University Of Chicago

NIH Research Projects · FY 2026 · 2017-04

PROJECT SUMMARY/ABSTRACT Innate immune factors are critical for the selection and maintenance of regional commensal gut microbiota that provide beneficial functions for the host and deterrence against pathogens and pathobionts. In the previous grant cycle, we discovered that peptide YY (PYY), an anorexigenic or satiety-related gut hormone of intestinal enteroendocrine L-cell origin, is expressed by innate immune intestinal Paneth cells (PC) and functions as an antimicrobial peptide (AMP) in its unmodified PYY1-36 form (henceforth referred to as PC-PYY1-36). PYY's structure differs from other PC-α and β−defensins but is remarkably similar to the amphibian AMP, magainin-2, that has both anti-fungal and anti-bacterial properties. PC-PYY1-36 further differs from other PC-AMPs by selectively targeting and killing the virulent (hyphal), but not commensal (yeast) forms of polymorphic gut fungi like Candida albicans, thereby playing an important role in maintaining fungal (and also bacterial) commensalism of the gut. PC-PYY1-36 also differs from the endocrine PYY3-36 that is derived from DPP-IV conversion of L-cell PYY in structure and function by being vectorally secreted into and retained by intestinal mucus where its activity is optimal and protected from dipeptidyl peptidase IV (DPP-IV) modification that would otherwise convert it to the endocrine PYY3-36 form. We subsequently discovered that PCs also express another anorexigenic peptide highly related to PYY, neuropeptide Y (NPY1-36), that also has AMP properties. However, its regulation, antimicrobial spectrum, and compartmentalization upon secretion in luminal fluid rather than overlying mucus differs from that of PC-PYY. Based on our findings, we hypothesize that PC-PYY and PC-NPY are a novel class of PC- AMPs that play important roles in maintaining gut microbial commensalism and conferring innate immune defense against certain pathogens and pathobionts. Two specific aims are proposed: (1) to define and differentiate the drivers, mediators, and signaling pathways involved in regulation of PC-PYY and PC-NPY gene expression, secretion, and regional compartmentalization under physiological and pathophysiological conditions, and (2) to determine and differentiate the antimicrobial spectra of PC-PYY and -NPY in vitro and in vivo and their impact on gene expression and adaptive responses of targeted bacterial and fungal species. These studies will employ newly established in vivo (PC-PYY and PC-NPY KO and PC-PYY transgenic mice) as well as in vitro and ex vivo approaches. The MPI team combines unique expertise and resources in experimental research, multi'omic technologies, and host-microbe interactions (Chang, UChicago), experimental physiology and pathophysiology (Pierre, UWisc-M), and microbiology, bioinformatics, and genetics (Peters, UTHSC). Thus, the whole is much greater than the sum of the parts. The MPI groups have collaborated productively over the past two years to acquire the preliminary and feasibility data, establish proof of concept, and set in motion new lines of inquiry. The initial findings from these studies are in press with the journal Science.

NIH Research Projects · FY 2025 · 2016-12

ABSTRACT Novel immunotherapies for cancer are having a major clinical impact, in particular anti-PD-1 mAbs which have been FDA-approved for 20 cancer entities. However, the mechanisms that explain why a subset of patients fails to respond to these therapies is incompletely understood. Understanding these mechanisms should lead to new therapeutic strategies for expanding efficacy further. Our prior data indicated that a baseline T cell-inflamed tumor microenvironment was predictive of response to anti-PD-1, which augments the functionality of CD8+ T cells already present within the tumor microenvironment. During the previous funding period, we made multiple novel discoveries that have been paradigm-shifting for the field, which have coalesced to motivate continued investigation into 5 research directions: investigation of novel T cell immune checkpoints, innate immune strategies to promote de novo T cell responses in the tumor microenvironment, tumor cell-intrinsic oncogenic events mediating immune resistance, regulation of anti-tumor immunity by the commensal microbiota, and germline variants influencing host anti- tumor T cell responses. Each of these directions is identifying novel therapeutic opportunities that are expected to expand the circle of efficacy for checkpoint blockade immunotherapy in the clinic.

NIH Research Projects · FY 2025 · 2016-09

Project Summary/Abstract Chemical modifications on mammalian messenger RNA (mRNA) have recently been shown to play critical and diverse regulatory roles in mRNA metabolism and translation. For example, the most abundant mRNA modification, N6-methyladenosine (m6A), is crucial for mammalian stem cell differentiation and tissue development in almost all systems tested so far. Dedicated proteins, many of which are essential in mammals, have evolved to install, recognize, and remove m6A marks (writers, readers, and erasers, respectively). Dysregulation of m6A methylation has been connected to a variety of human diseases and disorders. Functional roles have also been proposed for other internal modifications present in mammalian mRNA, including, but not limited to: pseudouridine (Ψ), 5-methylcytosine (m5C), 2’-O-methylation (Nm), N1-methyladenosine (m1A), N7- methylguanosine (m7G), and N3-methylcytosine (m3C). Our most recent research has uncovered modifications on chromosome-associated regulatory RNAs (carRNAs), such as promoter-associated RNA (paRNA), enhancer RNA (eRNA), and repeat RNAs, as well as frequent modifications in introns of pre-mRNA. The carRNA modifications have been shown to regulate chromatin state and transcription, and intron modifications may affect pre-mRNA processing. Despite rapid advances in the discovery and functional characterization of various RNA modifications and their effector proteins, a significant bottleneck limits the entire field of epitranscriptomics research: a dearth of quantitative sequencing methods that can comprehensively map most RNA modifications at base resolution with exact modification fraction information. The availability of such methods is critical for assessing the importance of these modifications in different regions of mRNA, examining the effects of dynamic changes in modification fraction, assigning modifications to different writers and analyzing their functional relevance, identifying target transcripts and sites of demethylation and analyzing their functional relevance, discovering new effectors for RNA modifications by overlapping with known RBP-binding sites or genomics features, and evaluating the physiological consequences of RNA modifications in biological processes. We have established both nucleic acid chemistry and directed protein evolution platforms to invent new technologies that transform RNA modifications to be read out as mutations or deletions that are universally compatible with extant sequencing platforms. Computational pipelines and RNA modification databases will be built to support the new method development and epitranscriptome research in the broad community. These new technologies will be optimized to work on low-input samples, particularly neuronal and clinical samples. We will focus on integrating new methods into robust protocols to map multiple RNA modifications in single experiments. Our proposed research will deliver high-throughput, high-resolution, and high-sensitivity methods that simultaneously map multiple RNA modifications in all biological areas.

NIH Research Projects · FY 2025 · 2016-07

Project Summary Protein lipidation is a dynamic post-translational modification (PTM) that affects subcellular trafficking, co-factor binding affinity, and membrane localization of proteins, which in turn influence downstream signaling cascades. In particular, cyclic S-acylation and -deacylation of proteins at specific cysteine residues is emerging as a key link between circulating lipid levels and the regulation of essential biological processes, including those involved in cellular growth, metabolism, and neurological health. In-depth study of this PTM, however, has proven technically difficult, in large part due to the paucity of selective, effective chemical inhibitors for the enzymes that catalyze its installation and removal. The proposed research program is designed to generate novel chemical technologies, namely small molecule probes and inhibitors, in the service of illuminating the involvement of regulated protein S-acylation in both normal and pathophysiological contexts. These goals will be realized through two complementary chemical and cellular biology research areas. One area will involve measuring, manipulating, and determining the targets of the “writers” of S-acylation, DHHCs. To do so, pan-active DHHC inhibitors will be identified using newly developed and optimized screening and selectivity profiling assays in combination with rationally and computationally designed molecules, as well as a library of putative inhibitors. Validated inhibitors and proteomics-based methods will then be used to identify the specific protein targets of DHHCs in live cells to more precisely describe their involvement in various disease states. The second research area will utilize our recently validated chemical tools to study the biological function of the “erasers” of S-acylation, APTs, with particular emphasis on the involvement of these enzymes in cellular redox homeostasis and metabolic disease. The expected outcome of this multidisciplinary research program is two-fold: generating a collection of chemical tools and assays for the study of this important PTM, and describing its biological function and influence in normal and disease states.

NIH Research Projects · FY 2026 · 2016-03

Project Summary Fatty liver disease has emerged as a major contributing factor to the increased incidence of hepatocellular carcinoma (HCC) in western societies in the past several years, and rates of HCC in the US are projected to increase further in the years ahead due to over-nutrition and the obesity epidemic. Thus, an understanding of how lipid levels in the liver are regulated and the underlying mechanistic basis of lipid accumulation in disease states is important to developing improved ways to prevent and treat fatty liver and HCC. The proposed research sets out to define the role of the BNIP3 and BNIP3L mitochondrial cargo receptors in lipid metabolism in normal liver and in preventing fatty liver disease and hepatocellular carcinoma. In the past cycle of this grant, we showed that BNIP3 is essential for mitophagy induced by nutrient deprivation and that this in turn promotes lipid droplet turnover. Further, we showed that BNIP3- dependent mitophagy sets up metabolic zonation in the liver through control of mitochondrial mass. We also showed that loss of BNIP3 promoted HCC due to lipid accumulation in both mouse models of liver cancer and in human liver, where loss of BNIP3 expression predicted HCC patient outcomes when combined with expression levels of genes involved in fatty acid metabolism. In this renewal application, our work aims to develop further our understanding of the mechanistic basis of how BNIP3 promotes lipid droplet turnover in concert with the turnover of mitochondria, how a second novel role for BNIP3 in modulating the mTOR signaling pathway contributes to progression of NAFLD and NASH to HCC, and how BNIP3 and BNIP3L (NIX) interact functionally in the liver. Specifically, in Aim 1 we seek to fully understand the role of BNIP3 in lipid droplet turnover and liver metabolism by investigating: (1) whether BNIP3 interacts with Rheb or other molecular partners at the lysosome and lipid droplet; and/or (2) how the Rheb-BNIP3 interaction modulates BNIP3-LC3 interactions and mitophagy. The key objective in Aim 2 is to define how BNIP3 suppresses steatosis and HCC via modulation of mTOR pathway signaling. In Aim 3, we propose to determine how BNIP3 and BNIP3L (NIX) differ in their role in hepatic steatosis and how this contributes to control of steatosis, mTOR activity, tumor cell growth and liver cancer. Throughout this renewal proposal, we make use of novel mouse models, human HCC cell lines and primary human liver samples to explain the role of BNIP3 and BNIP3L in lipid homeostasis and growth control in the liver.

NIH Research Projects · FY 2026 · 2016-01

Project Abstract: Membrane proteins are complex molecular machines whose functions are governed by sets of programed conformational transitions. Attempts to establish the fundamental molecular mechanisms that link membrane protein structure and dynamics to functions they induce have been thwarted by a number of seemingly insurmountable technical barriers. Principal among these barriers is that the conformational transitions are too transient to be studied using traditional structural biology techniques. To overcome these barriers, we have developed and implemented a set of novel methodologies and reagents based on phage display generated synthetic antibodies (sABs). Customize phage display selection strategies enable generation of sABs endowed with special properties, for instance, conformation and regio-specificity. These reagents have been used to study the molecular properties of transient states of membrane proteins at unprecedented detail. While sABs have demonstrated efficacy as crystallization chaperones, their use in cryo-EM as powerful fiducial marks, adding 50 kDa to the particle and their ability to trap conformation states, is especially impactful in studies linking conformational transitions and function. This is particularly relevant for smaller membrane proteins (< 50 kDa), which include ion channels transporters and receptors. These constitute the largest class of biomedically relevant target systems, but are recalcitrant to crystallization and are far too small for cryo-EM analysis. Building on our current technology platform, we propose to design and deploy a set of higher-order sAB constructions that will serve to increase the size, rigidity and, in some cases, the symmetry of the target membrane protein. These sAB-based entities will be engineered to serve as prefabricated modules of assembly. They are targeted to specific epitopes that have been introduced into the membrane protein and thus, can be universally employed irrespective of the system they are applied to. The power of the approach is that these “universal” sABs can be added to the molecule of interest in a “plug and play” fashion allowing any investigator access to the powerful technology without requiring generating target specific sABs. To test and evaluate these novel sAB modules, we will use a set of high value small membrane proteins provided by investigators from our collaborator network. These systems have been recalcitrant to structural analysis using traditional approaches and thus, will provide a good measure of the performance of the chaperone-assisted structure determination technologies. An important byproduct is that these structures will provide valuable information about linkages between structure and dynamics that had been out of reach previously.

NIH Research Projects · FY 2025 · 2015-09

ABSTRACT: This is the renewal U2R application for the “Bangladesh Center for Global Environmental and Occupational Health (GEOHealth)”- awarded along with the linked U01 grant in 2015 to the University of Chicago (UofC) and International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B). The project will be implemented in close collaboration with the Bangabandhu Sheikh Mujib Medical University (BSMMU) and selected US as well as local academic institutions to continue and augment complementary training expertise relevant to this proposal. The goal of the Bangladesh GEOHealth Center was to strengthen the research capacity of Bangladesh on pressing environmental and occupational health threats. During the current funding period, the Bangladesh GEOHealth Center has positively impacted on the foundation of environmental health research capacity in Bangladesh by training more than 200 students, young and mid-career researchers from diverse background through custom-designed in-country 2-day workshops and 2-week short-courses, and 16 fellows through US- based postdoctoral fellowships and 12-week (one academic quarter) training at the UofC. Evaluation of short courses and workshops in Bangladesh indicated increased knowledge and high satisfaction of the participants. The 16 fellows who received training at UofC have demonstrated outstanding research productivity in term of publications, presentations, grants, and career development. They produced 90 peer-reviewed publications during and after the training period. All 16 US-based fellows reported that they had cooperative and influential, mentor(s) during the training period and the trainees continued to receive guidance from the mentor(s) even after completion of the training. Despite these demonstrable impacts there remains a critical need for additional research capacity in Bangladesh to address the country’s huge environmental and occupational health burden given the acute shortage of researchers trained in modern environmental health research methods, especially data science and mHealth tools and techniques. In this renewal application, we propose to continue and augment our Bangladesh and US- based training activities through i. Workshops (2 day), ii. Short courses (1-2 week) iii. One academic quarter course work at UofC, iv. A master’s degree program (1-year) at UofC, and v. Postdoctoral training (1-year) at UofC. We will refine the training curricula that we used over the past 5 years to incorporate more materials on utilizing advanced technologies and analytical tools (artificial intelligence/machine learning (AI/ML), image analysis, mobile health (mHealth) data capture/analytics, exposure data science, etc.) for analyzing environmental health data with the existing materials, all of which will incorporate the linked U01 application’s research focus. A rigorous evaluation system will help the Administrative and External Oversight Committee to monitor and ensure program success. We have successfully implemented the current phase of the training grant, and we believe that our renewal training program will address critical gaps in environmental and occupational health research capacity, and bring benefit to a wide variety of researchers in Bangladesh.

NIH Research Projects · FY 2026 · 2015-04

SUMMARY Inflammatory diseases are often driven by inappropriate responses of effector CD4 T cells (Teff). IL17, IFNγ, or dual-producing polyfunctional effector Th1, Th17, or Th17.1 T cells can become imbalanced with suppressive Treg CD4 T cells in a variety of disease settings, including inflammatory bowel diseases (IBD). A key therapeutic objective in efforts to shift the immunologic balance towards tolerance, therefore, is to selectively inhibit Teff and promote Treg. We have shown that Teff and Treg subsets utilize different metabolic programs that represent fundamental features of T cell biology. Here we explore one carbon (1C) metabolism and related microenvironmental factors to modulate CD4 T cells in inflammatory diseases. 1C metabolism integrates multiple nutrient inputs to provide intermediates for de novo methionine and purine synthesis and is commonly targeted with anti-folate drugs. An in vivo CRISPR screen of primary T cells in IBD with a 1C metabolism enzyme-focused gRNA library identified the mitochondrial enzyme Methylene-tetrahydrofolate Dehydrogenase 2 (MTHFD2) as conditionally essential for effector T cell proliferation and inflammation. MTHFD2 was upregulated in T cells a variety of inflammatory conditions and while MTHFD2-deficiency impaired Teff, MTHFD-deficient Treg had increased FoxP3 expression in both mouse and human T cells. Consistent with a role as a metabolic checkpoint on inflammation, MTHFD2-deficiency protected against IBD and other inflammatory diseases. Mechanistically, MTHFD2 inhibition suppressed mTORC1 activity, possibly through reduced methionine and/or interrupted purine synthesis. Importantly, local nutrients play key roles in 1C metabolism and T cell fate. To quantify T cell access to nutrients in vivo, we established Positron Emission Tomography (PET) tracer-based methods to directly image and measure nutrient uptake in vivo. These studies showed a sharp increase in T cell glucose uptake in inflammation. In addition, IBD is often associated with folate-deficiency. The effects of dietary folate on T cell 1C metabolism, mTORC1 signaling, and fate, however, are unclear. Because inflammation is associated with fevers and enzymes are temperature-dependent, we also tested fever conditions on T cell metabolism. We found fever led to increased cytokine production from Teff and mitochondrial Reactive Oxygen Species (ROS) specifically in Th1 cells that, surprisingly, led to Tp53-dependent apoptosis. These findings support the hypothesis that 1C metabolism is limiting and serves as a metabolic checkpoint to integrate local nutrients and physical conditions through MTHFD2 and mTORC1 signaling to provide new immunometabolic targets to modulate effector and regulatory T cells. To test this, we will: (1) Test the role and mechanism of MTHFD2 as a limiting enzyme in methionine and nucleotide synthesis essential for mTORC1 signaling and effector T cells; and (2) Determine how nutrient and microenvironmental factors such as folate and fever influence effector and regulatory T cell metabolism and inflammation. These studies will test MTHFD2 and identify new immunometabolic mechanisms and targets that contribute to or may be exploited to treat inflammatory disease.

NIH Research Projects · FY 2026 · 2014-07

Abstract Understanding the molecular and organismal function of genetic variants in non-coding regions is crucial to dissect the genetic and evolutionary basis of variation in complex traits. As we and others have shown, the effect of a genetic variant on a molecular pathway, and ultimately on the individual's phenotype, may be modulated by the environmental context (gene-environment interactions, GxE). Most contexts considered to date focus on aspects of the physical environment that can be reproduced in vitro (e.g., pathogen particles, hormones, toxicants). We also pioneered the study of GxE with psychosocial experiences (e.g., socio-economic status, neighborhood stress). Importantly, psychosocial experiences can influence an individual's ability to respond to other environmental challenges and these effects can be measured at the cellular level. However the effects of psychosocial experiences on the transcriptional response to cellular stressors has not been fully evaluated. The response to environmental challenges is often mediated by changes in transcription factor activity. Single cell genomics technology has allowed us to discover that gene regulatory changes in response to environmental perturbations are often heterogeneous both across cell types, and across individual cells within a cell type, resulting in a dynamic response. This dynamic response can in turn be regulated through GxE, and it's an important contributor to disease risk. To study the dynamic gene regulatory response to chemical and social stressors, here we will focus on peripheral blood mononuclear cells from an inner city cohort of middle-aged adults collected as part of The Heart of Detroit Study (THDS) and consider psychosocial and cellular stressors. One of the main objectives of this project will be to develop functional genomics, computational, and statistical approaches to dissect the epigenetic mechanisms underlying the response to in vitro and in vivo stressors, their interactions with genetic variants, and ultimately their role in complex trait variation. The tools developed in this project will have broad applicability in the fields of functional genomics, systems biology, and GxE, ultimately allowing for a global and integrative analysis of genetic and environmental effects on biological systems.

NIH Research Projects · FY 2025 · 2014-04

Abstract The mission of the NRG Oncology Statistics and Data Management Center (SDMC) is to provide all necessary expertise and personnel for the design, conduct and analysis of clinical trials and associated research for NRG Oncology, a member group of the NCI National Clinical Trials Network (NCTN) program. The 2019-2024 cycle represents the second period of the NCTN program, which succeeded the NCI Cancer Cooperative Group program and of which NRG Oncology was formed by the unification of three of the Cooperative Groups. NRG Oncology has unique expertise in gender-specific malignancies (breast, gynecologic, and prostate cancer), as well as a major presence in several other cancer types (head & neck, lung, brain, upper GI) where multimodality therapy has a critical role. In the first cycle of the NCTN (2014-2019), the NRG SDMC and partner NRG Operations Center personnel expended significant effort in successfully transforming into a truly united and highly functioning group. Concurrently, the group made significant strides in advancing cancer treatment, ancillary care, and cancer biologic knowledge. During the first cycle, the group initiated 32 trials and obtained final approval on an additional 14 trials for development, while maintaining follow-up to primary and secondary endpoint reporting on over 80 additional trials that were active at the inception of NRG Oncology. Publications from the group through the initial 5 years number over 300, including 39 methodology articles by SDMC statisticians. To carry out its mission, the NRG Oncology SDMC will collaborate closely with NRG Oncology Operations leadership and trial investigators to design and conduct the highest quality clinical trials that will yield definitive conclusions. The SDMC will provide continuous trial conduct oversight through all stages from concept inception through protocol development, trial activation, trial monitoring, and dissemination of findings. Through its quality assurance program, the SDMC will provide quality control monitoring across the lifecycle of trials. The SDMC will continue to apply, or develop as needed, innovative statistical designs in all phases of clinical trials, using integrated developmental trial phases where advantageous, and incorporating biomarker-based information into trial designs and analysis plans. The SDMC will continue to Implement state-of-the-art data management and information technology systems to assure secure high-quality data collection, maintenance, and curation for additional discovery and data sharing with the larger research community.

NIH Research Projects · FY 2025 · 2013-09

Abstract: We are engaged in the systematic efforts to exploit catalytic carbon–carbon (C−C) bond activation for developing new, general and synthetically useful methods. Our objective, in the proposed funding period, is to focus on C−C activation and functionalization of ketones and amides, as they are easily accessible and widely found in feedstock chemicals. Through harnessing the power of transition-metal catalysts, sometimes assisted by an organic activator, a suite of new, selective, and efficient methods involving C−C activation of ketones and amides will be developed. Specifically, we will: (i) explore new C−C activation modes that allow rapid access to complex sp3-rich ring systems, (ii) enable a novel and broadly applicable cross-coupling reaction via deacylation of unstrained ketones, and (iii) allow selective activation of α-C−C bonds of unstrained amides. The research proposed is expected to offer an atom-economical approach for preparing novel complex sp3-rich molecular scaffolds from readily available substrates, which should help to access greater chemical space beyond the “flatland”. It is also expected to simplify synthesis through new strategies for bond disconnections and to enable skeletal editing of complex molecules under redox-neutral conditions, capitalizing on C−C activation/functionalization of ketones and amides.

NIH Research Projects · FY 2025 · 2012-08

Project Summary/Abstract DNA cytosine methylation (5-methylcytosine or 5mC) is a key epigenetic modification in the regulation of human gene expression. It plays critical roles in suppressing transcription of a large portion of human genome, including repetitive elements. 5mC can be reversed through oxidation by the human TET family enzymes, which utilize dioxygen to sequentially oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and finally 5-carboxycytosine (5caC). Both 5fC and 5caC can be recognized and excised by human thymine DNA glycosylase (TDG), followed by base excision repair (BER) to replace the modified cytosine with a normal cytosine, in an active demethylation process. Cell-type specific DNA methylation has been studied and applied as a biomarker for disease diagnosis and prognosis. Antibody-based MeDIP immunoprecipitation followed by high-throughput sequencing is a common method for genome-wide mapping of DNA 5mC distribution, but it is not quantitative and requires a significant amount of input material. Bisulfite sequencing is the gold standard approach, widely applied in quantitative 5mC sequencing; however, whole-genome bisulfite sequencing is expensive and causes severe DNA degradation. To overcome these critical barriers to progress, we propose to develop new methods that combine 5mC enrichment with quantitative 5mC sequencing, using limited starting material. The potential application of these new methods in circulating cell-free DNA (cfDNA) analyses stands to potentially revolutionize human disease diagnosis and prognosis. Whereas the 5mC modification suppress activation of a majority of human genome, studies from us and others revealed that 5hmC marks active loci. We previously developed robust procedures to map 5hmC genome-wide using 1,000 cells. However, quantitative methods that determine the presence and stoichiometry of 5hmC at the single-cell level are still lacking. Our ongoing efforts to overcome this challenge recently led to a chemical solution that yields base-resolution 5hmC information with modification stoichiometry, using limited input material. In this renewal, we propose base resolution sequencing of 5hmC at the single-cell level, enabling the detection of intercellular differences that are otherwise missed, including the epigenome remodeling that underlies the initiation of cell fates during early embryogenesis. The success of this program will provide new methods for 5mC and 5hmC mapping to enable breakthrough discoveries in both basic research and clinical applications.

NIH Research Projects · FY 2026 · 2012-07

Overall Summary Transplantation tolerance, a state of hyporesponsiveness to donor antigens after cessation of therapy, is an attractive approach for achieving life-long graft acceptance without global immunosuppression. Tolerance is rare in the clinic, and even when attained can be lost over time, sometimes after infections. Understanding the barriers to the induction of transplant tolerance in the clinic, and the vulnerabilities to durable tolerance, is essential to achieving the goal of one transplant for life. One barrier to the induction of transplant tolerance in the clinic is T cell memory (Tmem). The intrinsic independence of Tmem from costimulation and their resistance to Tregs can explain the difficulty in inducing tolerance. Project 2 has identified an additional hurdle by which Tmem can antagonize the induction of transplant tolerance: a small number of Tmem can “infect” naïve T cells into acquiring memory-like features and resisting costimulation blockade (CoB) via a process of ‘linked-sensitization’. Once established, transplantation tolerance may exhibit vulnerabilities to its maintenance especially during settings of proinflammatory infection. Project 1 has identified heterogeneity in states of dysfunction achieved by polyclonal alloreactive T cells following CoB, with T cells specific for alloantigens that are rapidly downregulated in the graft following transplantation, and T cells with low affinity/avidity to graft antigens, retaining function despite CoB. These functional T cells do not pose a threat to the graft at steady state because they are controlled by Tregs. However, inflammatory cytokines elicited by some infections are known to destabilize Tregs, activate APCs and upregulate graft MHC, such that these T cells that retain function may mediate graft rejection. Both projects have identified a solution to these barriers/vulnerabilities. Project 2 found that exposing donor- reactive Tmem to a semi-allogeneic pregnancy re-programs Tmem into becoming susceptible to CoB. Project 1 shows that repeated injections of donor splenocytes can induce dysfunction in a wider repertoire of alloreactive T cells, including Tmem. The molecular mechanisms underlying the acquisition of dysfunction will be investigated and compared between projects, thus underscoring the synergy of the projects. The global hypothesis of the current submission is that understanding the mechanisms by which linked sensitization and heterogeneity in alloreactive T cell dysfunction prevent tolerance induction or break established tolerance, as well as the mechanisms by which exposure to pregnancy or to repeated donor splenocyte injections overcome these barriers, will help identify critical molecular drivers of tolerance, markers of robust versus unstable tolerance, and aid in the design of new therapeutic approaches to induce durable transplantation tolerance. Project 1 addresses the mechanisms by which the duration of alloantigen expression determines the level of T cell dysfunction post- CoB (Aim 1) and tests the hypothesis that low affinity/avidity alloreactive T cells are poised to mediate rejection during infections (Aim 2). Project 2 investigates the mechanism behind linked sensitization (Aim 1), and how CD8+ (Aim 2) and CD4+ (Aim 3) alloreactive Tmem are re-programmed by pregnancy.

NIH Research Projects · FY 2026 · 2012-07

Project Summary Our long-term objectives are to understand genetic and phenotypic properties that allow the abundant human gut Bacteroidales species to survive in the host over its lifetime. In this proposal, we will build upon our work studying antibacterial toxins produced by the gut Bacteroidales that they use to antagonize other members of ecosystem. We will study important protective responses mounted by these bacteria upon exposure to antibacterial toxins and other stressors they encounter in the gut microbiota. We found that the genes of an unusual ECF-type sigma factor/antisigma factor pair, named EcfO-Reo, are induced upon exposure of Phocaeicola vulgatus to toxins and other stress-inducing compounds. The ensuing pleiotropic response protects these bacteria from various stressors that perturb the outer membrane. efcO-reo and the genes of its regulon are among the most highly expressed in vivo. The genes of this regulon include those encoding three families of proteins with unknown functions: the NigD proteins, a family of outer membrane porins, and proteins involved in the synthesis of long LPS. Our preliminary data suggest that these genes are regulated in different manners and provide distinct mechanisms of protection. In Aim 1, we will use a transcriptional reporter and phenotypic analyses to identify microbial, host and abiotic stressors that induce expression of the ecfO-reo operon and determine if induction of the response protects the bacteria from those stressors. In Aim 2, we will determine the molecular basis of long LPS (O-antigen) synthesis and the protection it confers to diverse stressors. In Aim 3, we will use genetic and phenotypic analyses combined with unbiased approaches to identify the protective functions of the NigD family of proteins. These proteins are are released in outer membrane vesicles when the EcfO regulon is induced by outer membrane perturbations. In Aim 4, we will study a family of outer membrane proteins that are highly upregulated when the EcfO-Reo operon is induced. We will use a combination of techniques to identify the unique mechanism of regulation and the contribution of these OMPs to the EcfO-Reo- induced protective response. For all aims, we will study the contribution of the different arms of the protective response to the fitness of the bacteria in the gnotobiotic mouse gut. The knowledge gained from this proposal will greatly increase our understanding of the molecular mechanisms that this order of bacteria use to survive in the human gut microbiota when exposed to various stressors of host, microbial and xenobiotic origin.

NIH Research Projects · FY 2025 · 2011-09

The CCDTR EP improves the prevention and management of diabetes and population health for all by advancing research in diabetes translation, promoting scientific exchange among Chicago-area and national investigators, and enhancing interactions between diabetes researchers and investigators from other fields with relevant expertise. Diabetes translation research encompasses a broad array of research methods from clinical trials to community-based participatory research to dissemination and implementation research to data analytics. Successful diabetes translation research also requires that investigators maintain intellectual relationships with the basic sciences and clinical investigation. In addition, successful translation requires robust interaction with clinicians and public health leaders who guide clinical care and shape policies. The enrichment program enhances all these interactions. The Specific Aims of the CCDTR EP are to: 1) Enhance and enrich the environment for diabetes translation research in Chicagoland through seminars, symposia and workshops for trainees, junior investigators, and senior investigators; 2) Create a Chicagoland network of diabetes translational investigators, and community, health care, and public health leaders who work together to improve diabetes population health.  The CCDTR EP uses existing UC and NU research and education infrastructure and complements these institutional strengths with outside resources to highlight innovative approaches to achieve both community and population health. The goals of the EP are achieved through seminars with local and guest speakers, special guest lectureships, the Annual Chicago Diabetes Day, the Annual Research Symposia of the Center for Chronic Disease Policy and Research, the Institute for Public Health and Medicine’s Population Health Forum, and regular workshops in medical decision making, health outcomes, implementation science, epidemiology, health services, and grant writing. The CCDTR EP also closely aligns with NIDDK supported T32 training programs in adult and pediatric endocrinology as well as other training programs relevant to the mission of the center such as NIDDK R25, NCATS K12, AHRQ and NHLBI T32 programs. The major benefit of the CCDTR EP is to provide diabetes researchers, ranging from trainees to senior investigators, as well as partners and stakeholders, with a forum to collaborate, innovate, and disseminate outstanding diabetes translation research. In concert with other CCDTR activities, the EP accelerates the conduct, dissemination, and implementation of translational diabetes research across Chicagoland.

NIH Research Projects · FY 2025 · 2010-12

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) latently infects one-fourth of the world’s population, causing pulmonary tuberculosis (TB) in ~10 million people and resulting in ~1.5 million deaths each year. The currently available TB vaccine, Mycobacterium bovis BCG (BCG), shows variable efficacy. In addition, Multi-Drug- Resistant (MDR) Mtb strains have recently emerged. Thus, there is a great need for new TB vaccines. Studies in the past decade have mainly utilized induction of T helper cell type 1 (Th1) responses and the production of the cytokine, Interferon-gamma (IFNγ), as readout for vaccine efficacy against TB. Our studies during the prior funding period demonstrated that Interleukin (IL)-17 and T helper type 17 (Th17) vaccine responses are critical for vaccine-induced immunity against TB. Importantly, we recently demonstrated that mucosal vaccination with the Mtb antigen with Th17-inducing adjuvants induced potent lung-resident Th17 cells and improved BCG vaccine-induced protection following Mtb challenge. Our mechanistic studies demonstrated that IL-17-induced chemokines, including CXCL-13, localize CXCR5- expressing T cells near Mtb-infected macrophages, resulting in the formation of lung lymphoid follicles and activating macrophages to mediate Mtb control. Despite these major advances in understanding the role of Th17 vaccine-induced cells in TB, our data show that upon Mtb infection, the accumulation of vaccine- induced Th17 immune responses in the lung is not accelerated enough to provide “sterilizing” immunity or complete Mtb control. In exciting new data generated during the prior funding cycle, we show that we can overcome this bottleneck by using Dendritic Cell (DC) based therapy by either activating endogenous DCs, or transfer of exogenously activated DCs into vaccinated hosts, to achieve superior Mtb control. Thus, the work proposed in this R01 renewal builds on these important and highly relevant findings with three Specific Aims: Specific Aim 1. Identifying the early cytokine pathways that modulate APC function to promote Th17 responses and induce superior immunity against TB. Specific Aim 2. Identifying the IL-23 and IL-17-dependent mechanisms that mediate early Th17 responses and Mtb control. Specific Aim 3. Identification of novel C-type lectin receptor agonists as Th17-inducing TB vaccines. The emergence of extensively drug-resistant strains (XDR) of Mtb, for which no treatments currently exist, makes the development of an effective TB vaccine incredibly urgent. The work proposed in this grant will continue to significantly impact the design and use of future vaccine strategies by allowing us to promote IL-17 responses to generate improved, long-term lasting vaccine-induced immunity against TB.

NIH Research Projects · FY 2024 · 2010-08

PROJECT SUMMARY/ABSTRACT The overall goal of the dual-track University of Chicago Paul Calabresi Career Development Award for Clinical Oncology is to increase the number of highly skilled clinicians (M.D.s, D.O,s, Pharm.D., nurses with PhD or equivalent) and non-clinician postdoctoral researchers who are capable of designing and testing innovative hypothesis-driven clinical therapeutic research protocols in clinical trial settings (pilot/Phase I, Phase II and Phase III trials). To do this, we have created a tightly structured and mentored education program within an academically rigorous training environment that prepares the most compelling senior fellows or junior faculty in clinical oncology for careers in patient oriented research. The program is anchored within our Comprehensive Cancer Center and led by Olufunmilayo Olopade, MD along with Walter Stadler, MD with strong support from an Executive Committee as well as Internal and External Advisory Committees. The 78 research training faculty preceptors have NIH or equivalent peer- reviewed funding, interact on a number of collaborative research and training efforts and are well qualified to serve as potential mentors for the five trainees per year participating in this K12 program. Each trainee is appointed for a minimum of two years. In this renewal application, we shall continue successful elements of the program in clinical pharmacology, genomics and immunotherapy while enhancing training opportunities in emerging fields of clinical informatics, data science and microbiome research. The Paul Calabresi K12 Scholars Program is our highly mentored, didactic coursework-intensive program, and “hands on” clinical research training which results in a Master of Science in Clinical Investigation. Leveraging clinical research infrastructure across University of Chicago Medicine, we have also created a flexible set of integrated interdisciplinary courses in translational science that blends entrepreneurships, cancer genomics, immunology, pharmacogenomics and community based clinical trials network. Of the 18 trainees who have completed the program since 2010, 13 (72%) are currently in academic careers, of whom 7 (54%) hold appointments at the Associate Professor level. Moreover, these 18 trainees have published a total of 176 oncology research papers, and are Principal Investigators or Co-Investigators on 56 oncology research grant awards. Of significance, 7 of 14 (50%) appointments to the program in the last funding cycle were women or members of underserved minority groups. An explicit goal of this Paul Calabresi Scholars program, as with all training programs in our institution, is that its training opportunities and benefits will extend far beyond the relatively few scholars whose stipends it will provide. The program has had a transformative and global impact and is reaching into the larger oncology trainee community in Chicago and to trainees in other countries who come to our Institution as Global Oncology Scholars. Thus, the benefit that accrues from the program's implementation and productivity is substantial and will increase the number of highly skilled clinicians and non-clinicians conducting cancer clinical trials.

NIH Research Projects · FY 2026 · 2010-02

Project Summary/Abstract Although many human cancers share similar metabolic alterations, including the Warburg effect, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. We identified phospholipase A2G7 (PLA2G7) as a “synthetic lethal” partner of Nras Q61K/R mutants in melanoma cells, which is selectively important for cell proliferation and tumor growth potential of melanoma cells expressing mutant Nras, but not in cells expressing BRAF V600E. PLA2G7 (a.k.a. platelet-activating factor acetylhydrolase (PAF-AH)) is a secreted enzyme produced by leukocytes including macrophages, T cells, and mast cells, which catalyzes the degradation of phospholipid platelet activating factor (PAF) and production of a biologically inactive phospholipid product Lyso-PAF, blocking PAF-induced inflammation and vascular permeability. Mechanistically, we found a surprising intracellular signaling function of PLA2G7. Knockdown of PLA2G7 results in decreased S338 phosphorylation of Raf-1 in cells, which is crucial for Raf-1 activation and consequently essential for mutant Nras-dependent MAPK activation, but dispensable for MAPK activation by BRAF V600E, which bypasses Raf-1. This explains the selective importance of PLA2G7 only in mutant Nras-expressing cells. Moreover, Lyso-PAF, a biological inactive form of PAF that has been suggested to be “functionless”, may contribute to p21-activated kinase 2 (PAK2)-dependent S338 phosphorylation of Raf-1, through direct binding to PAK2, likely in the catalytic cleft, leading to enhanced PAK2 kinase activity by stabilizing ATP binding. Thus, we hypothesize that “functionless” Lyso-PAF has an intracellular and signaling role that is selectively important for mutant Nras transformation by contributing to PAK2-dependent S338 phosphorylation of Raf-1, and PLA2G7 represents an alternative therapeutic target to selectively treat melanoma cells expressing mutant Nras. We have identified and validated a compound Succimer as a selective and potent PLA2G7 inhibitor. Three specific aims are proposed: (1) To determine the selective importance of the PLA2G7-Lyso-PAF axis in the proliferative and tumor growth potential of melanoma cells expressing mutant Nras, which is “bypassed” in cells expressing BRAF V600E, using diverse human melanoma cell lines and “isogenic” cell line pairs. (2) To explore the molecular and structural mechanisms by which Lyso-PAF contributes to PAK2-Raf-1 axis through directly binding to PAK2 catalytic cleft and consequently stabilizing ATP binding. (3) To evaluate PLA2G7 as an alternative target to selectively attenuate proliferative and tumor growth potential of mutant Nras-expressing melanoma cells in vitro, and in patient-derived xenograft (PDX) models of melanoma in vivo, respectively, using our newly identified PLA2G7 inhibitor, Succimer, and elucidate the underlying structural mechanism for further structure-activation relationship (SAR) studies.

NIH Research Projects · FY 2025 · 2009-09

Summary and Relevance of Proposed Research Humans have an impressive capacity to recognize the category membership of sensory stimuli. This ability, which is disrupted by a brain-based diseases and conditions such as Alzheimer’s disease, schizophrenia, stroke, and attention deficit disorder, is critical because it allows us to respond appropriately to the stimuli and events that we encounter in the environment. We are not born with an innate library of categories, such as “tables” and “chairs”, which we are preprogrammed to recognize. Instead, we learn to recognize familiar categories through experience. Our recent work has shown that both the posterior parietal cortex (PPC) and prefrontal cortex (PFC) are involved in categorical decisions. We recorded from neurons in PPC and PFC during performance of visual motion categorization tasks. These recordings revealed that neurons in both areas robustly encoded stimuli according to their learned category membership, suggesting that both regions are involved in computing and representing abstract categorical information about visual stimuli. We also showed that activity in PPC is causally related to categorical decisions, using reversible inactivation. This project uses novel brain recording techniques to monitor the activity of large neuronal populations of neurons in the lateral intraparietal area, frontal eye field, and superior colliculus during visual categorical decisions. This will allow us to gain a mechanistic understanding of how interactions between neurons in these three regions enable computations which transform visual feature encoding into categorical decisions. This work will also determine how multiple behavioral functions are mediated by this brain network, including eye movements and spatial attention, as well assess the causal significance of each brain area to categorical decisions. While much is known about how the brain processes visual features (such as color, orientation, and direction of motion), less is known about how the brain learns and represents the meaning, or category, of stimuli. A greater understanding of visual categorization is critical for addressing a number of brain diseases and conditions (e.g. stroke, Alzheimer’s disease, attention deficit disorder, schizophrenia, and stroke) that leave patients impaired in everyday tasks that require visual learning, recognition and/or evaluating and responding appropriately to sensory information. The long-term goal of this project is to guide the next generation of treatments for these brain-based diseases and disorders by helping to develop a detailed understanding of the brain mechanisms that underlie learning, memory and recognition. These studies also have relevance for understanding and addressing learning disabilities, such as attention deficit disorder and dyslexia, which affect a substantial fraction of school age children and young adults. Thus, a detailed understanding of the basic brain mechanisms of categorical decisions and attention will likely give important insights into the causes and potential treatments for disorders involving these cognitive and perceptual abilities.

NIH Research Projects · FY 2024 · 2009-05

Abstract The Summer Program to Increase Diversity in Biomedical Research and the Physician Workforce encompasses two training initiatives at the University of Chicago Pritzker School of Medicine. Both are designed to enhance the training experience of underrepresented minority and disadvantaged students who are interested in pursuing careers in medicine and research. The 8-week summer program for undergraduates is entitled “The Pritzker School of Medicine Experience in Research (PSOMER).” The 12-week Pritzker Summer Research Program (SRP) supports the participation of rising second year medical students. The goal is to provide strongly mentored summer experiences in research, as well as structured programming and mentoring that will encourage participants to continue into medicine and biomedical research careers. This grant has been ongoing at the University of Chicago for over 20 years. At the time of the initial award, the goal was to provide support for SRP participation by Pritzker medical students – a highly diverse group of students, including higher than average percentages of students who are underrepresented in medicine. Over the years, the University of Chicago has achieved great success in attracting its medical student graduates to careers in research and academic medicine, thus supporting efforts to increase the diversity of the faculty at our nation's medical schools. In 2008, the specific aims of the program were expanded to include PSOMER, developed specifically for high performing college students who were either members of groups underrepresented in medicine or were disadvantaged socio-economically. This expansion was prompted by concerns about the strength of the pipeline channeling such students into medicine and PSOMER was an effort to support and expand this pipeline. Over the last ten years, PSOMER has supported the preparation of these students for future success in medicine and biomedical research. In the current funding cycle 19 of the 27 PSOMER students who completed the program and applied (70%) have been admitted to top medical schools (two in PhD programs) with 11/19 (58%) of the admissions at the Pritzker School of Medicine. The Summer Program to Increase Diversity in Biomedical Research and the Physician Workforce has also benefited from a robust research environment at the University of Chicago, particularly in the mission areas supported by the NHLBI. Despite being a small institution, the University of Chicago has been highly successful in attracting grant support. The NHLBI currently funds over 40 research and training programs at the University of Chicago. This has given us a variety of scientists from whom to choose for student placements, with the goal of identifying as many mission-related subjects as possible. We are requesting continued support for the research training of 15 students/year. The program enjoys strong institutional support, evidenced by cost-sharing, provision of space, resources, and training by the BSD and Pritzker School of Medicine. Finally, the University of Chicago provides a robust research environment to support the goals of this NHLBI supported program.

NIH Research Projects · FY 2025 · 2008-06

Project Summary The core objective of the pre-doctoral Developmental Biology Training Program (DBTP) is to produce highly qualified, independent research scientists who are trained to take a broad interdisciplinary approach to developmental biology problems. This mission is consistent with the philosophy of the University of Chicago’s Biological Sciences Division (BSD), which seeks to avoid artificial boundaries between disciplines and encourage broad based interaction and collaboration. To produce researchers trained in a variety of areas relevant to human health and disease, the DBTP builds on both long-standing and burgeoning University of Chicago strengths in developmental biology. We have well-established strengths in the genetics of model organisms, the molecular and cellular basis of development, computation/modeling/systems level approaches in developing systems, and evolutionary developmental biology. During the ongoing third funding period, strategic new hires have enabled the DBTP to expand in areas of developmental neurobiology, stem cell biology, and biophysical approaches to development, and through affiliation with the MBL to exploit a wider array of non-traditional models. The DBTP trainers are a vibrant group of thirty-four well-funded researchers, including experienced senior faculty and talented junior faculty, based in eight BSD departments, the adjacent Chemistry department, the Pritzker School of Molecular Engineering, and at our affiliate organization the Marine Biological Laboratory (MBL). DBTP trainees are carefully selected from six interdisciplinary graduate training programs: training grant support begins as they enter their second year of studies and generally extends for two years, subject to competitive renewal. We propose to continue to support four trainees in year 1, and then increase that number to five trainees in years 2-5 to take advantage of the expanding pool of students with interests in our new joint graduate program with the MBL. This number of trainees will allow us to be highly selective, while maintaining a critical cohort size. Trainees benefit from a strategically designed curriculum that includes access to six dedicated formal courses, a unique new lab-based Embryology course at the MBL, a requirement for quantitative/computational training, and an extensive range of supplemental training-related activities. Among these activities are the DBTP sponsored developmental biology seminar series and data club (associated with our required communications course), an annual retreat, and student-run DBTP-sponsored symposia. Our training plan ensures students develop broad transferable skills—including communication, networking, teaching, computation, and rigorous critical analysis—key to their success in the biomedical research workforce. In summary, the DBTP integrates a range of training approaches to prepare future leaders in developmental biology research and education. The success of the DBTP is demonstrated by our funded tenure-track alumni.

NIH Research Projects · FY 2025 · 2008-05

PROJECT SUMMARY/ABSTRACT Given the aging of the population and concerns over the physician-scientist workforce, it is imperative to inculcate the next generation of physicians with the desire to understand and improve care for older adults through pursuit of scientific discovery in the basic, clinical, and social sciences. The purpose of this application is to renew support for the successful Short-Term Aging-related Research (STAR) T35 program which has provided summer training in basic, clinical, and social science research for medical students at the Pritzker School of Medicine to inspire and prepare them to pursue careers in aging research for the past 15 years. The STAR T35 program supports a culture of scientific inquiry throughout the school, and inspires its trainees to pursue additional years for research significantly more often than their peers. The proposed program will continue to be led by Dr. David Meltzer, a physician-economist who is a member of the National Academy of Medicine and an NIA-funded investigator, including PI of an NIA funded P30 Center on Healthy Aging Behaviors and Longitudinal Investigations (CHABLIS), and Dr. Vineet Arora, Dean for Medical Education at Pritzker and an NIH funded researcher who studies sleep in older persons. They will be joined by two accomplished assistant directors, including Dr. Stacie Levine, Section Chief of Geriatrics and Palliative Care, and Dr. Sangram Sisodia, an NIA-funded basic science researcher in Alzheimer’s disease. Building on the established processes of the successful long-running Pritzker Summer Research Program (SRP) and the Scholarship and Discovery (S&D) 4-year research infrastructure, we request funding to appoint ten T35 sponsored students each year with an additional ten supported by Pritzker. A cadre of nationally prominent NIH-funded principal investigators in basic, clinical, and social science relevant to aging who together have more than $23 million in funding will mentor individual trainees. In addition, trainees will take part in didactic instruction in responsible conduct of research, statistics, clinical geriatrics and an innovative course called Scholars in Translational Aging Research Training (START) Program which will expose them to concepts in biology, clinical geriatrics, and the social sciences that are central to the study of aging. For nearly 15 years, the START course has consistently increased interest in aging research careers among participants (both NIA T35 funded and other). Through rigorous evaluation and long-term tracking, we will be able to determine if participants show a greater propensity to continue pursuing research as a career. This program supports the imperative to inculcate the next generation of physicians with the desire to understand and improve care for older adults through pursuit of scientific discovery.

NIH Research Projects · FY 2025 · 2007-12

Plasmacytoid dendritic cells (pDCs) rapidly secrete type I interferon (interferon α/β, IFN) in response to virus- derived nucleic acids, facilitating both innate and adaptive antiviral responses. Conversely, aberrant IFN production by pDCs is associated with autoimmune diseases, establishing pDCs as important emerging therapeutic targets. The overall goal of this project is to elucidate the molecular control of pDCs lineage, including its development, homeostasis and function in various immune responses. In the previous award cycles, we have identified E protein transcription factor TCF4 (E2-2) and several other factors as important regulators of pDC development. The proposed work will build upon these findings to address major unanswered questions in pDC biology. In Aim 1, we will use high-dimensional single-cell analysis methods to characterize the stage and regulation of pDC lineage specification, as well as the functional heterogeneity of mature pDCs. In Aim 2, we will use genetic approaches to dissect transcriptional control and regulation of the unique IFN-producing capacity of pDCs. In Aim 3, we will explore the mechanism of virus recognition and IFN production by pDCs in vivo. Collectively, the proposed studies should yield a comprehensive molecular view of pDC development and function, paving the way for therapeutic approaches focused on this cell type.

NIH Research Projects · FY 2025 · 2007-01

ABSTRACT Bacillus anthracis, the anthrax agent, is a member of the Bacillus cereus sensu lato group, which includes invasive pathogens of mammals or insects as well as nonpathogenic environmental strains. Clade 1 members of the B. cereus s.l. group can exchange virulence-plasmids via horizontal transfer with B. anthracis to cause anthrax-like disease too. The natural route of infection is ingestion, often by grazing animals or, in humans, by the consumption of spore-contaminated food (gastrointestinal, GI anthrax). The proposal aims to determine the mechanisms of B. anthracis spore invasion across intestinal epithelia. The central hypothesis is that spores of B. anthracis and anthrax-causing strains germinate in the intestinal tract of infected animals. Our working model postulates that vegetative bacilli secrete several enzymes with mucin-binding and mucin-degrading activities to penetrate the thick mucin layer. Next, the adhesins BslA and BslB mediate uptake of B. anthracis into intestinal cells. BslA and BslB are two of 22 S-layer associated proteins (BSLs) that form the surface (S)- protein layer. BSLs associate via S-layer homology (SLH)-domains with secondary cell wall polysaccharide (SCWP), a peptidoglycan linked carbohydrate polymer. The integrity of the S-layer and SCWP is important for the activity of BSLs. Further, the bslA gene is located on the mobilizable pXO1 virulence plasmid. Thus, we propose that surface-protein layers contribute broadly to the pathogenesis of anthrax-causing organisms. Here, we will use mice and intestinal organoids to model GI anthrax and test our predictions. We propose to study the pathway for the synthesis of SCWP that is required to display the surface-protein layer and is essential for growth. We expect that the elucidation of the assembly pathway of SCWP will serve as a paradigm for the study of other complex polymers of Gram-positive bacteria and reveal new targets of antibacterial drugs. We anticipate that unravelling intestinal mucosa interactions with anthrax and anthrax-like pathogens will collectively advance the field of infectious diseases research.

NIH Research Projects · FY 2024 · 2005-09

PROJECT SUMMARY Celiac disease (CeD), a complex T cell-mediated enteropathy induced by dietary gluten in HLA-DQ2 or HLA- DQ8 individuals, currently affects 1% of the global population. While CD4 T cells are required for the development of villus atrophy (VA), the effector cells mediating intestinal epithelial cell destruction (IEC) are intraepithelial cytotoxic lymphocytes (IE-CTLS). Currently, the only effective CeD treatment is a lifelong gluten- free diet (GFD). However, 30-40% of adult CeD patients fail to restore a completely normal intestinal morphology on a GFD, and complete avoidance of gluten can be challenging. During the last funding period, we generated the first HLA and gluten-dependent mouse model of CeD (CeD-tg) that recapitulates the intricacies of CeD pathogenesis. We will take advantage of the CeD-tg mouse model and our expertise in human immunology, to (i) further dissect the mechanisms underlying tissue destruction, and (ii) profile the clinical spectrum of CeD using high dimensional single cell technologies with the goal of identifying new therapeutic avenues and biomarkers predicting tissue destruction. The central hypothesis emerging from our studies is that IE-CTL integrate signals from CD4 T cells and IEC to become licensed killer cells and mediate tissue destruction. Furthermore, while it is acknowledged that IEC play a role in IE-CTL activation, their role in CeD pathogenesis and how they impact on IE-CTL activation has not been investigated. The proposed specific aims are:1) Establish the role of epithelial cells in T cell-mediated CeD immunopathology using the CeD Tg mouse model; 2) Establish the Impact of γδ and CD4 T cells, IFNγ, IL-15 on IE-CTL activation in CeD-tg mice; and 3) Profile the heterogeneity of potential and active CeD. The goal is to provide a knowledge base that will help identify appropriate treatment strategies to individual patients, and help predict which potential CeD patients (patients who have develop inflammatory anti-gluten immunity but conserve a normal intestinal architecture) are at high risk of developing VA. The studies proposed will have a significant positive impact on human health because they will help define curative and preventive strategies that have the potential to prevent tissue destruction in celiac disease.