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Round 9: 2020-2021 Pilot Projects

Engineering T cells to target Klebsiella pneumoniae infections

Klebsiella pneumoniae infections cause millions of deaths worldwide each year, with a particular impact on immunocompromised patients such as those with diabetes or undergoing organ transplant. Due to increases in antibiotic resistance, therapeutic options toward K. pneumoniae are limited, underscoring the need for alternative treatment strategies. The objective of this work is to design an immunotherapeutic approach for clearing K. pneumoniae infection through genetically engineered T cells. We propose engineering T cells to express T cell receptors specific to K. pneumoniae antigens will provide targeted cellular therapy for these infections. To address this hypothesis, this work will 1) design and characterize engineered T cells expressing artificial TCRs that specifically recognize K. pneumoniae and 2) demonstrate efficacy of polyclonal and engineered T cells in an in vivo K. pneumoniae infection model. Overall,

Accomplishing these claims will establish an adaptable technique for development of therapies toward a multitude of bacterial pathogens.

Developing a bile acid biomarker to predict the efficacy of fecal microbiota transplantation in overt hepatic encephalopathy

Between 2013-2020, Ochsner Health System (“OHS”) diagnosed 18,321 patients with cirrhosis of the liver, and over 1 in 5 of those patients went on to develop hepatic encephalopathy (HE), a condition which causes impaired neurotransmission and inflammation that leads to cognitive impairment. Fecal microbiota transplantation (FMT) improves cognition in HE by reducing inflammation and increasing conjugation of primary BAs into secondary BAs [1-3]. At Ochsner, we are conducting an ongoing phase 2 clinical trial (NCT04155099) of an FMT oral formulation for patients with HE and banking serum samples at baseline, 1, 3, and 6 months. The proposed study will use banked samples to examine whether bile acids (BAs) can successfully predict recovery

following FMT. We hypothesize that in patients with HE, specific BAs or a BA signature (1) change in a dose-dependent manner following FMT, (2) can predict efficacy of FMT treatment, and (3) co-vary with inflammatory markers.

The role of macrophages colonization by Neisseria gonorrhoeae (N.g) during gonorrhea development

We propose to investigate the role of macrophages colonization by Neisseria gonorrhoeae (N.g) during infection and the impact on gonorrhea development. Rising incidence and increasing antibiotics resistance highlights the need to understand the molecular basis of N.g pathogenesis and the need for new therapeutic targets. Louisiana has the third highest rate of gonorrhea infection in the country, second only to Mississippi and Alaska. Because N.g is a highly human adapted pathogen, it has developed numerous mechanisms to counteract the immune responses. Macrophages are present in significant numbers throughout the genitourinary mucosae and represent 10% of total number of leukocytes recovered from these tissues. Thus, it’s likely that N.g encounters these cells during infection and that macrophages play an important role in N.g pathogenesis. Here, we will assess the presence and the function of macrophages in human cervico-vaginal lavage samples and test four repurposed drugs as novel therapy for gonorrhea development.

Epigenetic analysis of regulation of the inflammosome-activating NLRP3 gene in monocytes from atrial fibrillation patients and controls

The enhanced inflammatory response driving atrial fibrillation (AF) is associated with priming and activation of the NLRP3 inflammasome, which involves increased synthesis of the sensor protein NLRP3. Elderly women are most prone to AF. Our recent bioinformatic analysis indicates that the highly preferential expression of NLRP3 in leukocytes, and especially monocytes, is associated with a previously unreported formation of a super-enhancer. We found monocyte- specific DNA hypomethylation in two super-enhancer subregions. DNA hypomethylation can be a quantifiable marker of gene expression and can increase with age. We will examine the association of this DNA hypomethylation with NLRP3 expression in leukocyte subtypes and test its association in monocytes with age and AF status in females. DNA methylation and gene expression analysis will be performed by the newly developed Long-Range Enzymatic Methyl-seq and by qRT-PCR, respecti al for upregulation of NLRP3 in AF may offer new potential drug targets.

Assessing the Role of Transferable Exosomal PD-L1 in Renal Cell Carcinoma

Patients with metastatic renal cell carcinoma have dismal long-term survival, with only 15% of patients alive five years after diagnosis. Immuno-oncologic agents, which activate the patients' own immune system to fight the tumor, have provided durable long-term remissions in in a subset of patients with metastatic renal cell carcinoma, however identifying those patients most likely to benefit from these therapies remains imprecise as there are no biomarkers for response to therapy. Mechanisms of patient resistance to this therapy are poorly understood.

Exosomes are microvesicles derived from cancer cells which transfer both proteins and nucleic acid to other cells. It has been shown they can transfer resistance to cancer therapy between cells, allowing for disease progression. It is not known whether they can transfer agents which are used by tumors to hide from immunologic targeted therapy in kidney cancer. This proposal will use exosomes from patients with metastatic renal cell carcinoma undergoing immuno-onoclogic therapy to determine a specific mechanism for therapy resistance. In addition to determining the in vivo mechanism for therapeutic resistance, we will determine in the laboratory how these exosomes are transmitted between cells to create resistance to immunologic cancer therapy. These insights will broaden our understanding of how exosomes renal cell carcinoma to evade treatment so that therapeutic combinations can overcome this problem in the future.

The role of BID in NAFLD-related metabolic changes of hepatic macrophages

Non-alcoholic fatty liver disease (NAFLD) is intimately associated with the clinical features of me abolic syndrome. However, approved pharmacological therapy for NAFLD is still absent. Our unpublished study found that genetic deletion of BH3 interacting-domain death agonist (BID) can protect mice against diet-induced obesity and hepatic steatosis, which is intriguingly related to gut microbiota. Macrophages play essential roles in the progression of NAFLD. Our central HYPOTHESIS is that diet-induced gut dysbiosis changes hepatic metabolites which promotes BID- dependent metabolic signatures in hepatic macrophages, and thereby leading to a disrupted innate immune response and NAFLD. Successful completion of this study will identify a novel mechanism of BID in macrophage metabolism in the progression of NAFLD. We anticipate that this study will lead to innovative therapeutic approaches by targeting BID and/or through giving nutrient supplements in patients with NAFLD.

Community opinion of technology supported dietary pattern interventions (CoopTech)

Modern diet and disrupted chronobiology are independently linked to cardiometabolic dysfunction. We posit that certain dietary interventions, like intermittent fasting (IF), are proxies for enhanced eating regularity via manipulating inputs including meal timing, frequency, and quantity. Our work in IF demonstrates improvements in cardiometabolic health. This coupled with chronobiological literature and existing observations of eating regularity point to the importance of regular dietary patterns. The largest barrier to continued research is the lack of a singular method to quantify eating regularity. As part of our objective, we will develop an eating regularity index (E-REG) using near real-time dietary intake assessment via remote food photography, continuous glucose monitoring, and wrist actigraphy across ?-days of observation. Additionally, eating regularity will be used to cross-sectionally compare participants and qualitative data will ascertain their perceptions of eating regularity, acceptability of technology-supported assessment techniques, and willingness to engage in future interventions.