116 research outputs found
Hypophysitis Secondary to Cytotoxic T-Lymphocyte-Associated Protein 4 Blockade: Insights into Pathogenesis from an Autopsy Series
No full textHypophysitis that develops in cancer patients treated with monoclonal antibodies blocking cytotoxic T-lymphocyte-associated protein 4 (CTLA-4; an inhibitory molecule classically expressed on T cells) is now reported at an incidence of approximately 10%. Its pathogenesis is unknown, in part because no pathological examination of the pituitary gland has been reported to date. We analyzed at autopsy the pituitary glands of six cancer patients treated with CTLA-4 blockade, one with clinical and pathological evidence of hypophysitis, one with mild lymphocytic infiltration in the pituitary gland but no clinical signs of hypophysitis, and four with normal pituitary structure and function. CTLA-4 antigen was expressed by pituitary endocrine cells in all patients but at different levels. The highest levels were found in the patient who had clinical and pathological evidence of severe hypophysitis. This high pituitary CTLA-4 expression was associated with T-cell infiltration and IgG-dependent complement fixation and phagocytosis, immune reactions that induced an extensive destruction of the adenohypophyseal architecture. Pituitary CTLA-4 expression was confirmed in a validation group of 37 surgical pituitary adenomas and 11 normal pituitary glands. The study suggests that administration of CTLA-4 blocking antibodies to patients who express high levels of CTLA-4 antigen in the pituitary can cause an aggressive (necrotizing) form of hypophysitis through type IV (T-cell dependent) and type II (IgG dependent) immune mechanisms
DEVELOPING AND APPLYING SEQUENCING-BASED TOOLS TO UNDERSTAND ADAPTIVE IMMUNE RESPONSES IN MYOSITIS
No full textIdiopathic inflammatory myopathies (IIMs) are a group of heterogeneous, systemic diseases characterized by skeletal muscle pathologies, which most commonly result in chronic, progressive muscle weakness. Immune infiltrates, which includes B and T cells, are often found in the muscle of IIM patients. Currently, autoantibodies help stratify IIMs into clinical subtypes, however disease heterogeneity persists within subtypes of IIMs and the etiologies of IIMs remain unknown. Through the application of adaptive immune profiling methods: 1) Phage Immunoprecipitation Sequencing (PhIP-Seq) to understand antibody reactivities at cohort-scale and 2) B cell receptor sequencing, I have further characterized the antibody repertoires in IIMs. Using PhIP-Seq, I identified novel environmental and autoantibody reactivities in subtypes of IIMs. These findings may generate hypotheses about IIM etiologies, and improve IIM disease classifications. Using both PhIP-Seq and BCR sequencing, I characterized the diversity of the local antibody repertoire in muscle tissue of IIM patients, improving our understanding of the adaptive immune response within muscle. To improve our ability to perform immune receptor repertoire sequencing in a low-cost, high-throughput manner, I developed a method, INtraCEllular Reverse Transcription with Sorting and sequencing, or INCERTS, to efficiently link T cell phenotypes with T cell receptor sequencies. INCERTS is complementary to single-cell sequencing. Through the application of these sequencing- based methods, I have increased our understanding of the B cell immune response in IIMs. Through the development of INCERTS, I have improved our ability to perform immune receptor repertoire sequencing, which can be applied to further increase our understanding of the adaptive immune response in IIMs
DEVELOPING AND APPLYING SEQUENCING-BASED TOOLS TO UNDERSTAND ADAPTIVE IMMUNE RESPONSES IN MYOSITIS
No full textIdiopathic inflammatory myopathies (IIMs) are a group of heterogeneous, systemic diseases characterized by skeletal muscle pathologies, which most commonly result in chronic, progressive muscle weakness. Immune infiltrates, which includes B and T cells, are often found in the muscle of IIM patients. Currently, autoantibodies help stratify IIMs into clinical subtypes, however disease heterogeneity persists within subtypes of IIMs and the etiologies of IIMs remain unknown. Through the application of adaptive immune profiling methods: 1) Phage Immunoprecipitation Sequencing (PhIP-Seq) to understand antibody reactivities at cohort-scale and 2) B cell receptor sequencing, I have further characterized the antibody repertoires in IIMs. Using PhIP-Seq, I identified novel environmental and autoantibody reactivities in subtypes of IIMs. These findings may generate hypotheses about IIM etiologies, and improve IIM disease classifications. Using both PhIP-Seq and BCR sequencing, I characterized the diversity of the local antibody repertoire in muscle tissue of IIM patients, improving our understanding of the adaptive immune response within muscle. To improve our ability to perform immune receptor repertoire sequencing in a low-cost, high-throughput manner, I developed a method, INtraCEllular Reverse Transcription with Sorting and sequencing, or INCERTS, to efficiently link T cell phenotypes with T cell receptor sequencies. INCERTS is complementary to single-cell sequencing. Through the application of these sequencing- based methods, I have increased our understanding of the B cell immune response in IIMs. Through the development of INCERTS, I have improved our ability to perform immune receptor repertoire sequencing, which can be applied to further increase our understanding of the adaptive immune response in IIMs
Visualizing MIPSA: Exploring Web-based User Experience Design in Teaching and Promoting Molecular Technology
No full textThis thesis explores the potential of web-based user experience in teaching and promoting a novel molecular technology, Molecular Indexing of Proteins by Self Assembly (MIPSA), which is a scalable and low-cost solution for comprehensive antibody profiling. Despite its potential applications, there are no existing visuals that explain the complex science of MIPSA. To address this gap, a website was developed with animations, graphics, and concise content to educate and engage the target audience of researchers and investors in biotechnology.
The website is the first of its kind to explain and visualize the concept of MIPSA using multimedia and the multimedia learning theory. The website aims to deliver the didactic content in an engaging and comprehensible way by employing website design principles, multimedia and the multimedia learning theory. A focus group was held with researchers and students at Johns Hopkins to assess the effectiveness of the website, and results showed high ratings for overall effectiveness, navigation, content organization, and visual appeal. The project's implications for biocommunication and the promotion of a new molecular technology are discussed, along with suggestions for future studies
Visualizing MIPSA: Exploring Web-based User Experience Design in Teaching and Promoting Molecular Technology
No full textThis thesis explores the potential of web-based user experience in teaching and promoting a novel molecular technology, Molecular Indexing of Proteins by Self Assembly (MIPSA), which is a scalable and low-cost solution for comprehensive antibody profiling. Despite its potential applications, there are no existing visuals that explain the complex science of MIPSA. To address this gap, a website was developed with animations, graphics, and concise content to educate and engage the target audience of researchers and investors in biotechnology.
The website is the first of its kind to explain and visualize the concept of MIPSA using multimedia and the multimedia learning theory. The website aims to deliver the didactic content in an engaging and comprehensible way by employing website design principles, multimedia and the multimedia learning theory. A focus group was held with researchers and students at Johns Hopkins to assess the effectiveness of the website, and results showed high ratings for overall effectiveness, navigation, content organization, and visual appeal. The project's implications for biocommunication and the promotion of a new molecular technology are discussed, along with suggestions for future studies
Early Viral Suppression and the Antibody Response to HIV Infection in HPTN 071 (PopART)
No full textHigh HIV viral loads are associated with an increased risk of transmission and faster disease progression. Effective antiretroviral therapy (ART) reduces HIV viral load to low levels. Early HIV diagnosis and ART initiation are now recommended for all persons living with HIV, regardless of infection duration, CD4 cell count, or clinical status. HIV controllers naturally achieve low viral loads without ART. The antibody response to HIV infection evolves rapidly during the first year of infection, with dynamic changes in titer, avidity, and specificity. The HPTN 071 (PopART) trial provided a unique cohort to assess early HIV antibody responses in persons with natural and ART-induced viral suppression. This community-randomized trial, conducted in Zambia and South Africa, demonstrated that universal delivery of HIV counseling and testing decreased HIV incidence; HIV incidence was also reduced with provision of universal ART. In this dissertation research, we examined the impact of viral suppression on the HIV antibody response by evaluating the performance of serologic assays and algorithms designed for HIV cross-sectional incidence estimation. We also used a massively multiplexed antibody profiling system (VirScan) to characterize antibody reactivities in participants enrolled in HPTN 071. We identified HIV antibody specificities present prior to infection that were associated with HIV control and modulation of HIV viral load after infection. Our findings may inform future methods for identifying individuals with recent infection and estimating HIV incidence at the population level. They also provide new insights into factors associated with HIV control, which could inform future research on antibody-based interventions used for HIV treatment and prevention
The Development and Application of Self-Assembled DNA-barcoded ORFeome-scale Human Protein Library
No full textAntibodies play crucial roles in host defense by neutralizing viruses, bacteria, and foreign substances. However, a breakdown in immune tolerance can produce molecules known as “autoantibodies” that mistakenly attack our own tissues, organs, or cells and drive autoimmune pathology. Immunologists have studied numerous autoimmune diseases and identified autoantibodies as biomarkers and pathogenic molecules using traditional biochemical and immunologic techniques, such as western blotting, but these methods are not scalable. Given that the human genome contains approximately 20,000 protein-coding genes, profiling autoantibodies across large patient cohorts remains a substantial challenge.
The advent of nanotechnology, high-throughput biology and DNA sequencing has provided new tools to address this problem. Unbiased screening approaches, such as protein microarrays and phage display, have enabled the identification of novel autoantibody targets. More recently, molecular display techniques such as cDNA displayhave further advanced this effort by enabling the construction of barcoded protein libraries with very high complexity, which can be used to comprehensively profile autoantibody repertoires in patient samples..
This thesis presents the development of Molecular Indexing of Proteins by Self-Assembly (MIPSA) for high-throughput autoantibody profiling. The first part of the thesis describes proof-of-concept studies that characterize DNA–protein conjugates and a barcoded human ORFeome library, and it shows that MIPSA detects autoantibodies against TRIM21 in Sjögren’s syndrome, NT5C1A in inclusion-body myositis, and type I/III interferons in severe COVID-19. Concordance with data from phage immunoprecipitation sequencing (PhIP-Seq) and in-vitro confirmation of the interferon antibodies’ neutralizing activity further validate MIPSA platform.
The second part of this thesis focuses on refining the barcode-human ORF (BC-ORF) pair matching using Oxford Nanopore Technology (ONT) sequencing. We evaluated a set of read aligners including Minimap2, Winnowmap2, NGMLR, and Bowtie2 to assess their ability for ORF recovery from long reads and compared the constructed BC-ORF “dictionaries” to analyze their impact on enrichment analysis from a MIPSA screening data.
Lastly, the third part of this thesis showcases MIPSA’s potential future utility for ligand–receptor studies by examining the binding of epidermal growth factor (EGF) to its receptor, using both EGFR-overexpressing HEK cells and an EGFR-Fc recombinant protein
Nanopore DNA Sequencing to Expand Genetic Context in Non-Model Species Research
No full textAdvances in DNA sequencing technologies have dramatically transformed the scale and speed at which molecular evolution can be observed. As genome sequencing costs decrease and capacity becomes increasingly ubiquitous, new analytical capabilities are rapidly solving previously intractable biological problems. This is apparent with the rapid adoption of genomic surveillance during the COVID-19 pandemic, with more than one million SARS-CoV-2 genomes sequenced worldwide in just over one year from the initial declaration.
In this portfolio, I focus on genetic characterization of two non-model organisms through an initial deep characterization, followed by transition of an optimized application to standard practice that requires minimal user interaction. First, the genome of the grass Setaria viridis is assembled into an ultra-contiguous, chromosome scale reference genome. That genome is then used as the basis for rapid and reproducible identification of genetic modification events in newly generated transgenic plants. Second, as COVID-19 entered into the Baltimore-Washington DC region, genetic characterization of the SARS-CoV-2 virus was performed to characterize the early introduction. Methods developed on the primary effort are then used to implement genomic surveillance in low- and middle-income countries through a series of workshops and extended collaborations that enable widespread use in non-traditional genomics laboratories. All applications use a variety of genetic characterization technologies, with a primary focus on Oxford Nanopore sequencing for rapid and comprehensive analysis
Early Viral Suppression and the Antibody Response to HIV Infection in HPTN 071 (PopART)
No full textHigh HIV viral loads are associated with an increased risk of transmission and faster disease progression. Effective antiretroviral therapy (ART) reduces HIV viral load to low levels. Early HIV diagnosis and ART initiation are now recommended for all persons living with HIV, regardless of infection duration, CD4 cell count, or clinical status. HIV controllers naturally achieve low viral loads without ART. The antibody response to HIV infection evolves rapidly during the first year of infection, with dynamic changes in titer, avidity, and specificity. The HPTN 071 (PopART) trial provided a unique cohort to assess early HIV antibody responses in persons with natural and ART-induced viral suppression. This community-randomized trial, conducted in Zambia and South Africa, demonstrated that universal delivery of HIV counseling and testing decreased HIV incidence; HIV incidence was also reduced with provision of universal ART. In this dissertation research, we examined the impact of viral suppression on the HIV antibody response by evaluating the performance of serologic assays and algorithms designed for HIV cross-sectional incidence estimation. We also used a massively multiplexed antibody profiling system (VirScan) to characterize antibody reactivities in participants enrolled in HPTN 071. We identified HIV antibody specificities present prior to infection that were associated with HIV control and modulation of HIV viral load after infection. Our findings may inform future methods for identifying individuals with recent infection and estimating HIV incidence at the population level. They also provide new insights into factors associated with HIV control, which could inform future research on antibody-based interventions used for HIV treatment and prevention
The Development and Application of Self-Assembled DNA-barcoded ORFeome-scale Human Protein Library
No full textAntibodies play crucial roles in host defense by neutralizing viruses, bacteria, and foreign substances. However, a breakdown in immune tolerance can produce molecules known as “autoantibodies” that mistakenly attack our own tissues, organs, or cells and drive autoimmune pathology. Immunologists have studied numerous autoimmune diseases and identified autoantibodies as biomarkers and pathogenic molecules using traditional biochemical and immunologic techniques, such as western blotting, but these methods are not scalable. Given that the human genome contains approximately 20,000 protein-coding genes, profiling autoantibodies across large patient cohorts remains a substantial challenge.
The advent of nanotechnology, high-throughput biology and DNA sequencing has provided new tools to address this problem. Unbiased screening approaches, such as protein microarrays and phage display, have enabled the identification of novel autoantibody targets. More recently, molecular display techniques such as cDNA displayhave further advanced this effort by enabling the construction of barcoded protein libraries with very high complexity, which can be used to comprehensively profile autoantibody repertoires in patient samples..
This thesis presents the development of Molecular Indexing of Proteins by Self-Assembly (MIPSA) for high-throughput autoantibody profiling. The first part of the thesis describes proof-of-concept studies that characterize DNA–protein conjugates and a barcoded human ORFeome library, and it shows that MIPSA detects autoantibodies against TRIM21 in Sjögren’s syndrome, NT5C1A in inclusion-body myositis, and type I/III interferons in severe COVID-19. Concordance with data from phage immunoprecipitation sequencing (PhIP-Seq) and in-vitro confirmation of the interferon antibodies’ neutralizing activity further validate MIPSA platform.
The second part of this thesis focuses on refining the barcode-human ORF (BC-ORF) pair matching using Oxford Nanopore Technology (ONT) sequencing. We evaluated a set of read aligners including Minimap2, Winnowmap2, NGMLR, and Bowtie2 to assess their ability for ORF recovery from long reads and compared the constructed BC-ORF “dictionaries” to analyze their impact on enrichment analysis from a MIPSA screening data.
Lastly, the third part of this thesis showcases MIPSA’s potential future utility for ligand–receptor studies by examining the binding of epidermal growth factor (EGF) to its receptor, using both EGFR-overexpressing HEK cells and an EGFR-Fc recombinant protein
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