Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 83-98). Also available online.

Thesis (Ph. D.)--University of Hong Kong, 1995. / Includes bibliographical references (leaf 204-260).

Thesis (Ph. D.)--University of Chicago, Division of the Biological Sciences and the Pritzker School of Medicine, June 1999. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Shuker, Suzanne, Committee Chair ; Doyle, Donald, Committee Member ; Orville, Allen, Committee Member ; Barry, Bridgette, Committee Member ; McCarty, Nael, Committee Member.

No description available.

Asthma is a common chronic respiratory disease where patients suffer from restricted airways and airway inflammation (mainly eosinophilic type 2 (T2) inflammation). Inhaled (ICS)/oral corticosteroids (OCS) and, more recently T2-targeting biologics, are prescribed as mainstay therapies for asthma. Despite these therapies, a subset of asthma patients continues to have symptoms and airway inflammation, suggesting an underlying additional asthma pathology. We have observed multiple airway autoantibodies in 55% of moderate-to-severe asthma patients associated with inadequate response to corticosteroids and anti-T2 biologics. Increased airway degranulation (eosinophilic and/or infective) together with lymphopenia (low lymphocyte counts) underlie these self-reactive/autoimmune-like events. In healthy individuals, regulatory lymphocytes limit the development and activity of self-reactive cells, including those capable of producing autoantibodies. Lymphopenia can lead to skewed non-regulatory to regulatory lymphocyte subsets that support a microenvironment with reduced ability to limit self-reactivity. In this study, wanted to measure B cell subsets by flow cytometry and non-regulatory to regulatory B cell ratios in asthma patients and healthy controls. To understand B cell compartmentalization, we analyzed peripheral and sputum B cells. We identified decreased regulatory B cells (Bregs), in particular CD5+ Bregs, and skewed non-regulatory to regulatory B cell ratios in both circulation and airways of asthma patients. Compared to healthy controls, only patients requiring daily OCS had significantly lower CD5+ Bregs, suggesting a reduced regulatory component in more severe patients. Further, CD5+ Bregs, capable of producing immunomodulatory interleukin-10, were significantly lower in patients with a history of multiple lymphopenic events (70% requiring daily OCS). Together, this supports the need to investigate lymphopenia-induced dysregulation of Bregs, increase in autoreactive B cells and airway autoreactivities, and subsequent progression into a more-severe therapy refractory autoimmune pathology. This opens a new avenue for asthma treatment, particularly for the severe population with airway autoimmune responses often not targeted/controlled by current anti-inflammatory therapies. / Thesis / Master of Science (MSc) / Asthma is a common respiratory disease where patients have difficulty breathing and airway inflammation. Corticosteroids and/or targeting biologic therapies are prescribed to reduce inflammation. Despite these treatments, some patients still have inflammation. In patients that don’t respond well to treatment, we have found evidence of self-attacking antibodies that can further lead to disease severity. These antibodies are produced by self-attacking B cells normally suppressed by regulatory B cells (Bregs). We wanted to measure if asthma patients have reduced ability to limit self-attacking B cells because of reduced Bregs. We found that asthma patients have lower Bregs and an imbalance of non-regulatory to regulatory B cells in the blood and airways. Together, we show B cell subsets that suggest a self-attacking asthma component, not targeted by current asthma treatments. Understanding the involvement of Bregs in asthma opens a new therapeutic option in those that don’t respond well to current treatment.

Background: The presence of B cells in early stage non-small cell lung cancer (NSCLC) is associated with longer survival, however, the role these cells play in the generation and maintenance of anti-tumor immunity is unclear. B cells differentiate into a variety of subsets with differing characteristics and functions. To date, there is limited information on the specific B cell subsets found within NSCLC. To better understand the composition of the B cell populations found in NSCLC we have begun characterizing B cells in lung tumors and have detected a population of B cells that are CD79A(+)CD27(-)IgD(-). These CD27(-)IgD(-)(double-negative) B cells have previously been characterized as unconventional memory B cells and have been detected in some autoimmune diseases and in the elderly population but have not been detected previously in tumor tissue. Methods: A total of 15 fresh untreated NSCLC tumors and 15 matched adjacent lung control tissues were dissociated and analyzed by intracellular flow cytometry to detect the B cell-related markers CD79A, CD27 and IgD. All CD79A(+) B cells subsets were classified as either naive (CD27(-)IgD(+)), affinity-matured (CD27(+)IgD(-)), early memory/germinal center cells (CD27(+)IgD(+)) or double-negative B cells (CD27(-)IgD(-)). Association of double-negative B cells with clinical data including gender, age, smoking status, tumor diagnosis and pathologic differentiation status were also examined using the logistic regression analysis for age and student's t-test for all other variables. Associations with other B cell subpopulations were examined using Spearman's rank correlation. Results: We observed that double-negative B cells were frequently abundant in lung tumors compared to normal adjacent controls (13 out of 15 cases), and in some cases made up a substantial proportion of the total B cell compartment. The presence of double-negative cells was also found to be inversely related to the presence of affinity-matured B cells within the tumor, Spearman's coefficient of -0.76. Conclusions: This study is the first to observe the presence of CD27(-)IgD(-)double-negative B cells in human NSCLC and that this population is inversely correlated with traditional affinity-matured B cell populations.

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

Immunoglobulin (Ig) gene diversification plays an essential role in adaptive immunity. Faced with a continuous yet varied stream of self, non-self, and possibly harmful molecules, many organisms have mechanisms in their arsenal that have evolved to match the diversity of the antigens they encounter. In humans and mice, developing B and T lymphocytes go through a first round of genomic alteration — V(D)J recombination — in the bone marrow and the thymus, respectively. B cells can subsequently undergo two additional Ig gene diversification processes in secondary lymphoid tissues. Through somatic hypermutation (SHM), Ig variable regions of stimulated germinal center (GC)-forming B cells are mutated and further diversified, enabling affinity maturation. During class-switch recombination (CSR), on the other hand, B cells in the GC or prior to entering the GC recombine Ig constant regions, swapping the IgM-encoding locus for another isotype constant regions gene (e.g., IgG1, IgG3, IgE, IgA) to allow for different effector functions. Both B cell-specific genomic alterations are initiated when the single-stranded DNA (ssDNA) mutator enzyme activation-induced cytidine deaminase (AID) catalyzes the removal of the amino group off deoxycytidine residues, resulting in deoxyuridines and dU:dG mismatches. Low-fidelity cellular responses to the presence of dU, including the mismatch repair (MMR) and the base-excision repair (BER) pathways, are then thought to introduce mutations in SHM and CSR, as well as cause double-strand breaks (DSBs) repaired through canonical and alternative non-homologous end-joining in CSR. Though necessary for proper physiological function, these lymphocyte genome diversification processes are rife with danger for B cells and there is strong selective pressure to carefully orchestrate and target them so as not to threaten the genomic integrity of the cells through breaks or other mutations at non-Ig loci. Yet, these events can still occur, as demonstrated by the implication of AID with translocations found in some cancers (e.g., c- MYC:IGH in Burkitt’s lymphoma). Therefore, the mechanisms underlying AID mutagenic activity targeting to physiological deamination substrates have been the focus of several studies. Protein kinase A (PKA)-dependent phosphorylation of AID at its serine 38 residue has been shown to enable its interaction with replication protein A (RPA) before binding to ssDNA. Others have reported that SPT5 helps target AID to sites of RNA polymerase II (Pol II) stalling, such as the Ig switch sequences. Another cofactor, the RNA exosome complex, helps target the ssDNA mutator AID to both strands of DNA in vivo. The RNA exosome had hitherto been described in the context of RNA processing and degradation as 3’ → 5’ exoribonuclease. Sterile transcript-generating transcription at Ig loci was known to be required for proper AID catalytic activity; the newly described link between the RNA exosome and AID activity raised the prospect that RNA processing, and not mere transcription, might be playing a role in shaping the diversification of the immune repertoire in B lymphocytes. During CSR, transient three-strand structures called R loops are generated. R loops are formed as the nascent transcript invades the DNA duplex, hybridizing to the template strand, and displacing the non-template one. The G-rich nature of the non-template strand is posited to help stabilize the R loop, which allows the ssDNA mutator AID to use the exposed, non-template strand as a substrate. AID must then access the template strand. Here, we investigate the role that the RNA exosome and a potential cofactor, the putative RNA/DNA helicase senataxin (SETX), play in the sequence of biological events that result in CSR while protecting the cell from R-loop accumulation-associated genomic instability.