In summary, this study will be focused on Il-21 and its implications in the antibody response in influenza. The isotype classes primarily involved in this process will also be examined. This will be accomplished by looking at the serum of mice and analyzing the present influenza specific antibodies using ELISA. Another goal was to optimize the ELISA in order to make it sensitive enough to catch small differences in the results. This topic is important due to its implications for improving influenza vaccinations and preventions as current vaccines are not 100% effective. Influenza contributes to significant disease and death around the world every year and each piece of this puzzle is significant in order for the scientific community to be able to eventually make strides to improve the burden of this disease.

京都大学 / 新制・課程博士 / 博士(社会健康医学) / 甲第24806号 / 社医博第130号 / 新制||社医||12(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 松田 文彦, 教授 YOUSSEFIAN Shohab, 教授 永井 洋士 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

The development and refinement of FA has been adequately investigated with major emphasis on pathogenic microorganisms. The development of this technique has reduced both the time and number of biochemical tests necessary to identify a diversity of organisms. The organisms included are the protozoans, as described by Goldman (1953 and 1957) and by Ingram (1961), viruses, as reported by Liu (1955a) and Burgdorfer and Lackman (1960a), pathogenic bacteria which have been investigated by Moody, Goldman, and Thomason (1956), Moody and Winter (1959), Deason, Falcone, and Harris (1957) and Thomason, Cherry, and Moody (1957). Various fungi have been studied with FA by Kaufman and Brandt (1964), Kaufman and Kaplan (1961 and 1963) and Gordon (1958). Therefore, due to the success of the fluorescent antibody technique in many areas of microbiology in previous investigations, the logical assumption was that immunofluorescence might be incorporated into an efficient system in which a specific organism associated with fecal pollution, such as S. faecalis, could be rapidly identified. Based on this assumption, the feasibility of fluorescent antibody techniques, using S. faecalis was investigated as a means of rapid determination of bacterial pollution in water. Although much progress has been achieved in the study of cytochemical reactions by immunofluorescence, no attention has been focused on the application of this method as a determinative tool by which water contamination, due to the presence of the enterococci, could be demonstrated. Specifically, the purpose of the research reported in this dissertation was to devise an applicable, valid, and rapid method that could be employed in the detection and identification of S. faecalis.

No description available.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common cancer in the United States: in 2017 there will be around 54,000 new cases and 43,000 patient deaths. (SEER, 2017) The high mortality of PDAC is related to late disease presentation and aggressiveness; nearly 52% of patients present with metastatic disease at the time of diagnosis. (SEER, 2017) Current treatments have marginal improvements on survival, with the most efficacious treatment, gemcitabine and nab-paclitaxel, having a median survival of 12.2 months. (Wu 2018) Failure of current PDAC treatments is attributed to the inefficacy of systemic chemotherapeutics and the development of resistance. (Rahib, 2014) The Dual Endothelin1/Signal PeptideVEGF receptor (DEspR), represents a promising therapeutic target for the treatment of PDAC: it is a highly expressed, specific tumor antigen, which is involved in tumor vasculogenesis and cancer stem cell (CSC) survival. DEspR is a developmentally crucial receptor, responsible for early angiogenesis and neural crest migration, with minimal expression in normal adult tissue. In vitro and in vivo studies of anti-DEspR therapy in PDAC have shown efficacy in decreasing CSC survival, tumor angiogenesis, and improving overall survival in xenograft models of PDAC, with anti-DEspR therapy being a promising candidate for clinical use. Furthermore, anti-DEspR therapy seems to augment chemotherapeutic therapy in vitro and in vivo, suggesting that a DEspR-targeted antibody drug conjugate (ADC) would be highly effective. ADCs are a re-emerging drug class with significant promise. Initial failures of ADCs in clinic were related to poor antigen specificity and failures in drug conjugation chemistry to minimally impact the antibody. To develop our ADC, I have developed a novel method of site-specific conjugation that relies on a novel method of supramolecular assembly. My system employs two specific protein sequences that do not self-interact, and tightly assemble through coulombic and hydrophobic interactions, allowing site-specific, stoichiometric self-assembly. To facilitate stable drug delivery, I have synthesized a novel enzymatically cleavable tyrosine-clickable linker, which prevents drug release prior to tumor delivery. Both further investigation into the efficacy of anti-DEspR therapy, and the development of a stoichiometric, site-specific, stable method for drug loading will provide an advancement in anti-cancer therapy.

No / The isolation and characterisation of antiplatelet antibodies in autoimmune thrombocytopenia purpura patients (ITP) is described. Autoimmune thrombocytopenia purpura is an autoimmune disease, clinically defined by low platelet counts, normal or increased megakaryocytopoiesis and antiplatelet antibodies in serum. This study used phage display to isolate Fab antiplatelet antibodies to study the structure-function relationships of pathogenic antibodies in ITP. Out of six randomly selected colonies, four colonies reacted strongly with whole platelets in enzyme-linked immunosorbent assay (ELISA). Sequence analysis showed that all four colonies had the same DNA sequence and were the same antibody. Results of Western blotting against non-reduced human platelet lysate showed that the Fab reacted with platelet proteins with apparent molecular weights of 116, 92 and 39 kD. Furthermore, Western blotting assay against purified membrane glycoprotein IIIa demonstrated reactivity against a band with a molecular weight of 92 kD. Results from Western blotting against platelet lysate and pure platelet glycoprotein confirmed the Fab fragment recognised the platelet glycoprotein IIIa. Three out of the four phage colonies produced soluble Fab, which demonstrated reactivity against platelet autoantigens in ELISA. Further sequence analysis showed that the Fab was somatically mutated suggesting antigen drive and therefore T-cell assistance was important in the development of this antibody. One of the somatic mutations introduced an RSD amino acid sequence in the complementary determining region 1(CDR1) of the light chain, which may mimic the RGD motif of fibrinogen which binds integrin GPIIb/IIIa. This raises the possibility that somatic mutation and antigen drive have produced a pathogenic autoantibody.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院 / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第24837号 / 医博第5005号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤, 能永, 教授 生田, 宏一, 教授 上野, 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Monoclonal antibodies are powerful and versatile tools that enable the study of proteins in diverse contexts. They are often utilized to assist with identification of subcellular localization and characterization of the function of target proteins of interest. However, because there can be considerable sequence diversity between orthologous proteins in Xenopus and mammals, antibodies produced against mouse or human proteins often do not recognize Xenopus counterparts. To address this issue, we refined existing mouse monoclonal antibody production protocols to generate antibodies against Xenopus proteins of interest. Here, we describe several approaches for the generation of useful mouse anti-Xenopus antibodies to multiple Xenopus proteins and their validation in various experimental approaches. These novel antibodies are now available to the research community through the Developmental Study Hybridoma Bank (DSHB).

The development of safer and more accessible contraceptive options is necessary to reduce the high number of unintended pregnancies worldwide. As monoclonal antibody engineering continues to revolutionize drug development, a variety of strategies are being harnessed to establish antibody-based contraceptives. Human Contraception Antibody (HCA), an immunoglobulin G1 (IgG1) monoclonal antibody that potently agglutinates human sperm, is a promising candidate for nonhormonal immunocontraception in women. Our group recently established the safety and efficacy of a topical IgG1 HCA-formulated dissolvable vaginal film. Though successful, we are currently working to further optimize and improve the HCA product. In this study, we characterized engineered variants of HCA. Bioactivities, specifically agglutination and effector functions, of multimeric and fragment crystallizable (Fc)-mutated variants were compared and inform further engineering of an optimal clinical profile. We then established an atomized mRNA mechanism for delivery of HCA to the female reproductive tract (FRT). The use of mRNA could provide several advantages including: efficiency, reversibility, safety, durability, and cost-effectiveness. mRNA-encoded HCAs were expressed in several models of the FRT and were functional, sperm-specific, and safe. We also analyzed Fc N-glycans at the conserved glycosylation site on IgGs that regulate effector functions and compared the site-specific glycosylation on antibodies generated by two HCA expression platforms of interest, namely Nicotiana benthamiana and mRNA-transfected vaginal cells. Disparities in glycan site occupancy and glycoform populations between the two platforms were observed. Platform-specific HCA glycans resulted in differing levels of sperm phagocytosis, an Fc function. In summary, these studies provide a clearer understanding of engineered variants and delivery platforms to further advance the development of HCA as a novel, antibody-based female contraceptive.