Final publication is available at http://dx.doi.org/10.1016/j.bbrc.2015.12.114 CreativeCommonsAttribution Non-Commercial No Derivatives License の記載が必要 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19924号 / 医博第4144号 / 新制||医||1017(附属図書館) / 33010 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 野田 亮, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Levels of Immunoglobulin G (IgG)-subclasses are analyzed when patients have reoccurring infections and in order to follow the treatment of IgG4 related disease. The measured quantity of IgG-total can be within the reference interval even if the patient has a deficiency in one of the IgG-subclasses. Due to this Sundsvall’s hospital plans to begin analyzing IgG-subclasses. The aim was to verify the performance level of the analysis IgG-subclasses (IgG1-4) with Siemens Atellica NEPH 630. The method was verified by evaluating the method’s precision and by comparing the sum of IgG-subclasses with the quantity of IgG-total analyzed on Cobas c502. In addition, the reference intervals provided by Siemens were evaluated using samples from blood donors. The study evaluated whether there was a correlation between infection with SARS-CoV2 and a deficiency in IgG-subclasses. The verification began by performing quality control twice daily over a period of four weeks. The IgG-subclasses test was performed on blood donor samples with Siemens Atellica NEPH 630 for evaluation of the reference values. The coefficient of variation was less than 6 % for all four subclasses. The reference values for IgG1, IgG2, IgG3, and IgG4 are all in alliance with the reference values provided by Siemens. The sum of IgG-subclasses corresponded well with IgG-total with a correlation value (R) 0.82. No correlation was found between IgG deficiency and infection with SARS-CoV2. The validation of the analysis of IgG-subclasses was successful with quality measurements within the supplier’s intervals. No adjustments will be needed for the reference intervals.

Calves are born agammaglobulinemic and rely on colostrum consumption for the transfer of maternal passive immunity. Calves that fail to absorb adequate amounts of maternal antibodies from colostrum are commonly referred to as having failed transfer of passive immunity (FTPI). The overall aim of this dissertation was to explore the usefulness of FTPI testing in neonatal beef calves to predict their risk for subsequent illness or death. The objectives were to evaluate the impact of FTPI on pre-weaning disease in beef and dairy calves, quantify and compare the variance in IgG concentrations measured by radial immunodiffusion and serum total protein (STP) values measured by optical refractometry, and evaluate the correlation between herd-level prevalence of FTPI and herd-level prevalence of pre-weaning disease in beef calves. Evaluation of literature relevant to FTPI was compiled and assessed to quantify the impact of FTPI on pre-weaning disease in beef and dairy calves. A series of randomized trials were used to evaluate the variance in IgG concentrations and STP values from banked serum. Health records from multiple farms and IgG results were used to evaluate the relationship between FTPI and disease at the individual and herd-level. Failed transfer of passive immunity had a variable association with pre-weaning disease in beef and dairy calves. IgG concentrations were less precise than STP values especially when dilution was required. IgG concentrations and STP values were associated with an increased risk of disease in pre-weaned beef calves, but FTPI cut-off values poorly classified the risk for subsequent disease. The proportion of calves with FTPI was not correlated with the proportion of calves that developed pre-weaning disease. Using a single immunological factor, such as IgG concentration or STP, to predict disease results in the misclassification of disease risk and does not consider additional component causes of disease.

The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD. An on-line preconcentration-CE method that can selectively preconcentrate any protein for which an antibody is available would be very useful for the analysis of low abundance proteins and would establish CE as a major tool in biomarker discovery. To accomplish this, an on-line protein G monolithic preconcentrator CE system for enrichment and separation of proteins was developed. This system proved effective for on-line sample extraction, clean-up, preconcentration, and CE of IgG in human serum. IgG from diluted (500 and 65,000 times) human serum samples was successfully analyzed using this system. The approach can be applied to the on-line preconcentration and analysis of any protein for which an antibody is available. The desire to separate all proteins present in human tissues, cells and biological fluids has challenged the separation research community for many years. The difficulty of this task resides in the complexity of the sample. Blood serum, for instance, may express up to 10,000 proteins with an estimated dynamic range of 9 orders of magnitude. Additionally, most of these proteins are present at very low concentrations (ng/mL). Identification and quantification of low abundance proteins is hindered by the presence of high abundance proteins, such as human serum albumin (HSA) and immunoglobulins (IgG). Therefore, in most cases, removal of the high abundance proteins or enrichment of low abundance proteins is necessary prior to the analysis of low abundance proteins. To address this, a coupled affinity-hydropobic monolithic column for the simultaneous removal of IgG, preconcentration of low abundance proteins, and separation by capillary zone electrophoresis was designed. The system proved to be very reproducible. The run-to-run %RSD values for migration time and peak area were less than 5%, which is typical of CE. Finally, a new method was developed to prepare monoliths with anion exchange functionality. Polymer monoliths were prepared by in situ polymerization of methacrylate monomers. The monoliths were coated with a water soluble polymer and used for the analysis of proteins. Using this approach, a model monolith was prepared. Subsequent coating yielded a monolith with quaternary ammonium groups on the surface, which was confirmed by strong anodic electroosmotic flow. Analysis of standard proteins by ion exchange LC and CEC was demonstrated. This simple and rapid method for surface modification opened new avenues for the preparation of monoliths with a broad range of functionalities.

Immunolocalization of proteins within the cell is a significant and powerful tool that improves understanding of cellular functions and processes, such as molecule secretion during immune responses. Immunogold electron microscopy (IEM) is an immunohistochemistry technique that uses gold-conjugated antibodies and electron microscopy (EM) to identify and localize antigens at the ultrastructural level. Here, we are trying to develop and optimize an IEM staining protocol that targets glomerular proteins of interest in Minimal Change Disease (MCD), and eliminates background staining, and preserves tissue morphology. Using this optimized protocol, we hope to learn more about the relationship between IgG and Nephrin in MCD. Kidney biopsies diagnosed with MCD, Membranous Nephropathy (MN), and Thin Basement Membrane Disease (TBMD) and previously embedded in paraffin blocks were retrieved from the tissue archive of the Renal Pathology Laboratory at Boston Medical Center. MN and TBMD were selected as positive controls for IgG and Nephrin staining protocols, respectively. Co-staining of IgG and Nephrin was performed after the protocols for each target were optimized. During protocol development, it was observed that section quality is significantly affected by the angle and sharpness of the knife, and the thickness of the section. Moreover, section quality highly impacted gold particle localization. Ultimately, co-staining of IgG and Nephrin was successful in MCD cases. However, further improvements are needed to optimize IgG and Nephrin staining, and in turn, our understanding of MCD.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24991号 / 医博第5025号 / 新制||医||1069(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 生田 宏一, 教授 椛島 健治, 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

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<p>Among food allergies, peanut has attracted the most research attention because the allergy is typically lifelong, often severe and potentially fatal. Furthermore, other than epinephrine, there are no treatments available to date. A decade of research has provided a great deal of insight into the factors that promote and regulate the <em>development </em>of allergic responses. However, less in known about the factors involved in the <em>elicitation</em> of the most common and severe manifestation of peanut allergy, namely anaphylaxis. The research in this thesis centers on the investigation of cellular and molecular pathways leading to peanut-induced anaphylaxis (PIA) as well as potential therapeutic targets. Specifically presented are: i) the development and characterization of a mouse model of PIA (Chapter 2), ii) the role of molecules including histamine, leukotrienes (LT) and platelet-activating factor (PAF) (Chapter 3) and, iii) the relative contribution of mast cells, basophils and macrophages as well as IgE and IgG₁(Chapter 4). Our data show that oral sensitization to peanut in C57BL/6 mice generated local and systemic markers of type-2 immunity that was associated with robust and consistent clinical anaphylaxis following antigen challenge. In this context, concurrent blockade of PAF and histamine receptors markedly decreases the severity of these reactions. Moreover, they demonstrate that distinctive immune effector pathways involving activation of mast cells (via IgE and IgG₁) and macrophages (via IgG₁) cooperate to elicit a broad range of systemic reactions to peanut. These findings highlight that concomitant and progressive recruitment of immune-effector pathways leads to a full range of anaphylactic reactions and therefore, therapeutic strategies for PIA may need to target several pathways or, alternatively shared components within these pathways. Combination therapy blocking both PAF and histamine may represent such as a therapeutic approach.</p> / Doctor of Philosophy (Medical Science)