Browsing by Subject "antibodies"

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    Investigating the mechanisms underlying antibody-mediated effector functions in malaria and Multisystem Inflammatory Syndrome in Children (MIS-C)
    (2024-06) Dick , Jenna
    Antibodies have multiple functions within the body to provide host protection, including interacting with Fc receptors on innate immune cells to destroy pathogens or cells infected by pathogens. We studied antibody-mediated cellular responses in the context of two infections: malaria, a parasitic infection, and SARS-CoV-2, a viral infection. Plasmodium falciparum (P.f) is the main cause of malaria worldwide and is major cause of morbidity and mortality. Deaths occur because of the blood stage of the P.f. life cycle. To date, there is no effective blood stage malaria vaccine. Natural killer (NK) cells inhibit the growth in the blood through interacting with antibodies via antibody dependent cellular cytotoxicity (ADCC). A subset of NK cells in malaria, known as adaptive NK cells, lack the FcR chain and have enhanced ADCC. However, it is unclear if the lack of FcRchain is the reason for increased functionality or if it only serves as a marker. Using CRIPSR/Cas9, we found that ablating the FcR chain did not enhance ADCC. Using cohort of subjects from a malaria clinical study in Mali, we then searched for other alterations in malaria NK cells that could explain the enhanced functionality. We found that the expression of Sigelc-7, an inhibitory receptor, is decreased on NK cells from individuals with malaria and that decreased expression of Siglec-7 correlates with increased ADCC. These data provide evidence that the host may increase the number of Siglec-7 negative NK cells to aide in the clearance of infected RBCs through ADCC. I then investigated antibody-mediated effector functions in Multisystem Inflammatory Syndrome in Children (MIS-C), a rare and severe complication of SARS-CoV-2 infection that is characterized by multi-organ involvement and substantial inflammation. Little is known about antibody-mediated cellular responses in MIS-C. We show that monocytes in MIS-C were hyperfunctional while NK cells were hypofunctional for antibody-dependent cellular destruction and cytokine production. We also show multiple ways leading to decreased cytotoxicity for NK cells, including phenotypic exhaustion of NK cells and cytokine-induced associations. Together, our results reveal unique dysregulation in antibody-mediated responses in MIS-C that contribute to the immune pathology of this disease and may be amenable to immunomodulation. Overall, this thesis provides insight into the mechanisms underlying antibody-mediated cellular functions in malaria and MIS-C that can be utilized in developing therapeutics and vaccine targets for these diseases .
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    Measuring Binding Kinetics of Therapeutic Antibodies to Membrane Receptors Using Nanohole Array SPR Biosensors
    (2016-04) Jordan, Luke
    In the field of drug discovery, two important metrics of candidate drugs are their binding affinity and kinetics to target receptors. Dr. Moses Rodriguez and his colleagues at the Mayo Clinic have found monoclonal IgM antibodies exhibiting therapeutic effects for multiple sclerosis and amyotrophic lateral sclerosis in animal models, and therefore desired to obtain the kinetic profiles of these antibodies to their targets. Dr. Sang-Hyun Oh’s lab at the University of Minnesota specializes in designing and fabricating plasmonic devices, and have developed a nanohole array sensor coated with silicone dioxide which permits formation of cell mimicking supported lipid bilayers. The focus of this dissertation has been to build these devices and develop assays to measure the binding between these antibodies and receptors in cell extracts and supported lipid bilayers. The first antibody to measure was rHIgM22, which binds to myelin membrane. We did not know the receptor, so we used myelin extracts which would include the unknown receptors, and attached these particles to the sensor surface by passive immobilization. To reduce particle size into the sensor detection window, we extruded the particles through pores of known dimensions. After immobilization we measured binding with antibodies. Unfortunately, binding with rHIgM22 was undetectable, but a similar antibody, mouse IgM O4, which also binds to myelin and has a therapeutic effect, did bind consistently and gave KD, apparent = 2.6 ± 3.6 nM, ka = 2.5 ± 0.0l × 10^4 M^-1^s-1, and kd,slow = 6.6 ± 0.3 × 10^-5 s^-1. The second antibody to measure was rHIgM12, which binds to neuronal membranes. We found rHIgM12 binds to the gangliosides GT1b and GD1a, but not GM1. These gangliosides were incorporated into supported lipid bilayers (5 mol %) and binding to the antibodies was measured. Binding of rHIgM12 to GT1b gave KD, apparent = 24.8 ± 7.9 nM, ka = 2.19 ± 0.196 × 10^4 M^-1s^-1, and kd,slow = 4.72 ± 1.15 × 10^-4 s^-1. Binding of rHIgM12 to GD1a gave KD, apparent = 42.3 ± 20.6 nM, ka = 1.79 ± 0.516 × 10^4 M^-1s^-1, and kd,slow = 4.43 ± 1.38 × 10^-4 s^-1.