Thesis (M.S.P.H.)--University of Michigan, 1932.

Thesis (PH. D)--University of Chicago, 1912. / Reprinted from the Journal of infectious diseases, July 1912. Includes bibliographical references. Also available on the Internet.

Thesis (M.S.P.H.)--University of Michigan, 1932.

Thesis (M.S.P.H.)--University of Michigan, 1942.

Thesis--University of Michigan. / "From the Department of Pediatrics and Infectious Diseases of the University of Michigan." "Reprinted from the Journal of Allergy ... vol. 11, no. 6 ... September, 1940." Bibliography: p. 11.

M.Tech.

Type I hypersensitivities are mediated by the IgE antibody. The effector functions and synthesis of IgE result from interactions with a network of proteins that include a high affinity (FcRIα) and a low affinity (CD23, FcRII) Fc receptor in conjunction with the B lymphocyte receptor, CD21. CD23 is a multifunctional type II transmembrane protein that binds its known ligands through its ectodomain either as a membrane-bound or soluble receptor generated in vivo by specific proteolytic cleavages. IgE production is primarily regulated by interactions between IgE, CD23 and CD21. Despite its importance for development of strategies to limit hypersensitivity, precise information about the molecular interactions remains limited. During this study, I engineered, expressed and purified from bacteria three soluble human CD23 fragments that are normally formed in vivo and shed from the cell surface (1) derCD23, amino acids 156-298 (2) sCD23, amino acids 150-321 and (3) the entire ectodomain, exCD23, amino acids 48-321 to examine the comparative binding of recombinant human CD21 SCR 1-2 and native human IgE to these fragments. Gel filtration HPLC revealed that derCD23 and sCD23 were monomeric whereas exCD23 assembled as a heterogeneous mixture that included trimers and monomers. At the concentrations utilized, CD23 fragments sCD23 and exCD23 bound CD21 with similar affinity, whereas interaction between derCD23 and CD21 was minimal when analyzed by surface plasmon resonance (SPR) spectroscopy. These findings suggest that penultimate “tail” amino acids between 298 and 321 stabilize CD21 attachment, although it cannot be ruled out, the region between Met 150 and Ser 156 may also play a role in binding CD21 SCR 1-2. In contrast, there is a progressive increment in the affinity of soluble fragments (exCD23>sCD23>derCD23) for IgE, upon increasing length of the proximal CD23 “stalk” domain. These findings highlight the differences in both the structural basis and affinity of the three physiological fragments of human CD23 for the ligands CD21 and IgE and underscore the complexity of CD23-mediated regulatory networks. It was found that B-cells only make up ~5% of the PBMC population, and that these cells were able to be activated, via STAT-6 phosphorylation, to enter class switch recombination (CSR) by the addition of switch factors (IL-4 and anti-CD40). Titration experiments dictated that 25 ng/mL of CD23 was the most efficient concentration to up-regulate IgE synthesis in PBMCs; furthermore, soluble CD23 proteins were incubated with PBMCs in the presence and absence of CD21 SCR 1-2 to investigate the effect that these recombinant proteins have on IgE synthesis. Results showed that the influence of recombinant proteins (both CD23 and CD21) on IgE synthesis was slight. It was shown that while derCD23 had no significant effect, monomeric sCD23 down-regulated, and the mixture of monomeric and oligomeric exCD23 up-regulated IgE synthesis. On addition of CD21 SCR 1-2 to the cells switched and treated with soluble CD23, it was found that in both cases for sCD23 and exCD23, IgE synthesis was increased, while for derCD23, there was no noticeable difference in IgE synthesis. This confirmed previous data showing the lack of binding between derCD23 and CD21 SCR 1-2. The exact binding site for CD21 on the CD23 molecule is unknown, and incompletely represented in the NMR and crystal structures. It is thought that CD21 binds to the C-terminal tail section, not present in derCD23. It is therefore likely that only a negative-feedback mechanism operates with derCD23 to regulate IgE synthesis. Further investigation of the binding of CD23 fragments to SCR 5-8 of CD21 and the effect of this on IgE synthesis may lead to a potential therapeutic role for derCD23 in the treatment of allergic disease. Data accumulated in this study suggests that investigating the modulation of oligomeric state and thus the activity of soluble CD23 fragments may be important in the construction of new regulators of IgE synthesis.

Studies made on the lipid components of Microsporum quinckeanum, have shown that 6 day old fermenter-grown mycelia and that of 21 day old static cultures grown in Sabouraud's glucose liquid medium have a lipid content of approximately 16% by dry weight. Thin layer chromatography showed that the lipid fractions obtained from both methods of cultivation were identical. During the purification of the lipid extracts, a lipoprotein was isolated having 16.45$ protein content, and after hydrolysis and chromatography this material demonstrated the presence of eleven amino acids. Dried mycelia, purified lipid and lipoprotein were used separately in complete and incomplete Freund's adjuvant to sensitize guinea pigs and rabbits by various routes. Sensitization of guinea pigs was achieved also by Bloch's method. All animals were challenged by separate intradermal injections of total lipid extracts, lipoprotein and the six lipid fractions. Delayed hypersensitive responses of varying intensities were demonstrated by all sensitized animals. Further investigations were made using lymph nodes removed from rabbits separately sensitized with dermatophyte lipid extracts and lipoprotein. Protein extracts of lymph nodes from these animals were examined electrophoretically. The presence of precipitating antibodies in sera and lymph node extracts were demonstrated by immunodiffusion and precipitin ring tests. Passive transfer tests were carried out successfully to conclusively demonstrate the presence of antibodies in the lymph node extracts. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Approximately 10% of North Americans have at least one food allergy. Allergic reactions to foods are mediated by IgE antibodies which, upon allergen exposure, are crosslinked on the surface of mast cells and basophils, resulting in degranulation. The granule contents of mast cells and basophils are responsible for the diverse signs and symptoms of food allergy, including hives, itchiness, edema, vomiting, diarrhea, and a potentially lethal form of systemic shock called anaphylaxis. The clinical recommendation for food allergic Canadians is strict allergen avoidance, and emergency epinephrine use upon an accidental exposure. There are presently no disease modifying therapeutic options for allergic Canadians. The work in this PhD thesis focuses on the pathways which result in the production and memory of allergen-specific IgE. Using murine models of food allergy and anaphylaxis, we have delineated pathways of early allergic sensitization which may occur prior to allergic patients presenting in the clinic during the first allergic reaction. We have demonstrated that CD4+ T cell activation can occur in the absence of B cell activation, but can hold the memory of IgE responses persistently, potentially for a lifetime. Upon re-exposure to the allergen, CD4+ T cells can activate naïve B cells, which pursue either direct or sequential isotype switching to IgE, both resulting in clinical reactivity against food allergens. Our findings will inform future diagnostic and prognostic tests in pre-allergic patients, and to design novel therapeutics with disease modifying capacity in humans. / Thesis / Doctor of Philosophy (PhD)

House dust mite (HDM) is an indoor aeroallergen which is commonly associated with the development of allergic rhinitis and allergic asthma. In fact, up to 70% of allergic asthmatic individuals have been shown to demonstrate reactivity towards HDM allergens. The downstream adaptive immune response to HDM is well characterize and is described as being largely T helper cell type 2 (Th2) and partly T helper cell type 17 (Th17) dominated. However, less is known about how resident antigen presenting cells (APCs) and structural cells such as airway epithelial cells recognize and respond to HDM. Unlike other microbial antigens, recognition of HDM is not primarily mediated through pattern recognition, rather by an intrinsic enzymatic or lipid-binding ability or by specific prost-translational modifications such as glycosylation. Various innate immune receptors, such as toll-like receptors (TLRs), C-type lectin receptors (CLRs), and protease-activated receptors (PARs), found on the surface of APCs and airway structural cells, have been demonstrated to be important in response to HDM. Nonetheless, the exact mechanisms by which these receptors or other undescribed receptors or molecules promote adaptive immunity, particularly Th2 immunity, is unclear. Using knock-out mice, we have shown that the kinase RIP2, which functions downstream of the peptidoglycan receptor NOD2, is involved in the early response to HDM likely within AECs and that its activation promotes airway inflammation. In follow-up work, we also demonstrate that early and acute pharmacologic inhibition of this kinase in a HDM asthma model has lasting effects on the reduction of airway inflammation and type 2 immunity associated with the pathology. The third project describes the identification of a novel receptor for HDM, LMAN1. We find that LMAN1 on dendritic cells functions to inhibit inflammatory responses to HDM and that loss of LMAN1 enhances airway inflammation in an asthma model. Altogether, my work has contributed to our understanding of the early events and molecules involved in responding to HDM. Our hope is that these findings will be useful in the discovery and testing of new therapies for treatment of allergic disease.