 as a potential pro-invasive extracellular receptor as a target in antibody cancer therapy. In summary, this thesis aimed to confirm the activity and the binding specificity of the IFITM1 antibody. Additionally, this thesis demonstrated a promising application of the recombinant antibody in the ADEPT technology. Characterization of IFITM1 mAb effector functions indicated that the antibody was cytotoxic to cervical cancer cells. This highlights an important element in the immune suppressive tumour microenvironment. And finally, this thesis also provides the basis for the production of recombinant mouse human chimeric antibodies that are a part of a new group of immunotherapeutic molecules paving the way for cancer therapeutics.

Trois bactéries du genre Yersinia sont pathogènes pour l’homme : Yersinia pestis (bacille de la peste), et les bactéries entéropathogènes Yersinia pseudotuberculosis et Yersinia enterocolitica. Yersinia pestis est responsable de plus de 20 000 cas humains de peste déclarés à l’Organisation Mondiale de la Santé (OMS) ces dix dernières années dans différents foyers en Afrique, Asie et Amériques. Considérée aujourd’hui à tort comme une maladie du passé, elle est au contraire classée parmi les maladies réémergentes Même si elle ne se présente plus sous la forme d’épidémies massives, elle pose encore au monde actuel d’importants défis de par son extrême gravité, sa rapidité de dissémination, une apparition de résistances aux antibiotiques et une éventuelle utilisation terroriste du bacille. Dans ce contexte, l’immunothérapie contre Y. pestis pourrait être une bonne alternative pour traiter la peste bubonique et pulmonaire. Un des objectifs de cette thèse était de produire des anticorps monoclonaux murins contre trois protéines de l’injectisome (YscF, YscC et LcrV), un facteur de virulence clé des Yersinia. Les anticorps obtenus ont été caractérisés et pour certains leurs épitopes identifiés. Par la suite, en collaboration avec Elisabeth Carniel à l’Institut Pasteur, leur pouvoir neutralisant a été évalué in vivo dans un modèle murin de peste bubonique. Ces mêmes anticorps monoclonaux, produits contre les protéines de l’injectisome sont en cours d’évaluation pour la mise au point d’un test de diagnostic rapide de Y. pestis dans différents fluides et échantillons biologiques. Yersinia pseudotuberculosis et Yersinia enterocolitica sont présentes dans le monde entier et sont transmises par contamination à partir de viande de porc mal cuite, de lait ou produits laitiers et de végétaux, ou par contact avec des animaux porteurs sauvages ou domestiques. Une transmission interhumaine par voie fécale-orale est également possible. Ces bactéries sont responsables très fréquemment d’infections entériques. Cependant leur recherche dans les coprocultures n’est pas réalisée de façon systématique en laboratoires d’analyses médicales du fait de leur croissance lente et difficile sur les milieux usuels, ce qui rend leur isolement à partir de fèces difficile. De plus, les procédures de routine sont coûteuses et longues. Cela entraine probablement une sous-estimation de l’incidence des infections à Yersinia entéropathogènes, la prescription de traitements non adaptés et la réalisation d’appendicectomies non nécessaires, d’où la nécessité de développer des tests de diagnostic rapides, spécifiques, sensibles et faciles à utiliser. Un des objectifs de cette thèse était de produire un panel d‘anticorps monoclonaux murins contre les principaux biotypes et sérotypes pathogènes de Y. pseudotuberculosis et Y. enterocolitica pour le développement de tests de diagnostic immunologiques (ELISA et tests bandelettes) répondant aux caractéristiques recherchées et utilisables directement avec des échantillons biologiques humains. / Three bacteria of the genus Yersinia are pathogenic for the human: Yersinia pestis (the plague bacillus) and the enteropathogenic bacteria: Yersinia pseudotuberculosis and Yersinia enterocolitica. Yersinia pestis is responsible for more than 20,000 human cases of plague declared to the World Health Organization (WHO) during the ten last years in different areas from Africa, Asia and America. Mistakenly considered today as a disease from the past, on the contrary, the plague is re-emerging. Even if it doesn’t occur as a massive epidemic, it still lays down a challenge to the world for its important severity, its quick spreading, the appearance of antimicrobial resistance and a potential use for terrorism. Under the circumstances, the immunotherapy against Y. pestis could be a good option to treat bubonic and pneumonic plague. One the aims of this thesis was to produce murine monoclonal antibodies against the three proteins of the injectisom (YscF, YscC, LcrV), a key virulence factor of Yersinia. The obtained antibodies were characterized and for certain, the epitopes were identified. Then, in collaboration with Elisabeth Carniel from Institut Pasteur, their therapeutic effect was evaluated in vivo with a bubonic plague model in mice. The antibodies generated against the proteins from the injectisom are now evaluated in a diagnosis test for a fast detection of Y. pestis in different biological samples. Yersinia enterocolitica and Yersinia pseudotuberculosis, the two enteropathogenic Yersinia species for humans, have a worldwide distribution and are among the most frequent agents of human diarrhea in temperate and cold countries. However, research of enteropathogenic Yersinia is not consistently performed in medical laboratories because of their specific growth characteristics, which makes their isolation from the stool samples difficult. Moreover, current procedures for isolation are expensive and time consuming, which leads to underestimation of the incidence of yersiniosis and prescriptions of inappropriate antibiotic treatments. One the aims of this thesis was to produce different murine monoclonal antibodies against the main pathogenic biotypes and serotypes of Y. pseudotuberculosis and Y. enterocolitica for the development of fast, sensitive, specific and easy-to-use immunoassays (ELISA and dipsticks), useful for both human and veterinary diagnosis.

Typescript. / Digitized by Kansas Correctional Industries

Thesis. 1977. Ph.D.--Massachusetts Institute of Technology. Dept. of Biology. / Microfiche copy available in Archives and Science. / Bibliography : leaves 272-276. / Ph.D.

Le mélanome est la forme la plus agressive des cancers de la peau. Si sa prise en charge à des stades précoces est de bon pronostic, l’espérance de vie des patients chute dramatiquement pour les stades métastatiques. Malgré les avancées thérapeutiques spectaculaires récentes, le problème majeur réside dans la résistance aux traitements et la récidive et le défi principal est désormais de tendre vers un contrôle efficace et durable. Les anticorps monoclonaux (AcM) ont la capacité de cibler et éliminer spécifiquement la cellule tumorale tout en recrutant des cellules du système immunitaire, permettant de développer et/ou renforcer l’immunité de l’hôte avec le développement d’une réponse immune anti-tumorale de type vaccinale. Dans un modèle de tumeur solide de mélanome murin après greffe sous-cutanée des cellules B16F10, nous avons étudié le potentiel immunomodulateur de l’AcM TA99 qui cible un antigène de surface TYRP-1 surexprimé dans les mélanocytes tumoraux. Nos résultats montrent qu’environ 30% des souris sont protégées sur le long-terme et présentent une réponse immunitaire humorale et cellulaire mémoire. Par ailleurs, l’analyse de l’infiltrat immunitaire chez les souris qui échappent au traitement par l’AcM TA99 et qui développent une tumeur à plus ou moins long terme, montre une surexpression de PD-1 et Tim3 associée à une perte de fonctionnalité des cellules effectrices au sein de la tumeur. Ce même phénotype a été observé sur des biopsies de patients atteints de mélanome métastatique. Nous montrons aussi dans le cadre de ce travail que, le blocage de l’axe PD1/PD-L1 par inoculation d’un AcM anti-PD1 au moment de l’échappement, potentialise la réponse immunitaire anti-tumorale et entraîne une augmentation de la survie. Cependant, l’absence de régression complète suggère la mise en place d’autres voies immunosuppressives. En effet nous avons observé une surexpression des ectonucleotidases CD39 et CD73 dans le micro-environnement tumoral suggérant l’implication de l’adénosine dans la résistance au traitement de l’AcM TA99 plus l’α-PD-1. Ce résultat ouvre des perspectives intéressantes pour le blocage concomitant la voie de l’adénosine et de l’axe PD1/PD-L1. Une autre stratégie a consisté à améliorer les effets immunomodulateurs précoces de l’AcM TA99 en le combinant avec l’oxaliplatine, chimiothérapie favorisant la mort immunogénique. Bien que les combinaisons thérapeutiques testées dans cette étude montrent des effets in vivo encourageants avec un délai significatif dans la survie globale, aucune augmentation significative de la réponse anti-tumorale sur le long terme n’a pu être observée, suggérant la mise en place d’autres voies immunosuppressives non redondantes ou des stratégies de combinaisons non adaptées. Une analyse dynamique approfondie, tant phénotypique que fonctionnelle, des différents acteurs cellulaires du micro-environnement tumoral sera une étape clé dans la mise en place de combinaisons pertinentes en association avec l’AcM TA99. Ce travail prend d’autant plus d’intérêt qu’un essai clinique de phase I (IMC-20D7S) utilisant le flanvotumab (équivalent humain de l’AcM TA99) réalisé chez 27 patients atteints de mélanome métastatique, montre des effets cliniques intéressants sans effets secondaires sévères, ouvrant la voie au développement de combinaisons thérapeutiques associées à cet AcM. / Melanoma is the most aggressive form of skin cancer. Although early management is of good prognosis, the survival of patients decrease dramatically for metastatic stages. Despite the recent spectacular therapeutic advances, the major problem lies in resistance to treatment and relapse and the main challenge now is to develop an effective and sustainable control. Monoclonal antibodies (mAbs) have the ability to specifically target and eliminate tumor cells while recruiting cells from the immune system, to develop and / or enhance the immunity of the host with the development of a vaccinal immune response. In a solid tumor model of murine melanoma after subcutaneous transplantation of B16F10 cells, we investigated the immunomodulatory effect of TA99 mAb targeting a TYRP-1 surface antigen overexpressed in tumor melanocytes. Our results showed that about 30% of mice are protected in the long term and have an antitumoral humoral and cellular immune response. Moreover, the analysis of the immune infiltrate in mice that escape to the treatment with TA99 mAb and develop a tumor, shows an overexpression of PD-1 and Tim3 associated with a loss of effector cell functions within the tumor. This same phenotype has been observed in biopsies of patients with metastatic melanoma. Thus, blocking the PD-1 / PDL-1 axis by inoculation of an anti-PD1 mAb at the time of tumor escape potentiates the anti-tumor immune response and results in increased survival. However, the absence of complete regression suggests the establishment of other immunosuppressive pathways. Indeed we have observed an overexpression of CD39 and CD73 ectonucleotidases in the tumor microenvironment suggesting the involvement of adenosine in the resistance mechanisms observed and opening interesting perspectives for the concomitant blocking of this pathway and the PD1 / PDL-1 axis. Another strategy has been to improve the early immunomodulatory effects of TA99 mAb by combining it with oxaliplatin, a chemotherapy that promotes immunogenic death. Although the therapeutic combinations tested in this study showed encouraging in vivo effects with a significant delay in overall survival, no significant increase in the long-term anti-tumor response was observed, suggesting the establishment of other non-redundant immunosuppressive mechanisms or unsuitable combinations strategies. Both phenotypic and functional analysis of the different cellular actors of the tumor microenvironment will be a key step in the implementation of relevant combinations in association with the TA99 mAb. This work is highlighted by a phase I clinical trial (IMC-20D7S) using flanvotumab (human equivalent of mAb TA99) in 27 patients with metastatic melanoma that shows interesting clinical outcome without severe side effects, opening the way for the development of therapeutic combinations associated with this mAb.

by Frankie Kar-Ming Wong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 101-107). / Acknowledgements --- p.IV / Abstract --- p.V / Abbreviations --- p.VI / Chapter Chapter 1 --- Introduction to Lipoprotein and Apolipoprotein --- p.1 / Chapter 1.1 --- Lipoprotein structure and classification --- p.1 / Chapter 1.2 --- Apolipoprotein A-I and B100 --- p.1 / Chapter 1.2.1 --- Apolipoprotein A-I (apoA-I) --- p.1 / Chapter 1.2.2 --- Apolipoprotein B100 (apoB100) --- p.3 / Chapter 1.2.3 --- Biological functions of apolipoprotein --- p.4 / Chapter 1.3 --- Evidence linking apoA-I and B100 with atherosclerosis --- p.4 / Chapter 1.4 --- The roles of apoA-I and B100 in the development of atherosclerosis --- p.6 / Chapter 1.5 --- Measurement of human serum lipoproteins as an assessment of risk for coronary heart disease (CHD) --- p.8 / Chapter 1.6 --- Aims of this study --- p.10 / Chapter Chapter 2 --- Purification of ApoA-I and B100 and Production of Polyclonal Antibodies --- p.12 / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.1.1 --- Purification of apoA-I and B100 from human serum --- p.12 / Chapter 2.1.2 --- Immunization for polyclonal antibodies production against apoA-I and B100 --- p.14 / Chapter 2.1.3 --- Antibody purification --- p.15 / Chapter 2.1.3.1 --- Ammonium sulfate precipitation --- p.17 / Chapter 2.1.3.2 --- DEAE and QEAE Sepharose --- p.17 / Chapter 2.1.3.3 --- Protein A and Protein G --- p.17 / Chapter 2.1.3.4 --- Affinity chromatography --- p.18 / Chapter 2.2 --- Methods --- p.20 / Chapter 2.2.1 --- Purification of HDL and LDL --- p.20 / Chapter 2.2.2 --- Purification of apolipoproteins --- p.22 / Chapter 2.2.3 --- Immunization of rabbit with apoA-I and B100 --- p.23 / Chapter 2.2.4 --- Enzyme-linked immunosorbent assay (ELISA) --- p.24 / Chapter 2.2.5 --- Purification of lipoprotein specific immunoglobulin from antisera --- p.25 / Chapter 2.2.5.1 --- Salt fractionation --- p.25 / Chapter 2.2.5.2 --- Purification of immunoglobulin by Protein A affinity chromatography --- p.25 / Chapter 2.2.5.3 --- Isolation of specific antibody by lipoprotein-coupled affinity chromatography --- p.26 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Purification of apoA-I and B100 --- p.27 / Chapter 2.3.2 --- Purification of immunoglobulins from rabbit anti-apolipoprotein sera --- p.32 / Chapter 2.4 --- Discussion --- p.38 / Chapter Chapter 3 --- Production of monoclonal antibodies against apoA-I and B100 --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.1.1 --- What is monoclonal antibody? --- p.48 / Chapter 3.1.2 --- The basic methodology --- p.49 / Chapter 3.1.2.1 --- Immunization of host --- p.49 / Chapter 3.1.2.2 --- Cell lines required for fusion --- p.49 / Chapter 3.1.2.3 --- Fusion --- p.51 / Chapter 3.1.2.4 --- Selection of hybrids --- p.52 / Chapter 3.1.2.5 --- Screening assay --- p.54 / Chapter 3.1.2.6 --- Cloning --- p.54 / Chapter 3.1.2.7 --- Bulk production of monoclonal antibody --- p.55 / Chapter 3.1.2.8 --- Monoclonal antibody purification --- p.55 / Chapter 3.2 --- Methods --- p.55 / Chapter 3.2.1 --- Immunization of mice with apoA-I and apoB100 --- p.55 / Chapter 3.2.2 --- Preparation before fusion --- p.58 / Chapter 3.2.2.1 --- Preparation of tissue culture working solutions --- p.58 / Chapter 3.2.2.2 --- Preparation of spleen cells --- p.59 / Chapter 3.2.2.3 --- Preparation of myeloma cells --- p.60 / Chapter 3.2.3 --- Fusion --- p.60 / Chapter 3.2.4 --- Screening assay for positive clones --- p.61 / Chapter 3.2.5 --- Limiting dilution cloning --- p.61 / Chapter 3.2.6 --- Determination of isotype --- p.62 / Chapter 3.2.7 --- Cryopreservation of myeloma and established hybridoma cell lines --- p.62 / Chapter 3.2.7.1 --- Freezing cells --- p.62 / Chapter 3.2.7.2 --- Thawing cells --- p.63 / Chapter 3.2.8 --- Bulk production of monoclonal antibodies from ascites --- p.63 / Chapter 3.2.9 --- Purification of monoclonal antibodies from ascites --- p.63 / Chapter 3.2.10 --- Western blot analyses of the monoclonal antibodies --- p.64 / Chapter 3.2.11 --- Iodination of apolipoproteins --- p.64 / Chapter 3.2.12 --- Binding of the monoclonal antibody to iodinated apolipoprotein --- p.65 / Chapter 3.2.13 --- Competitive displacement analyses --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Development of monoclonal antibodies --- p.66 / Chapter 3.3.2 --- Purification of monoclonal antibody from ascites --- p.69 / Chapter 3.3.3 --- Western blotting analyses of AB6 and BE8 --- p.69 / Chapter 3.3.4 --- Monoclonal antibody titration curve for apolipoproteins by radioimmunoassays --- p.75 / Chapter 3.3.5 --- Competitive displacement analysis of AB6 and BE8 --- p.75 / Chapter 3.4 --- Discussion --- p.79 / Chapter Chapter 4 --- Enzyme-linked immunosorbent assay (ELISA) for ApoA-I --- p.84 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.1.1 --- Alkaline phosphatase (ALP) --- p.84 / Chapter 4.1.2 --- Conjugation methods --- p.85 / Chapter 4.1.3 --- Design of the immunoassay format --- p.87 / Chapter 4.1.4 --- Modified solid-phase: Protein A antibody-capture ELISA (PACE) --- p.87 / Chapter 4.2 --- Materials and Methods --- p.90 / Chapter 4.2.1 --- Conjugation of AB6 with maleimide activated alkaline phosphatase --- p.90 / Chapter 4.2.2 --- Titration curve of AB6-ALP conjugate --- p.90 / Chapter 4.2.3 --- Calibration curve of apoA-I sandwich ELISA --- p.91 / Chapter 4.2.4 --- Measurement of apoA-I by Protein A antibody-capture ELISA --- p.91 / Chapter 4.3 --- Results --- p.92 / Chapter 4.3.1 --- Characterization of AB6-ALP conjugate --- p.92 / Chapter 4.3.2 --- Calibration curve for the measurement of apoA-I --- p.92 / Chapter 4.4 --- Discussion --- p.95 / Chapter Chapter 5 --- General Conclusions --- p.99 / References --- p.101

Orientador: Wilma Aparecida Starke Buzetti / Banca: Solange Maria Gennari / Banca: Rosangela Zacarias Machado / Banca: Maria Conceição Zocoller Seno / Banca: Gilson Pereira de Oliveira / Resumo: O objetivo do presente estudo foi analisar a resposta imune humoral, pelo monitoramento dos anticorpos anti-Babesia bovis, anti-Babesia bigemina e anti-Anaplasma marginale, em búfalos (Bubalus bubalis) naturalmente infectados. Para esta pesquisa, utilizaram-se amostras de soro e de colostro/leite de búfalas adultas do periparto aos 11 meses após, e de soros dos seus bezerros, durante o primeiro ano de vida nos anos de 1999/2000 e 2005. Para determinar o perfil da resposta imune humoral destes animais, utilizou-se o método ELISA indireto e os dados foram apresentados e analisados como a média de um grupo de animais, em diferentes faixas etárias e, individualmente. Após a leitura e interpretação dos dados, os resultados dos animais analisados em grupos apresentaram baixa concentração de anticorpos, ou seja, abaixo do ponto de corte (D.O. = 0,265 e NE=3) de anticorpos anti-A. marginale nos soros, durante os primeiros 90 e 105 dias após o parto e nascimento, respectivamente, para búfalas e seus bezerros. Em seguida, a concentração de anticorpos anti-A. marginale no soro dos bezerros búfalos aumentou ligeiramente acima do ponto de corte e manteve-se assim até atingirem aproximadamente um ano de idade, indicando uma imunidade adquirida, após o contato com a bactéria. Nas búfalas ocorreu soroconversão (NE acima de 3) para Babesia de ambas as espécies, por quase todo o período analisado, com uma elevação acentuada (NE=4 a NE=6) entre os dias 91 e 335 dias após o parto, fato não verificado para os bezerros, no mesmo período. No colostro/leite, os anticorpos anti-B. bovis e anti-B. bigemina foram detectados nos primeiros sete dias pós-parto, mas não foram observados no teste anti-A. marginale. Quando os animais foram analisados individualmente (duas búfalas e seus bezerros), observou-se em um dos bezerros, uma forte imunidade humoral... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of the present study was to analyze the humoral-immune response of water buffaloes (Bubalus bubalis) naturally infected with Babesia bovis, B. bigemina and Anaplasma marginale. For this work, colostrums/milk and blood samples were weekly, fortnightly and monthly harvested prior and after partum (buffalo cows) and from birth to 365 days after birth (buffalo calves). The antibodies in the colostrums/milk and serum samples from these animals were determined using an ELISA indirect method and the data were analyzed as a mean of a group of animals with matched ages during the period of 1999/2000 or individually during the year of 2005. The data from animals analyzed in group showed that the antibodies against A. marginale were in low concentration (below the cut off point, D.O. = 0.265 and ELISA levels, EL = 3), in the sera of buffalo, during the first 90 and 105 days, respectively for cows and calves. Then, the concentration of anti-A. marginale in the serum samples of buffalo calves, slightly raised to above the cut off point and kept in higher levels up to approximately 365 days after birth, indicating acquired immunity. Serum conversion for Babesia occurred in high levels and above the cut off point only for buffalo cows for all period of experimentation. The antibody levels against Babesia for both species and Anaplasma increased in the sera of buffalo cows between the days 91 and 335 after partum. In the colostrums, anti-B. bovis and anti-B. bigemina antibodies were detected in high levels during the first seven days after partum, but then abruptly declined to zero. Anti-A. marginale, on the other hand were not detected in the colostrums of these animals. When four animals (two buffalo cows and their calves) were individually analyzed it was observed an individual variation in the immune response: in one buffalo calf there was a strong passive... (Complete abstract click electronic access below) / Doutor