• In vitro Methods to Better Evaluate Drug Responses in Cancer

      Michael J. Lee; Schwartz, Hannah (UMass Chan Medical School, 2022-09-08)
      Evaluating anti-cancer drugs in vitro is an important aspect of the drug development pipeline. When evaluating anti-cancer drugs, two different measurements are used: relative viability, which scores an amalgam of proliferative arrest and cell death, and fractional viability, which specifically scores the degree of cell killing. These two metrics are often used interchangeably despite measuring different aspects of a drug response. This study explored the relationship between drug-induced growth inhibition and cell death, and found that most drugs affect both proliferation and death, but in different proportions, and with different relative timing. This causes a non-uniform relationship between relative and fractional response measurements. To unify these measurements, I created a data visualization and analysis platform, called drug GRADE, which characterizes the degree to which death contributes to an observed drug response. GRADE captures drug- and genotype-specific responses, which are not captured using traditional pharmaco-metrics. Current in vitro anti-cancer drug evaluation practices measure drug responses with cancer cell lines in mono-culture. However, many cell types in the tumor microenvironment influence cancer’s drug response and disease progression. Therefore, current drug evaluation practices overlook complex cell-cell interactions that influence cancer’s drug response. In this study, I developed a high-throughput assay to study the effect of another cell type (cytotoxic T cells) on cancer viability in co-culture, in vitro. Further, I developed a reference framework to model the complex interaction between cancer cells and cytotoxic T cells, and to model how T cell-mediated cell death is modulated by anti-cancer drug treatment. Taken together, this study highlights two new methods which enable better in vitro evaluation of drug responses in cancer.
    • Cellular heterogeneity and gene regulatory network coordination during thymic epithelial cell development

      René Maehr; Magaletta, Margaret (UMass Chan Medical School, 2022-08-23)
      Thymic epithelial cells, derived from the pharyngeal endoderm, perform essential functions for establishing a self-tolerant immune system. Unsurprisingly, dysfunction of thymic epithelial cells resulting from maldevelopment of the pharyngeal endoderm causes immunodeficiency or autoimmunity syndromes, some of which cannot be fully explained according to known genetic errors. Despite the functional significance and disease-relevance of pharyngeal endoderm with respect to thymic epithelial cells, we lack a comprehensive understanding of the gene regulatory networks driving pharyngeal endoderm differentiation. To close this gap, we applied transcriptome and chromatin accessibility single cell profiling to generate a multi-omic developmental resource covering pharyngeal differentiation toward organ-specific epithelia in the mouse embryo. We identified cell-type specific gene regulation of developing organ domains and characterized the role of an immunodeficiency-associated forkhead box transcription factor, Foxn1, during early thymus development. Furthermore, analyses of the pharyngeal endoderm multi-omics atlas led us to discover a novel gene associated with thymus development, namely Grainyhead-like3 (Grhl3). We assessed the expression pattern and the functional importance of Grhl3 in the prenatal and postnatal thymus, uncovering a putative role in a specialized medullary thymic subtype. In conclusion, this dissertation provides insight on the molecular basis of pharyngeal endoderm differentiation and subsequent development of the thymus.
    • Regulated Gene Therapy Towards Glycosphingolipid Biosynthesis Deficiencies

      Guangping Gao; Robert H. Brown Jr.; Yang, Huiya (UMass Chan Medical School, 2022-08-22)
      Glycosphingolipids (GSLs) are a group of amphipathic glycolipids essential for maintaining the normal ultrastructure and function of neural and oligodendrocyte cell membranes throughout the mammalian central nervous system (CNS). De novo GSL biosynthesis defects cause severe neurological diseases such as GM3 synthase deficiency (GM3SD) and hereditary sensory and autonomic neuropathy type 1A (HSAN1A), each lacking effective treatment. Here, we developed two distinct potential therapeutic approaches for these neurological diseases. For GM3SD that is caused by loss-of-function mutations in ST3GAL5, we employed a recombinant adeno-associated virus (rAAV)-mediated human ST3GAL5 gene replacement therapy. First, using ST3GAL5 mutant patient iPSC-derived neurons and St3gal5 knock-out mouse models, we have achieved ST3GAL5 gene normalization and restoration of the functional products, cerebral gangliosides. Importantly, we revealed the hepatic toxicity caused by ubiquitous expression of ST3GAL5 and optimized a CNS-restricted rAAV gene replacement therapy for the safe and efficacious rescue of the severe neurodevelopmental phenotypes and early lethality in disease mouse models, given by both intracerebroventricular and intravenous routes of administration. These results support for further clinical development of ST3GAL5 gene therapy. On the other hand, to target gain-of-function SPTLC1 mutation caused HSAN1A, we screened antisense oligonucleotides (ASOs) and achieved efficient reduction of mutant SPTLC1 transcripts and its toxic products in patient-fibroblasts. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in GM3SD and allele-specific ASO silencing in HSAN1A, highlighting the significance of personalized gene therapy for monogenic neurological disorders.
    • Investigating Proteolytic Processing of Ataxin 2, a Neurodegenerative Disease Associated Protein

      Patrick Emery; Chitre, Monika (UMass Chan Medical School, 2022-08-08)
      Ataxin 2 (ATXN2) is a ubiquitously expressed mRNA binding protein involved in the development and progression of spinocerebellar ataxia 2 (SCA2) and amyotrophic lateral sclerosis (ALS). In the context of both neurodegenerative diseases, its N-terminal polyglutamine (polyQ) domain is mutated and expanded in length. Several other polyQ proteins, such as huntingtin (Htt), ataxin 3 (ATXN3), and ataxin 7 (ATXN7), undergo proteolytic processing that produces toxic fragments containing their polyQ domains. Investigating how ATXN2 is regulated by proteolysis is hindered by the lack of available molecular biological tools such as N-terminal ATXN2 antibodies to target and analyze the endogenous N-terminus of ATXN2. To circumvent this challenge, I developed a transient overexpression model of N-terminally tagged ATXN2 in HEK293E cells. Here, I demonstrate that both wild-type and mutant ATXN2 are targets of N-terminal proteolysis. I confirmed that ATXN2 produces an independent polyQ cleavage fragment like other polyQ proteins through basic molecular biology approaches such as Western blotting and immunoprecipitation. Additionally, I identified the specific region that is both necessary and sufficient for cleavage to occur via deletion mapping with multiple truncated ATXN2 mutants and reporter constructs. Further definition of ATXN2 as a target of proteolytic cleavage aligns it with other neurodegenerative polyQ proteins, and proteolysis is currently a less explored avenue of research for ATXN2-related disease development, progression, and therapeutic modalities. This work reveals a novel site that directs cleavage of ATXN2 and provides a potential avenue of investigation for how ATXN2 posttranslational modifications contribute to the progression of SCA2 and ALS.
    • High bacillary burden and the ESX-1 type VII secretion system promote MHC class I presentation to CD8 T-cells during Mycobacterium tuberculosis infection

      Samuel M. Behar; Mott, Daniel (UMass Chan Medical School, 2022-07-26)
      T-cell mediated immunity is required for optimal protection against Mycobacterium tuberculosis (Mtb) infection, but often fails to completely clear the pathogen. Mtb has evolved strategies to subvert host immunity so it can persist in host cells despite pressure from innate and adaptive immunity. While cytotoxic CD8 T-cells should recognize and clear infected host cells, eliminating Mtb’s intracellular niche, previous findings have demonstrated otherwise [1]. In fact, we have shown that CD8 T-cells specific to the immunodominant antigen TB10.4 poorly recognize Mtb infected macrophages in vitro. Here we extend our initial findings to show that class I MHC-restricted epitopes other than TB10.44-11 are inefficiently presented by Mtb-infected macrophages to CD8 T cells. The only exception we find is for heavily infected macrophages. During high burden infections, macrophages cross-present TB10.4 antigen to CD8 T-cells. These high burden infections result in considerable cell death, and we find that uninfected macrophages effectively scavenge dead cellular debris and cross-present this antigen to CD8 T cells. Furthermore, we find that cross-presentation by heavily infected cells is dependent on the ESX-1 type VII secretion system, suggesting that phagosomal membrane damage and host cell death are crucial for effective class I MHC cross-presentation during Mtb infection.
    • The Role of Age-Associated B Cells (ABC) in Combating Influenza Infection

      Susan L. Swain; Kugler-Umana, Olivia (UMass Chan Medical School, 2022-07-12)
      With age, follicular helper T cell (TFH) dependent B cell responses erode, reducing B cell memory and long-term antibody responses. However, aged mice and humans develop an alternative B cell population, termed age-associated B cells (ABC), that may produce TFH independent antibodies. ABC lack CD21 and CD23 expression, and some express transcription factors and adhesion molecules indicative of antigen exposure. Some of these have been implicated in autoimmunity. We found a unique population of responding B cells following influenza A virus (IAV) infection, which was Fashi/GL7neg. We postulated that the CD21-CD23-ABC might be progenitors of these non-follicular B cells, that we called induced ABC (iABC). Using T-deficient RAG KO and TFH deficient SAP KO hosts, we found that the CD21-CD23-ABC can become iABC (FasHiGL7-) upon IAV infection. These iABC share the same phenotype of iABC found in infected aged mice and can become Ab-producing cells without T cell help. We showed that CD21-CD23-ABC can be separated into IgD+ (putative naïve B cells) vs. IgD- (memory-like B cells). We followed their ability to become iABC in SAP KO hosts. The IgD+ABC were most efficient at giving rise to iABC. Further transfer studies revealed that iABC generation from donor IgD+ABC requires extrinsic TLR signaling, and IgD+ABC can provide Ab-medicated protection. We concluded that upon T-independent stimulation, IgD+ABC (CD21-CD23-) become iABC (Fashi/GL7neg), some of which can secrete IAV-specific Ab and may provide protection against IAV.
    • Novel Genetic Pathways in Functional Regulation of Hematopoietic Stem Cells

      Shaoguang Li; Desouza, Ngoc (UMass Chan Medical School, 2022-07-08)
      Hematopoietic stem cells (HSCs) are a rare population of bone marrow cells that have self-renewal and differentiating capabilities enabling them to produce all blood lineages during normal hematopoiesis. Many molecular pathways are involved in the regulation of HSCs, and the survival, maintenance and proliferation of these cells must be tightly controlled to avoid aberrant activities that can cause blood diseases, such as hematopoietic malignancies. Therefore, additional factors involved in the functional regulation of HSCs must be discovered to provide new therapeutic treatments for hematopoietic diseases. In chapter I, I briefly introduce the hematopoietic system and hierarchy through which HSCs can produce all mature blood cells in the lifespan. I also describe various methods to identify different hematopoietic cells, with a focus on using cell surface antigen markers. I additionally discuss an important method to study HSCs in mice by using bone marrow transplantation in which the donor cells are manipulated to examine the role of a gene of interest. I also briefly describe several signaling pathways important in HSCs, such as the Bmp, Wnt, Hedgehog and Notch signaling pathways. I provide known relevant information to my thesis work on c-Kit and Sca-1 receptors, as well as on LSK and LSK- cells. In chapter II, I describe my findings regarding a novel mechanism in which Ikzf3 plays a suppressive role in regulating HSC survival and maintenance. Ikzf3 suppresses the population of LSK (lineage-Sca-1+c-Kit+) cells that contains HSCs, and increases the LSK- (lineage-Sca-1+c-Kit-) population, which is highly apoptotic and derived from the LSK population. The DNA binding domain of Ikzf3 is required for its inhibition of HSCs, and Ikzf3 downregulates the expression of Bcl-2, Bcl-xL and c-Myc. Ikzf3 expression in HSCs is maintained at low levels by the c-Kit pathway. In chapter III, on the basis of data from microarray analysis previously performed to compare gene expression profiles between Hif1a knockout HSCs and wild type HSCs, the effects of both Hif1a and Notch1 deletion on HSC regulation are examined. Loss of both Hif1a and Notch1 induces the development of myelodysplastic/myeloproliferative diseases, which are clonal hematopoietic stem cell neoplasms characterized by abnormal regulation of the myeloid pathways for cellular proliferation, maturation and survival. Loss of both Hif1a and Notch1 also leads to a loss of HSC function. These findings indicate a mechanism through which the hypoxia pathway acts in coordination with the Notch pathway in HSCs. In chapter IV, I summarize the findings from chapter II and III and discuss the importance of these results in the field. I also provide the future directions that can answer more questions to expand our knowledge on these pathways. Together, the findings reveal two novel pathways involved in functional regulation of HSCs: (i) the Ikzf3 pathway, which involves c-Kit, Icsbp, Bcl-2, Bcl-xL and c-Myc, and suppresses normal HSCs to maintain homeostasis and (ii) the synergy of Hif1a and Notch1 in regulating HSCs. The loss of both genes can cause myelodysplastic/myeloproliferative-like diseases.
    • COVID-19: a gray swan's impact on the adoption of novel medical technologies

      Dunlap, Denise; Santos, Roberto S.; Lilly, Craig M.; Teebagy, Sean; Hafer, Nathaniel S.; Buchholz, Bryan O.; McManus, David D. (2022-07-08)
      The COVID-19 pandemic offers a unique context and opportunity to investigate changes in healthcare professional perceptions towards the adoption of novel medical technologies, such as point-of-care technologies (POCTs). POCTs are a nascent technology that has experienced rapid growth as a result of COVID-19 due to their ability to increase healthcare accessibility via near-patient delivery, including at-home. We surveyed healthcare professionals before and during COVID-19 to explore whether the pandemic altered their perceptions about the usefulness of POCTs. Our network analysis method provided a structure for understanding this changing phenomenon. We uncovered that POCTs are not only useful for diagnosing COVID-19, but healthcare professionals also perceive them as increasingly important for diagnosing other diseases, such as cardiovascular, endocrine, respiratory, and metabolic diseases. Healthcare professionals also viewed POCTs as facilitating the humanization of epidemiology by improving disease management/monitoring and strengthening the clinician-patient relationship. As the accuracy and integration of these technologies into mainstream healthcare delivery improves, hurdles to their adoption dissipate, thereby encouraging healthcare professionals to rely upon them more frequently to diagnose, manage, and monitor diseases. The technological advances made in POCTs during COVID-19, combined with shifting positive perceptions of their utility by healthcare professionals, may better prepare us for the next pandemic.
    • Gliotransmission Orchestrates Neuronal Type-specific Axon Regeneration

      Yang Xiang; Wang, Fei (UMass Chan Medical School, 2022-06-30)
      Why closely related neuronal types differ in their axon regenerative abilities remains elusive. Here, I demonstrate gliotransmission determines such a difference in Drosophila larval sensory neurons. Axotomy activates ensheathing glia, which signal to regenerative neurons through the gliotransmitter adenosine, to mount regenerative programs including neuronal activity and Ras. Surprisingly, ensheathing glia do not signal to non-regenerative neurons. Such neuronal type-specific responses to gliotransmission result from specific expression of adenosine receptors in regenerative neurons. Disrupting gliotransmission impedes regeneration of regenerative neurons. Strikingly, reconstitution of gliotransmission in non-regenerative neurons enables them to regenerate. Furthermore, activation of an adenosine receptor in adult mice promotes both regeneration and survival of retinal ganglion cells, uncovering a conserved pro-regenerative role of adenosine receptors. My studies demonstrate gliotransmission as a novel mechanism by which glia instruct axon regeneration, with neuronal type-specificity, and suggest targeting purinergic signaling as a new strategy for mammalian central nervous system repair.
    • Updating and Improving the Capstone Course Experience for Learners and Teachers

      Bronwyn Cooper; Lin, Ashley; Cooper, Bronwyn (2022-06-30)
      Background:The Capstone Scholarship and Discovery Course (“Capstone course”) is a required four-year course which aims to support students in the design, execution, writing, and presentation of a longitudinal scholarly project. Currently, the Capstone course meets criteria for “underperforming” designation (<75% approval) designationin two areas: Overall, how would you rate this course? | 2.52/4 | 52.77% Feedback on reports supported progress/learning | 2.95/4 | 74.07% The overall medical school curriculum is transitioning to a new curriculum (“Vista”) in which Capstone will fit into the context of student-selected Pathways. As the Capstone student representative responsible for communicating student feedback to Capstone leadership, I became interested in taking advantage of this opportunity to improve the course. I believe that every medical student should feel supported in their Capstone research and finish medical school with a foundational understanding of how to conduct longitudinal scholarly work. Objectives: 1) Identify student and faculty concerns about the Capstone course 2) Propose potential changes to the Capstone course to address the most significant concerns 3) Measure student approval towards these proposed changes 4) Summarize and present the most highly student-approved proposed changes to Capstone Course Leadership Team. Methods: I reviewed all Capstone oasis evaluations from the 2020 – 2021 Academic Year, which included 109 faculty evaluations of students, 133 student evaluations of Capstone faculty (~80% response rate), and 136 student evaluations of the Capstone course (~81% response rate). I also created a pre-survey to assess the most unclear aspects of the Capstone course as well as top barriers to meeting deadlines. I received 44 survey responses from third-year students (~22% response rate) and 47 responses from fourth-year students (~30% response rate). From the oasis evaluations and survey responses, I identified the most significant concerns. I generated twenty-two proposed changes to address these concerns, and created a slideshow that summarizes these proposed changes to the Capstone course. This was distributed to current third- and fourth-year medical students, along with a post-survey where students could vote on the proposed changes and indicate whether these changes addressed the unclear aspects of the Capstone course and top barriers to meeting deadlines. (Terminology note: learning community assigned affiliate = “affiliate”, faculty advisor specific to project = “advisor”). Results: I received 29 post-survey responses from third-year students (~15% response rate) and 37 responses from fourth-year students (~23% response rate). Between the pre- and post-surveys, the average % of students finding certain aspects of Capstone “unclear” decreased by 26.1%, and the average % of students rating certain barriers as significant for meeting Capstone deadlines decreased by 15.5%. Notably, the post-survey found that some students believe the proposed changes would not address their lack of interest in doing a longitudinal project (53.1%), and that certain projects students are interested in would still not meet Capstone requirements (31.3%). An abbreviated summary of the most popular proposed changes (>50% of students agree, and/or >10% of students indicated it as one of their top three changes, highlighted in bold) is below. Proposed Solution | % top 3 | % disagree | % neutral | % agree 1a) Create research project database | 30.4 | 3.1 | 12.3 | 84.6 1b) Create roster of past highly-rated advisors (based on oasis evals) | 7.1 | 4.6 | 24.6 | 70.8 1d) MS1 Capstone curriculum focused on developing research skills | 23.2 | 9.2 | 18.5 | 72.3 1e) Affiliate must give feedback on project feasibility as a Capstone project within a month of project proposal. | 5.4 | 6.2 | 32.3 | 61.5 2a) Add additional, optional Capstone poster presentation session before ERAS deadline so presentation can be on ERAS app | 10.7 | 6.2 | 24.6 | 69.2 2b) Optional workshops (or asynchronous videos) on common issues | 5.4 | 15.4 | 32.3 | 52.3 3a) In place of report submissions, create a live document that can be accessed by affiliate and mentor | 16.1 | 10.8 | 26.2 | 63.1 3b) Flexible format (such as Senior Scholars format: background, objectives, methods, results, conclusion) with no length requirement | 21.4 | 1.5 | 12.3 | 86.2 3c) Rework deadlines to avoid exam dates, Step 1 period, holidays, and flexible range of deadlines provided during clinical rotations | 33.9 | 0 | 3.1 | 96.9 3d) Timeline of deadlines given to affiliates and mentors, with exam dates and Step 1 period marked on timeline | 5.4 | 1.6 | 15.6 | 82.8 3e) No punishment for those who get their work done early | 23.2 | 0 | 4.7 | 95.3 3f) Meeting with affiliate can substitute for an update with affiliate approval | 1.8 | 4.7 | 25 | 70.3 4a) Projects that take place over >1 month should be accepted as “longitudinal” and all affiliates should be on the same page. | 42.9 | 1.5 | 16.9 | 81.5 5a) Students may submit outside project to their affiliate to be approved for Capstone credit as long as they worked on this project during med school | 44.6 | 1.5 | 10.8 | 87.7 5c) Website should be simplified. | 7.1 | 0 | 21.9 | 78.1 Conclusion: Students enter medical school with widely differing levels of experience in conducting scholarly research, and the current Capstone course does not have flexibility for different types of projects, or the resources needed to accommodate student needs. Capstone affiliates and advisors also face difficulties providing optimal assistance to students. Through this work, I have identified solutions that are popular amongst students, which address unclear aspects of the Capstone course and top barriers to meeting deadlines. Bringing these solutions to the Capstone Course Leadership Team is the next step to addressing these concerns. Future goals include asking Capstone affiliates for their input, as this group was not included either in the oasis evaluations or the surveys. Another limitation of this study was the low survey response rates, ranging from 15-30%. I hope that ultimately some of these proposed changes to the Capstone course will be adopted and integrated into the new Vista curriculum.
    • Surveillance of Host and Pathogen Derived Metabolites Activates Intestinal Immunity

      Read Pukkila-Worley; Peterson, Nicholas D. (2022-06-30)
      Intestinal epithelial cells function, in part, to detect infection with pathogenic organisms and are key regulators of intestinal immune homeostasis. However, it is not fully understood how intestinal epithelial cells sense pathogen infection and coordinate the induction of protective immune defenses. Here, we define two new mechanisms of innate immune regulation in a metazoan host. First, we characterize the first bacterial pattern recognition receptor and its natural ligand in Caenorhabditis elegans. We show that the C. elegans nuclear hormone receptor NHR-86/HNF4 directly senses phenazine-1-carboxamide (PCN), a metabolite produced by pathogenic strains of Pseudomonas aeruginosa. PCN binds to the ligand-binding domain of NHR-86/HNF4, a ligand-gated transcription factor, and activates innate immunity in intestinal epithelial cells. In addition, we show that C. elegans NHR-86 senses PCN, and not other phenazine metabolites, as a marker of pathogen virulence to engage protective anti-pathogen defenses. Second, we show that a phase transition of the C. elegans Toll/interleukin-1 receptor domain protein (TIR-1) controls signaling by the C. elegans p38 PMK-1 MAPK pathway. Physiologic stress, both P. aeruginosa infection and sterol scarcity, induce multimerization of TIR-1 within intestinal epithelial cells. Like the mammalian homolog of TIR-1, SARM1, oligomerization and phase transition of C. elegans TIR-1 dramatically potentiate its NAD+ glycohydrolase activity. TIR-1/SARM1 multimerization and NAD+ glycohydrolase activity are required for activation of C. elegans p38 PMK-1 pathway signaling and pathogen resistance. These data uncover a mechanism by which nematodes interpret environmental conditions to prime innate immune defenses and promote survival in microbe rich environments. C. elegans animals augment these immune defenses by surveying for ligands specifically associated with toxigenic pathogens that are poised to cause disease. These findings define a new paradigm of intestinal immune control that informs the evolution of innate immunity in all metazoans.
    • Integrated Multi-omics Characterization of Human Disease Models

      Erik J. Sontheimer; Rodríguez, Tomás (UMass Chan Medical School, 2022-06-07)
      Animal and cell-based models of human disease offer simplified biological systems for studying the basis of more complex pathologies under well-controlled conditions. An ever-expanding suite of genomic and transcriptomic tools allows us to thoroughly characterize these models, highlighting disease-driving molecular features and exposing novel therapeutic targets. Here, we integrate diverse DNA- and RNA-sequencing strategies to describe the gene-regulatory chromatin landscape of models for hepatoblastoma and retrovirally-infected CD4+ T-cells. We first developed a conditional hepatoblastoma mouse model using doxycycline-inducible YAP1 overexpression and constitutive β-cateninDelN90. We found that YAP1 withdrawal alone is sufficient to trigger tumor regression and substantially increase survival. We reasoned that a thorough chromatin profile of this tumor model during YAP1 withdrawal could reveal YAP1-driven mechanisms of hepatoblastoma tumorigenesis. Our integrated approach revealed 31 novel YAP1-targeted cis-regulatory element-gene pairs. Subsequent validation confirmed that regulation of Jun-Dimerization Protein 2, among others, is both YAP1-dependent and functionally consequential for the hepatoblastoma phenotype in human cells and in hepatic malignancies. To expand our efforts to apply multi-omics technologies to disease models, we next engineered a fluorophore-containing murine leukemia virus (MLV-GFP) stably integrated into Jurkat CD4+ T-cells to report on defective transcriptional silencing by the retroelement-silencing complex, HUSH. A CRISPR knockout screen identified DHX29 as essential for HUSH-mediated silencing of newly-integrated retroviruses. Profiling genomic and transcriptomic features of MLV-GFP Jurkat cells after HUSH and DHX29 knockout revealed their epistatic roles in silencing, and revealed a suite of loci targeted by HUSH. Finally, we used site-specific proteomics and chromatin profiling to identify HUSH-associated factors at the newly integrated proviral reporter.
    • The Development of a Skin-Targeted Interferon-Gamma-Neutralizing Bispecific Antibody for Vitiligo Treatment

      John Harris; Hsueh, Ying-Chao (2022-06-06)
      Despite the central role of IFNγ in vitiligo pathogenesis, systemic IFNγ neutralization is an impractical treatment option due to strong immunosuppression. However, most vitiligo patients present with less than 20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFNγ signaling during vitiligo, I hypothesized that tethering an IFNγ neutralizing antibody to keratinocytes would limit anti-IFNγ effects to the treated skin for the localized treatment. To that end, I developed a bispecific antibody (BsAb) capable of blocking IFNγ signaling while binding to desmoglein expressed by keratinocytes. I characterized the effect of the BsAb in vitro, ex vivo, and in a mouse model of vitiligo. SPECT/CT biodistribution and serum assays after local footpad injection revealed that the BsAb had improved skin retention, faster elimination from the blood, and less systemic IFNγ inhibition than the non-tethered version. Furthermore, the BsAb conferred localized protection almost exclusively to the treated footpad during vitiligo that was not possible by local injection of the non-tethered anti-IFNγ antibody. Thus, keratinocyte-tethering proved effective while significantly diminishing off-tissue effects of IFNγ blockade, offering a new treatment strategy for localized skin diseases, including vitiligo.
    • The Effect Cognate Antigen Has on T Cells Responding to Influenza Infection

      Susan L. Swain; Jones, Michael C. (2022-06-03)
      The contributions of peptide antigen affinity for TCR in driving T cell memory is unclear. Effector CD4 T cells must recognize cognate antigen again at an effector checkpoint, 5-8 days post-infection, to generate an optimal memory population. In this thesis, we examined whether peptide affinity for the TCR of effectors impacts the extent of memory and degree of protection against rechallenge. We used an influenza A virus (IAV) nucleoprotein (NP)-specific TCR transgenic strain, FluNP, and generated NP- peptide variants that bind FluNP TCR with a broad range of avidity. Varying peptide avidity in vivo at the effector checkpoint revealed that higher affinity interactions yielded greater numbers of FluNP memory cells in the spleen and most dramatically in the lung and dLN. The major impact of avidity was on memory cell number, not cytokine production, and was already apparent within several days of transfer. These memory cells demonstrated enhanced protection against lethal IAV infection with a robust early day 5 secondary effector response in the lung. We previously showed that autocrine IL-2 production during the effector checkpoint prevented default effector apoptosis and supported memory formation. Here, peptide avidity determined the level of IL-2 produced by effectors while IL-2R expression was unaffected. However, IL-2Ra expression by APC drove more memory cell formation, suggesting that transpresentation of IL-2 by APC at this checkpoint enhanced CD4 memory generation. Secondary memory generation was also avidity-dependent. We propose this pathway selects CD4 effectors of highest affinity to progress to memory and can instruct future vaccine design.
    • A Feasibility Study of Therapeutic Conversations with Family Members to Reduce the Symptoms of Post-Intensive Care Syndrome

      Susan Sullivan Bolyai; Tehan, Tara (2022-05-25)
      PURPOSE: The purpose of this feasibility study was to explore the use of a nurse-centered intervention, the Critical Caring Program, with family members of critically ill adults. The intervention was a series of therapeutic conversations with a family member, beginning in the ICU and following patient discharge from the ICU. FRAMEWORK: The Family Adjustment and Adaptation Response Model (Patterson, 1988) provided the conceptual framework; the intervention was adapted from the Calgary Family Assessment and Intervention Model. DESIGN: A randomized, controlled design with two groups (usual care and intervention) was used to assess the feasibility of the intervention. A convenience sample of 19 adult family members were recruited from an 18-bed ICU from October 2021–January 2022. RESULTS: 151 family members were screened for participation; 40 who were eligible and 19 were enrolled. Overall retention was 58% for the intervention group; 62% for the usual care group. Outcomes revealed no statistically significant differences between groups or changes within groups. The nurses viewed the training and conversations as positive but identified incorporating the visits into routine practice as challenging. CONCLUSION: The Calgary Family Intervention Model is a useful model for addressing families’ need for communication and support. Additional research is needed on incorporating therapeutic conversations into critical care nursing practice.
    • Identification of a Detoxification Requirement During De Novo Sphingolipid Biosynthesis in Cancer Cells

      Dohoon Kim; Spears, Meghan E. (2022-05-25)
      Sphingolipids are a class of lipid molecules that function both as structural membrane components and as bioactive signaling molecules. Sphingolipids can be produced de novo or salvaged and recycled. Despite the established roles of sphingolipids such as sphingosine 1-phosphate and ceramides in regulating signaling involved in pro- and anti-tumorigenic cellular processes, the role of the de novo sphingolipid biosynthesis pathway in cancer is unclear. The main objective of this thesis study was to determine whether there is an essential role for this pathway in cancer and whether its disruption can be a cancer-specific metabolic vulnerability. Here, we find that de novo sphingolipid synthesis through the rate-limiting enzyme serine palmitoyltransferase (SPT) is not required in cancer cells due to their salvage capacity. However, upregulation of SPT in cancer cells creates a requirement to detoxify its product, 3-ketodihydrosphingosine (3KDS), via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). We demonstrate that KDSR is essential in cancer cells both in vitro and in vivo to restrain the levels of its substrate 3KDS, the accumulation of which can disrupt ER structure and function, resulting in proteotoxic stress and cell death. Our findings also reveal that KDSR is essential specifically in cancer cells and not normal cells and that upregulation of SPT in cancer may act as a biomarker for sensitivity to targeting KDSR. Altogether, this thesis study provides new insights into the role of KDSR in the de novo sphingolipid biosynthesis pathway in both cancer and ER homeostasis and demonstrates the potential to exploit this for therapeutic purposes in a cancer-specific manner.
    • Barriers to Perinatal Depression Care Access in Women with and without a Self-reported Psychiatric History

      Tiffany Moore Simas; McNicholas, Eileen (2022-05-19)
      Background: Perinatal depression affects 1 in 7 childbearing individuals and remains underdiagnosed and undertreated. Individuals with a psychiatric history are at increased risk of perinatal depression, and little is known about how experiences with the mental health care pathway may differ between these individuals and those without a psychiatric history. Methods: This was a secondary analysis of data from the PRISM (PRogram in Support of Moms) study, a cluster randomized controlled trial of two interventions for perinatal depression. Care access and barriers to care were evaluated in perinatal individuals who screened positive for depression using the EPDS (N=280). Results: Individuals with no psychiatric history prior to pregnancy (N=113), compared to those with such history (N=267), were less likely to be screened for perinatal depression, and less likely to be offered a therapy referral, though equally likely to attend when referred. In adjusted models, those without a psychiatric history had 0.59 times the odds of attending therapy (95% CI 0.28-1.25), 0.23 times the odds of utilizing medication (95% CI 0.11-0.47), and overall, 0.22 times the odds of receiving any depression care (95% CI 0.11-0.43). Participants reported on average 3 barriers as preventing them from receiving care “a lot” or “quite a lot”. The proportion of individuals endorsing each barrier was similar between groups, excepting “concerns about treatments available” and “thinking the problem would get better by itself”, which were more prevalent in those without a prior psychiatric history. Conclusions: There exist meaningful differences in the way perinatal individuals access care for depression based on psychiatric history. An understanding of these differences is crucial in addressing gaps between mental health care need and care receipt.
    • Prevention of Ulnar Collateral Ligament Reconstruction: A Systematic Review

      Shao-Hsien Liu; Cellurale, Adam (2022-05-19)
      Background: Medial ulnar collateral ligament reconstruction surgery (UCLR) is a common surgical procedure performed on elite level baseball players. Physical signs and symptoms of ulnar collateral ligament (UCL) injuries requiring UCLR along with treatment have been clearly defined, however, an exact etiology of UCL injuries and methods of preventing UCLR surgery remain unclear. Objective: Systematically review and qualitatively provide an in-depth summary of recent literature about the relationships between changes in advanced pitching metrics for potential warning signs of UCL injury prior to requiring UCLR. Methods: We searched two electronic databases (PubMed, Scopus) from inception to October 2021 using a keyword search. Data extracted included author and year of publication, study design, sample size, study location, and primary outcome variables. Articles that met inclusion criteria were then evaluated using a modified Downs and Black criteria. Results: The key word search returned 51 articles of which a total of seven articles were included in the review. For the papers that met the inclusion criteria, four noted changes to velocity as potential an indicator for UCLR surgery, two identified lateralization of arm angles as an indicator of UCLR surgery, and one reported change to spin rates of specific pitches as an indicator for UCLR surgery. Conclusions: The results of this review show that changes in certain advanced pitching metrics such as spin rate, velocity, and lateralization of arm angles could be potential indicators of UCL damage. Further evaluation is needed to continue to improve our understanding of how these trends could be used as predictors of UCLR.
    • Binge Alcohol Drinking Alters the Differential Control of Cholinergic Interneurons over Nucleus Accumbens Medium Spiny Neurons

      Gilles Martin; Kolpakova, Jenya (UMass Chan Medical School, 2022-05-06)
      Striatal cholinergic interneurons (ChIs) play a central role in basal ganglia function by regulating associative learning and reward processing. Drug addiction, such as alcoholism, is often described to hijack the natural reward system. In the nucleus accumbens (NAc), a brain region that mediates rewarding properties of substance of abuse, ChIs regulate glutamatergic, dopaminergic, and GABAergic neurotransmission. However, it is unclear how ChIs orchestrate the control of these neurotransmitters to determine the excitability of medium spiny neurons (MSNs), the NAc output neurons that translate accumbens electrical activity into behavior. Combining ex vivo electrophysiology, fast scan cyclic voltammetry and optogenetics approaches, I have demonstrated that stimulating NAc ChIs decreases the spontaneous excitatory postsynaptic currents (sEPSCs) frequency of both D1- and D2-MSNs through different mechanisms. While this effect in D1-MSNs was mediated by dopamine, it resulted from a direct control of glutamate release by ChIs in D2-MSNs. Interestingly, after two weeks of binge alcohol drinking, the effect of ChI stimulation on glutamate release was reversed in D1-MSNs, while its effect on D2-MSNs remained unchanged. Finally, in vivo optogenetic stimulation of NAc ChIs significantly increased alcohol consumption compared to unstimulated mice, but failed to alter mouse locomotor activity and saccharine or water consumption. Together, these results identify ChIs as a key modulator of NAc circuit activity and as a potential therapeutic target for alcohol use disorder.
    • Assaying Microglial Function within Neural Circuits: Implications for Regulating Neural Circuit Excitability

      Dorothy P. Schafer; Feinberg, Philip A. (2022-04-29)
      Microglia are the resident macrophage in the central nervous system (CNS) that actively survey their environment and participate in shaping neuronal circuits. Among the transcription factors necessary for microglia development, interferon regulatory factor 8 (IRF8) is a known risk gene for multiple sclerosis and lupus and it has recently been shown to be downregulated in schizophrenia. These studies suggest that lack of microglial IRF8 can subsequently impact neuronal function in disease, but the mechanisms underlying these effects remain unknown. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. To interrogate the impact of disrupted microglial IRF8 signaling on brain circuits, I first show by RNAseq that several genes known to regulate neuronal function are dysregulated basally in Irf8-/- brains. I then found that these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to chemically-induced lethal seizures in Irf8-/- mice. Importantly, I also show that developmental synaptic pruning, a key function for microglia, proceeds normally in Irf8-/-mice. Finally, I identified that these IRF8-dependent effects on circuits are due to elevated TNF-α in the CNS as genetic or acute pharmacological blockade of TNF-α in the Irf8-/- CNS rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. The next steps are to use cell-specific genetic approaches to manipulate this signaling, which I have further developed over the course of this project.