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The antibody Immunoglobulin G (IgG) main function is to protect and prevent the body from infections, and it is normally found in human serum. This study is about IgG glycosylation, which is associated with different types of diseases such as neurological diseases, cancers and immunodeficiency etc. This study attempts to optimize IgG glycopeptide enrichment in a 100 μL micropipette tip set up, and to separate the enriched glycopeptides using capillary electrophoresis (CE). Matrix-assisted laser desorption/ionization – mass spectrometry (MALDI-MS) was used for data acquisition and glycopeptide profiling.  In this study, loading solutions with different combinations of acetonitrile (ACN) and trifluoroacetic acid (TFA), together with various precondition and sample preparation procedures were evaluated on IgG digest samples. Best enrichment performance, particularly regarding the selectivity, was achieved using the parameters as follows: loading solution of 83% ACN/16% H2O/1% TFA, sample solution in H2O containing 83% ACN, using a 100 μL micropipette tip packed with 1 mg cotton wool. A re-enrichment step was carried out on enriched glycopeptide samples, and improved selectivity of glycopeptides could be observed. Enriched glycopeptides could be separated into three major groups by CE using an acidic background electrolyte of 50 mM formic acid and 50 mM acetic acid, pH 2.5. / Huvudfunktionen för antikroppen Immunoglobulin G (IgG) är att skydda kroppen och förhindra infektioner och det finns normalt i mänskligt serum. Denna studie handlar om glykosylering, som är kopplad till olika typer av sjukdomar såsom neurologiska sjukdomar, cancer och immunbrist etc, och är en potentiell biomarkör för sjukdomar. Denna studie försöker optimera en IgG glycopeptidanrikningsmetod i en 100 μL mikropipettspets och separera de anrikade och intakta glycopeptiderna med hjälp av kapillärelektrofores (CE). Matrisassisterad laserdesorption/jonisering – masspektrometri (MALDI-MS) användes för datainsamling och glykopeptidprofilering  I denna studie utvärderades lösningar med olika kombinationer av acetonitril (ACN) och trifluororättiksyra (TFA) tillsammans med olika föberedelseförfaranden och provberedningsprocedurer på IgG prover. Anrikningsprestanda, särskilt selektiviteten, uppnåddes bäst med användning av följande parametrar: lösningen av 83% ACN / 16% H2O / 1% TFA, provlösning i H2O innehållande 83% ACN, med användning av en 100 mikroliter mikropipettspets fylld med 1 mg bomull. Återreningssteget genomfördes på anrikade glykopeptidprover och förbättrad selektivitet för glykopeptider kunde observeras. Anrikade glykopeptider kunde separeras i tre huvudgrupper med CE med användning av sur bakgrundselektrolyt med 50 mM myrsyra och 50 mM ättiksyra, pH 2,5.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24994号 / 医博第5028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 上野 英樹, 教授 椛島 健治, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Serum cortisol and immunoglobulin G (IgG) concentrations were measured to investigate the relationship between vitamin A status and immune function in lambs. Twenty-four crossbred ewe lambs were each fed 900 g·d-1 of a carotene—deficient diet composed of 95.5% whole oats, 3% molasses, .5% trace mineral salt and 1% limestone. All lambs were injected monthly with vitamins D and E and with selenium. The 12 control lambs also received a 100,000 IU oral dose of vitamin A palmitate in capsule form every 2 wk. All lambs were challenged by injecting them with 1 mg ovalbumin in 1 ml of Freund’s complete adjuvant. At the time of challenge, serum vitamin A levels for the control and A-deficient (A—def) lambs were 33.3 and 3.1 ug·dl-1 respectively. Blood was collected prior to and 6, 13, 20 and 34 d post—challenge. The lambs were then reschallenged using the same antigen and blood was obtained 1, 2, 6 and 22 d post—challenge. Lambs were sacrificed at the end of the second challenge period. Spleen weights were obtained and gross post—mortem observations were made at this time. / Master of Science

Conventional housing environments for broiler chickens and commercial laying hens are often barren, high-density environments with an emphasis on production efficiency. These housing conditions limit birds' ability to display species-specific behaviors, can negatively impact health, and may contribute to negative cumulative experience. Cumulative experience is the culmination of all positive and negative experienced during an animal's lifetime. However, cumulative experience is difficult to quantify, as no validated measures of cumulative experience exist. Additionally, existing measures of negative animal experience mostly rely on interpretations of animal behavior which can be subjective, time consuming, and difficult to interpret. Therefore, there is scientific need for objective measures that can detect cumulative experience in poultry. Secretory and plasma Immunoglobulin A (IgA), telomere length, feather corticosterone concentrations, and attention bias testing all seem to respond to positive and negative experiences in humans or other non-human animal species, indicating that they may be useful as measures for poultry. Therefore, the objective of this thesis was to determine if these novel measures could be used as indicators of cumulative experience in broiler chickens and laying hens. In chapter 3, secretory and plasma IgA concentrations were measured in broilers raised in either high-complexity or low-complexity environments under either high or low stocking density over three replicated experiments. Birds housed in highly complex environments showed higher concentrations of plasma IgA compared to birds housed in low-complexity environments at day 48 of age, indicating reduced chronic stress in the former. Additionally, day 48 secretory IgA concentrations were decreased in birds housed in high-density environments compared to birds housed in low density environments, indicating birds from high-density environments were more chronically stressed. In chapter 4, gonad and kidney telomere length was measured to determine cumulative experience in broilers raised in the same housing conditions and replicated experiments of chapter 3. Treatment did not impact gonad telomere length, in line with expectations as gonads contain stem cells which produce high concentrations of telomerase. Birds housed in high-complexity pens had longer kidney telomeres compared to birds in low-complexity pens, indicating high-complexity birds had more positive cumulative experience. Stocking density did not impact kidney telomere length. In chapter 5, attention bias, tonic immobility, plasma and secretory IgA concentrations, and feather corticosterone concentrations were determined in laying hens raised in conventional cages or enriched floor pens. Birds in enriched floor pens showed increased attention bias, decreased tonic immobility, increased secretory IgA concentrations at week 22 of age, and decreased feather corticosterone concentrations compared to caged hens. These results indicate that compared to conventional cages, enriched pens in this study improved immune systems, reduced chronic stress, reduced fear, but increased anxiety in hens. In conclusion, secretory and plasma IgA and telomere length show appropriate contrast in response to broiler chicken housing conditions. However, additional work needs to be done before these measures can be widely used as measures of cumulative experience in poultry. Furthermore, attention bias, secretory IgA, and feather corticosterone showed an appropriate contrast between chronic stress responses in laying hens, but confirmation is needed in other contexts. Overall, the results indicate a beneficial relationship between environmental complexity and poultry welfare physiology and affective state, with the exception for anxiety in laying hens. Thus, providing an enriched environment can improve the welfare of commercial poultry and result in positive cumulative experience in most situations. Additionally, these results indicate that stocking density is a negative environment in broilers but potentially less intense than previously thought under experimental conditions. The assessment of behavioral and physiological measures of cumulative and positive animal experience should be included in experiments seeking to determine the impacts of environmental or management conditions to determine the broader impacts on poultry welfare. / Doctor of Philosophy / Conventional housing systems of broiler chickens (raised for meat) and laying hens (raised for egg production) can negatively impact their welfare. Animal welfare, defined as an animal's ability to interact with and cope with their environment, is an individual experience for each animal and fluctuates on a scale from very negative to very positive. Traditionally, measurements of animal welfare have focused only on avoiding the negative aspects of animal welfare such as fear, distress (negative stress), hunger, thirst, pain, and suffering. However, it is important that animals are provided opportunities to experience positive animal welfare to provide a life worth living. So, when measuring animal welfare, all positive and negative experiences (termed cumulative experience) should be included to form an accurate picture of an animal's welfare. However, no validated measures of cumulative experience exist in non-human animals. However, recently, several potential measures of cumulative experience have been proposed in human and non-human animals including secretory and plasma IgA, telomere length, feather corticosterone, and attention bias testing. So, the objective of this thesis was to determine if these proposed measures can be used to determine cumulative experience in commercial broilers and laying hens. In chapters 3 and 4, we investigated if secretory and plasma IgA concentrations (measure of chronic stress; chapter 3) and telomere length (measure of cumulative experience; chapter 4) responded to environmental complexity (positive stimulus) and stocking density (negative stimulus) over three replicated experiments. Broilers were housed in a 2 × 2 factorial study of either high or low complexity or high or low density. This resulted in four treatment groups of high-complexity/high-density, low-complexity/low-density, high-complexity/low-density, and low-complexity/high-density. During chapter 3, environmental complexity increased concentrations of plasma IgA, indicating that birds from high-complexity pens were under less chronic stress compared to birds from low-complexity pens. Alternatively, high density decreased secretory IgA, indicating that birds from high-density pens were under a more chronic stress than birds from low density pens. In chapter 4, environmental complexity increased telomere length in broilers compared to low-complexity pens indicating that environmental complexity positively impacted cumulative experience. However, stocking density did not impact telomere length, indicating that high density did not negatively impact cumulative experience. In chapter 5, we investigated if attention bias (measure of anxiety), tonic immobility duration (measure of fear), plasma and secretory IgA (chronic stress), and feather corticosterone (chronic stress) responded to environmentally enriched floor pens (positive housing system) and conventional caging (negative housing system). We found that birds housed in enriched floor pens were more anxious (increased attention bias), less fearful (decreased tonic immobility duration), and less chronically stressed (increased SIgA concentrations at week 22 and increased feather corticosterone concentrations) compared to birds housed in conventional cages. Overall, IgA concentrations and telomere length (broilers) and attention bias, secretory IgA concentration, and feather corticosterone concentrations (layers) seem useable as measures of animal experience in commercial poultry. Additionally, these results indicate that positive experience has a positive impact on cumulative experience in commercial poultry. Stocking density also seems to contribute to chronic stress in broilers, indicated by decreased SIgA concentrations, but only during the last few weeks of life. These findings should be confirmed by additional studies before common use as measures of cumulative experience in animals. However, the inclusion of measures of cumulative and positive animal experience should be included in experiment which wish to determine the broad impacts of housing system on non-human animals.

Immunoglobulins are key mediators of humoral immunity and can be appreciated in an isotype-dependent manner in autoimmune diseases, and to an extent, immune-mediated metabolic diseases. The most common isotype is Immunoglobulin G (IgG), yet there is little understanding of the role IgG plays in the pathogenesis of macrophage-driven metabolic disorders like atherosclerosis, metabolic dysfunction-associated steatotic liver disease (MASLD), and metabolic dysfunction-associated steatohepatitis (MASH). The contents of this dissertation introduce IgG as an activator of the macrophage inflammasome and its deposition in atherosclerotic plaques and fatty livers as a driver of disease progression. The presence of IgG in these depots and its accumulation is dictated by the neonatal Fc receptor (FcRn) in macrophages. IgG levels are known to be determined by recycling, especially in macrophages, rather than by production, and FcRn, encoded by the gene Fcgrt, is the sole receptor responsible. Interestingly, we uncover a myriad of roles for FcRn involved in regulating the biological properties of macrophages, such as their transcriptional and secretory profiles and their polarization amidst an immune response. Taken together, we identify FcRn as a hitherto unknown contender in the manipulation of macrophage function and regulation of IgG in the development of macrophage-associated cardiometabolic diseases using a multitude of methodologies. These findings highlight the importance of macrophage IgG recycling in metabolism and may warrant the potential to explore this phenomenon for therapeutic pursuits.