Browsing by Subject "Antibodies"
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Item Implications of Dysregulated Antibody Hypermutation Patterns in Multiple Sclerosis Patients(2014-01-06) Ligocki, Ann Jolanta; Ward, E. Sally; Monson, Nancy L.; Vitetta, Ellen S.; Scheuermann, Richard H.; Diamond, Betty A.The potential role of B cells in contributing to the pathology of the human autoimmune neurological disease multiple sclerosis (MS) has recently become of significant interest. MS is a complex disorder of the central nervous system (CNS) with various presentation types, symptoms, and damage that involves many different components of the immune system. Historically, the role of B cells was considered minimal and overshadowed by the impact of T cells. The main effector function of a B cell is mediated by the downstream production of antibodies, that bind to their cognate antigens.. Thus, detailed investigation of antibody genomics reveals important information regarding the response of a B cell to its environment. We previously discovered a pattern of somatic hypermutation in the antibody genes expressed isolated from cerebrospinal fluid (CSF) B cells of patients with MS and those at high risk for developing MS. The focus of this thesis project was to further characterize this pattern of mutation termed the antibody gene signature (AGS) and determine the biological significance of this shared AGS among patients. Secondly, I aimed to discover differences in B cell subtypes and antibody genomics in the two of the most common initial presentations of clinically isolated syndrome (CIS) patients at high risk for converting to MS: optic neuritis (ONCIS) and transverse myelitis (TMCIS). Through my work in analyzing antibody genomics, I demonstrated that the AGS is present at the site of MS disease, within the CNS tissue. This provides support for the study of CSF B cells since it recapitulates what is present within the parenchyma. I also determined that both ONCIS and TMCIS patients are enriched for AGS positive B cells within the CSF. This exemplifies that regardless of initial presentation, patients at high risk for converting to MS share this AGS. Further characterization of TMCIS patients revealed that a subset harbor an expansion of plasmablasts in both the periphery and the CSF compared to ONCIS patients. Furthermore, in depth analyses of the peripheral plasmablasts uncovered altered genomic selective pressures in the periphery. This differential expansion of plasmablasts may offer insight for future studies of the possible varied underlying biological processes between these two patient groups. I aimed to determine the biological significance of this shared genomic pattern of the AGS by examining the CNS targeting potential. Immunohistochemical experiments revealed that AGS-enriched antibodies from ONCIS, TMCIS, and MS patients target neurons and astrocytes within the gray matter. These novel findings provide future directions for elucidating the auto-antigen(s) responsible for eliciting the shared AGS. Furthermore, identifying the AGS-enriched antibody binding patterns could aid in identifying potential therapeutic targets to help reduce CNS damage in both CIS and MS patients.Item Mechanisms Controlling Virulence Thresholds of Mixed Viral Populations and Identification of Novel Host Barriers to Poliovirus Neuropathogenesis(2012-07-20) Lancaster, Karen; Pfeiffer, Julie K.Neurotropic viruses comprise some of the worlds most widespread and deadly pathogens, including West Nile virus, rabies virus, and poliovirus. Poliovirus, as a model neurotropic virus, is also an RNA virus. RNA viruses have high mutation rates and a propensity to revert attenuating mutations, contributing to disease and complicating treatment and vaccine development. Despite worldwide epidemics in the early nineteenth century, paralysis from poliovirus is a rare event occurring in less than 1% of poliovirus infections. This suggests the presence of viral and host barriers limiting disease. Here we examined viral barriers by exploring the concept of virulence thresholds using mixtures of virulent and attenuated viruses in a transgenic mouse model of poliovirus infection. We determined that 1000-fold excess of an attenuated strain of poliovirus was protective against disease induced by the virulent strain. Protection was induced locally, was a poliovirus specific effect, and inactivated virus conferred protection. Treatment with a poliovirus receptor-blocking antibody phenocopied the protective effect of inactivated viruses in vitro and in vivo, suggesting virulence thresholds may be modulated by competition for viral receptor. Furthermore, we found the attenuated virus became virulent in immune-deficient mice due to enhanced replication and reversion of attenuating mutations. We also identified additional host barriers limiting pathogenesis using a novel hybridization-based viral diversity assay to quantify the efficiency of poliovirus transport from the periphery to the central nervous system. We found viral replication in peripheral axons is limited and the type I interferon response limits viral replication in peripheral tissues, protecting against disease. Significantly, we discovered that retrograde axonal transport of poliovirus in the sciatic nerve was inefficient and only 20% of viral pool members reaching the brain. The efficiency of viral transport increased upon muscle damage, leading to increased viral diversity and pathogenesis. In summary, we identified a viral induced mechanism controlling virulence of mixed viral populations, and characterized three host barriers that restrict poliovirus pathogenesis in the nervous system. The identification of these barriers restricting virulence may help explain the rare incidence of neurological complications following poliovirus infection and aid in our understanding of viral population dynamics and pathogenesis.Item Pathogenic and Protective Potential of B Cell Dysregulation in Systemic Lupus Erythematosus(2014-07-25) Mayeux, Jessica; Stüve, Olaf; Satterthwaite, Anne B.; Mohan, Chandra; Monson, Nancy L.; Davis, LaurieSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by loss of tolerance to nuclear antigens. Hyperactive B cells are present in SLE patients and murine models of lupus, many of which have defects in inhibitors of B cell receptor (BCR) signaling or plasma cell differentiation. Autoantibodies against a wide range of self antigens contribute to the pathogenesis of SLE and are used to diagnose SLE, determine prognosis, and predict specific disease manifestations. Autoantibodies form immune complexes which deposit in the kidney and joints, resulting in glomerulonephritis and arthritis, respectively. Autoantibodies against antigens in the CNS can cause Neuropsychiatric SLE manifestations, such as psychosis, memory loss, seizures, strokes, and mood disorders. Human SLE patients and murine models of lupus are used here to identify novel autoantibodies in SLE and to better understand the mechanisms by which autoantibodies accumulate in SLE. Protein arrays can be used to identify autoantibodies and autoantigens that are targeted in SLE patients. Using this approach, we identified Stress Induced Phosphoprotein 1 (STIP1) as an autoantigen in a subset of SLE patients. Those patients with elevated levels of anti-STIP1 IgG autoantibodies in their serum were less likely to have parameters associated with more severe disease, suggesting a protective role for anti-STIP1 IgG. In addition, I defined a genetic interaction between the src tyrosine kinase, Lyn, and the Ets family transcription factor, Ets1, in autoimmunity. Lyn is both a positive and negative regulator of BCR signaling; however its net effect is inhibitory. Lyn deficiency results in hyperactive B cells and Lyn-/- mice serve as a murine model of SLE. Ets1 is a regulator of plasma cell differentiation, and Ets1-/- mice have a similar phenotype to Lyn-/- mice. Lyn and Ets1 are in a shared pathway in which Lyn maintains Ets1 levels, thus limiting plasma cell accumulation. Compound heterozygotes of Lyn and Ets1 were used to determine whether partial loss of Lyn and Ets1 results in accelerated autoimmunity. Lyn and Ets1 were found to synergize in limiting the accumulation of activated and memory T cells, myeloid dendritic cells, age associated B cells, and IgM, but not IgG autoantibodies.Item [Southwestern News](2005-05-15) Heinzl, ToniItem [Southwestern News](2003-08-11) O'Brien, StephenItem [Southwestern News](1997-07-25) Steeves, Susan A.Item Targeting the Neonatal Fc Receptor, FcRn, to Treat Autoimmunity and Elucidation of Sites of FcRn Function(2016-10-13) Challa, Dilip Kumar; Eberhart, Robert C.; Ward, E. Sally; Greenberg, Benjamin M.; Satterthwaite, Anne B.; Stüve, OlafThe neonatal Fc receptor, FcRn, is expressed in many different cell types and serves several functions, some of which are cell type-specific. A function common to most cell types that express this receptor is salvage of IgG from cellular degradation which is responsible for the long in vivo half-life of IgG. This property of IgG is responsible for its indispensable role in humoral immunity and also contributes to the successful use of IgGs as therapeutics. In autoimmunity, however, autoantibodies are generated that can contribute to pathology. FcRn-mediated salvage is also responsible for the long half-life of autoreactive IgGs. Therefore, this study employed an engineered antibody (Abdeg, a novel FcRn inhibitor) that lowers endogenous IgG levels by competing for binding to FcRn, to directly assess the effect of decreased antibody levels in an autoantibody-dependent murine model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Essentially, Abdeg delivery reduced the accumulation of autoantibodies in the target organs and ameliorated the disease. Autoreactive CD4+ T cells are also known to play an important role in the pathogenesis of autoimmune diseases. Thus, with the goal of inducing antigen-specific T cell tolerance, the current study employed immunoglobulin Fc engineering to develop a novel platform for the delivery of peptide epitopes as Fc-peptide fusions with different dynamic properties. Using very low doses of these engineered antigens to avoid anaphylactic shock, the study demonstrates that the longevity of the peptide antigen is the primary determinant of tolerance induction in a murine EAE model that is driven by autoreactive CD4+ T cells. Long-lived Fc-antigen fusions are effective tolerogens in both prophylactic and therapeutic treatments, although distinct mechanisms lead to tolerance in these two settings. Further, to identify the FcRn-expressing cell types that mediate the effects of Abdegs and Fc-antigen fusions on autoantibodies and autoreactive T cells, respectively, cre-loxp technology was used to generate multiple cell type-specific FcRn knockout mice. This study indicates that macrophages are the primary sites among hematopoietic cells where IgG homeostasis occurs. Collectively, these studies have led to an improved understanding of FcRn function at both the level of its sites of functional activity and targeting this receptor for therapy.Item Using Advanced Microscopy Techniques for the Study of Macrophage-Cancer Cell Interactions in the Presence of Therapeutic Antibodies(2017-07-19) Velmurugan, Ramraj; Li, Wen-Hong; Ward, E. Sally; Ober, Raimund J.; Alexandrakis, Georgios; Pasare, ChandrashekharThe use of monoclonal antibodies represents a rapidly expanding area for cancer therapy. One of the main mechanisms of action of these antibodies is Fcγ receptor-mediated engagement of macrophages and other immune cells. When macrophages engage tumor cells opsonized with antibody molecules, they can perform trogocytosis, the process of internalizing fragments of the target cell, or phagocytosis, the internalization of entire cancer cells. This study first establishes whether the process of trogocytosis can lead to cancer cell death. A variety of microscopy and flow cytometric assays were used to quantify the levels of trogocytosis and cell death, in co-cultures of macrophages and cancer cells. Using HER2-overexpressing breast cancer cell lines and anti-HER2 antibodies, we show that persistent trogocytosis can lead to the killing of cancer cells. The mechanism of trogocytosis was also explored using multifocal plane microscopy (MUM). Imaging the process of trogocytosis using MUM revealed that it proceeds through the macrophage-mediated extrusion of tubular structures of the target cell membrane. This membrane-tubulation results in the preferential uptake of the membrane components from the target cell. The study also investigated the maturation pathway followed by phagosomes containing entire cancer cells. A vacuole-like structure associates with these phagosomes, which whilst also lysosomal in nature, displays characteristics distinct from the phagosome itself. The interface between the vacuole and the phagosome is impermeable to certain solutes as observed through microscopy. Further, the size of the phagosome-associated vacuole is affected by inhibition of the mTOR pathway. Use of advanced microscopy techniques such as MUM in these and other biological problems provides mechanistic insight at the spatiotemporal level. To further develop the algorithms involved in MUM data processing, I have therefore also explored various non-parametric methods of estimating the axial location of point sources from MUM data. A new non-parametric method is proposed, which uses multiple intensities calculated from each image of a point source in MUM data. The performance of this approach is compared with other non-parametric methods through simulations and Fisher information calculations. The effectiveness of this method on experimental data is also evaluated.Item Using B Cell Characteristics as Predictors of Multiple Sclerosis in Clinically Isolated Syndrome Patients(2009-06-15) Cameron, Elizabeth; Monson, Nancy L.Clinically isolated syndrome (CIS) is the diagnosis of patients who have experienced a single event due to nerve demyelination of the white matter of the central nervous system. This can be due to numerous causes, both autoimmune and infectious. We hypothesized that CIS patients with B cell characteristics like those seen in multiple sclerosis (MS) patients would develop clinically definite MS. We have determined that, like MS patients, several CIS patients have an increased frequency of VH4-expressing CD19\super +\nosupersub B cells in their cerebrospinal fluid (CSF) compared to peripheral B cells from healthy donors (HCPB) or CSF B cells from patients with neurological diseases not related to MS. However, VH4 bias was a moderate predictor for conversion to MS. Nevertheless, detailed analysis of antibody V-gene repertoires revealed eight codons that are significantly more mutated in the MS CSF than HCPB VH4-expressing B cells. This MS-specific antibody signature includes 25% of all mutations within the repertoires of CSF-derived B cells from MS patients. We then used the prevalence of this signature to predict if CIS patients converted to MS within two years of repertoire sampling. Indeed, we accurately predicted conversion to MS in 10 of 11 CIS patients. The B cell VH4 antibody signature can potentially be used as a diagnostic and prognostic tool for MS.Item [UT Southwestern Medical Center News](2007-09-26) Rian, RussellItem [UT Southwestern Medical Center News](2008-03-01) Piloto, ConnieItem [UT Southwestern Medical Center News](2008-04-04) McKenzie, AlineItem [UT Southwestern Medical Center News](2006-03-29) McKenzie, Aline