LMP student seminars: 14 January
Each week during term time, MSc and PhD candidates in the Department of Laboratory Medicine and Pathobiology present their research.
Anyone is welcome. No need to register.
Location: Medical Sciences Building, rooms 4171 or 4279, see below.
As part of the core research curriculum, students taking LMP1001/2/3: Graduate Seminars in Laboratory Medicine and Pathobiology will present their projects. Please see abstracts below.
Group 3: Cardiovascular, Physiology and Metabolism
Location: MSB 4171
Isabel Nie Xin Lim
- Title: Modeling human macrophage biology in the cardiovascular system using a humanized mouse model
- Supervisor: Dr. Clinton Robbins
Mavis Lee
- Title: Optimization of Cervical Extravillous Trophoblast Isolation and Secretome Analysis from First Trimester Pregnancies
- Supervisor: Dr. Sascha Drewlo
Group 5: Infectious Diseases, Inflammation and Immunology
Location: MSB 4279
Yuying Zhang
- Title: Human CD44 transgenic mice demonstrate Fc-dependent protection by anti-human CD44 antibodies in experimental immune thrombocytopenia
- Supervisor: Dr. Alan Lazarus
Madison Denney
- Title: Examining the impact of the gut microbiome on the tumour immune response
- Supervisor: Dr. Dana Philpott
Abstracts
Isabel Nie Xin Lim: Modeling human macrophage biology in the cardiovascular system using a humanized mouse model
Atherosclerosis is the leading risk factor for cardiovascular disease including heart attack and stroke. This disease is driven by macrophages that uptake cholesterol and accumulate into fatty plaques within arterial walls. In healthy arteries, monocytes, which are the precursors to macrophages are rare, however, hypercholesteremia triggers an inflammatory recruitment of monocytes that differentiate into plaque-forming macrophages. Over time, excessive immune cell accumulation at the lesion site narrows arteries, restricts blood flow, and increases the risk of heart attacks and strokes. Murine models such as ApoE-/- and LDLR-/- have been instrumental in defining the role of immune cells in atherosclerosis, yet there are fundamental differences between the human and murine immune systems, which limits translational relevance. A key limitation is the lack of models to study human macrophages in vivo during atherosclerosis development. This project aims to establish a human-mouse xenograft model of atherosclerosis using the MISTRG6 humanized mouse, which supports robust engraftment of human myeloid cells. We hypothesize that human macrophage populations will be faithfully recapitulated in the murine aorta and contribute to atherosclerotic plaque formation under hypercholesterolemic conditions. In addition, emerging murine studies suggest that high cholesterol diet reprograms bone marrow hematopoietic stem cells to bias towards pro-atherogenic macrophages. However, it is unknown if this phenomenon occurs in human. By directly interrogating human stem cell and macrophage responses in vivo, my thesis aims to provide the first mechanistic insight into how hypercholesteremia shapes human hematopoiesis and immune cell driven atherosclerosis. Ultimately, these findings may reveal novel therapeutic targets beyond cholesterol lowering.
Mavis Lee: Optimization of Cervical Extravillous Trophoblast Isolation and Secretome Analysis from First Trimester Pregnancies
Background: The complex process of placental development begins around 7 days post-fertilization. By the end of the 1st trimester, the placenta assumes its critical role in nutrient and gas exchange for the fetus. However, early stages of placentation pose challenges for sample accessibility, delaying studies on placental disease. Consequently, placenta-mediated complications often go undetected until symptoms become clinically apparent, limiting opportunities for effective intervention. The trophoblast retrieval and isolation from the cervix (TRIC) method was established to collect cervical extravillous trophoblasts (cEVTs) as early as 5 weeks gestation (GA) for genetic and proteomic analysis. To further evaluate TRIC as an approach for early assessment of trophoblast molecular biology, and therefore, placental health, it was applied to samples from a high-risk pre-term birth clinic. In parallel, the cervical fluid secretome was investigated as a novel cell-independent window into placental biology.
Methods: Cervical samples were collected from patients ≤20 weeks GA in low-risk (LR) early anatomy (n = 173; cytobrush), high-risk (HR) pre-term birth (n = 68; cytobrush, broom & hydraflock), and termination clinics (n = TBD; cytobrush). Immunocytochemistry (ICC) staining against EVT marker, HLA-G, was performed on 19 clinical samples (HR = 16, LR = 3), 4 HR samples spiked with HLA-G overexpressing (OE) cell lines, and EVTs extracted from 1st trimester placental explants. Immunomagnetic purification techniques were validated with ICC staining and flow cytometry using HLA-G OE cells. Enzyme-Linked Immunosorbent Assay (ELISA) for placental growth factor (PlGF, n = 40), epidermal growth factor (EGF, n = 40), human chorionic gonadotropin (hCG, n = 40) and galectin 14 (LGALS14, n = 80) were performed.
Results: Placental explant-derived EVTs and HLA-G OE cells stained positively for HLA-G. The predominant cell types observed in clinical samples were ectocervical squamous epithelial cells (intermediate and superficial), with few endocervical columnar cells and many polymorphonuclear leukocytes. HLA-G positive EVT cells were rarely detected with a maximum ratio of 1 HLA-G positive cell per 100,000 cervical cells. Immunomagnetic beads adhered to HLA-G positive cell surfaces on slides and in suspension. ELISA revealed a qualitative correlation between sample turbidity and protein concentration. Additionally, LGALS14 and hCG concentrations were more consistently quantifiable compared to other markers assessed.
Conclusion: As cEVTs are posited to localize in the endocervical mucus plug, the observed bias of clinical sampling towards ectocervical cells may limit TRIC recovery of HLA-G-positive EVTs in this cohort. Importantly, cervical fluid secretome profiling is feasible and provides a practical, cell-independent method to assess early placental biology.
Yuying Zhang: Human CD44 transgenic mice demonstrate Fc-dependent protection by anti-human CD44 antibodies in experimental immune thrombocytopenia
Monoclonal IgG antibodies targeting CD44 show potent anti-inflammatory activity in several murine autoimmune models. In mouse immune thrombocytopenia (ITP), anti-CD44 antibodies mirror the therapeutic effects of intravenous immunoglobulin (IVIg) at one-thousandth the dose, identifying CD44 as a possible target for IVIg-replacement strategies. The protective activity of anti-CD44 in ITP has been linked to effects on macrophage Fc gamma receptor (FcγR)-driven clearance of antibody-opsonized platelets. Whether antibodies to human CD44 share these properties has not been determined. We generated mice expressing human CD44 (huCD44 mice) and tested the human-specific CD44 antibody BJ18. BJ18 protected both heterozygous and homozygous huCD44 mice from thrombocytopenia in vivo. Deglycosylated BJ18, which has impaired FcγR engagement, failed to protect, indicating that therapeutic activity requires an intact IgG Fc region. Myeloid-specific reduction of human CD44 using LysM-Cre lowered BJ18 activity and correlated with reduced human CD44 expression in monocytes and macrophages. The extent of protection tracked with the level of CD44 expression on myeloid cells. Together, these findings show that anti-human CD44 antibodies require a functional Fc region for activity in vivo and support a model in which macrophage FcγR pathways contribute to protection. This work introduces a human CD44 mouse model for functional testing of anti-human CD44 antibodies and provides a framework for developing antibody-based IVIg-replacement therapies in autoimmune disease.
Madison Denney: Examining the impact of the gut microbiome on the tumour immune response
The development and progression of colorectal cancer (CRC) has long been shown to be affected by dysbiosis of the gut microbiome. While previous research has focused on specific bacterial species, there has been less investigation into whole community effects. Utilizing human-derived community (HDC) mice, a gnotobiotic mouse model with a minimal microbiome of 8 human-sourced bacteria species, we investigated how the immune response to tumours differed compared to mice with a specific pathogen-free (SPF) microbiome. The HDC mice have normally developing immune systems with similar intestinal characteristics to mice with an SPF microbiome. However, the HDC mice have increased tumour weight and volume over time compared to SPF or germ-free mice when injected subcutaneously with MC-38 tumour cells sourced from mouse CRC. The HDC mice also have differential immune cell infiltration in the tumours, with fewer T cells and dendritic cells at 15 days post-injection, measured by flow cytometry. Using HEKBlue and luciferase reporter assays, we demonstrate higher bioavailabilities of ligands for key pattern recognition receptors (PRRs) TLR4 and TLR2, as well as a decreased bioavailability of ligands for TLR5 within the digestive tract. Additionally, metabolomic analysis revealed that the HDC mice exhibited a significant loss of bacterial products, including inosine and indole, while also showing increased production of putrescine. These metabolites and ligands are crucial for the activation of host immune cells, suggesting that these differences may contribute to the reduced efficacy of the HDC tumour immune response. Future work will focus on how this microbiome affects the response to anti-PD1 immunotherapy.
Contact
No need to register.
Contact lmp.grad@utoronto.ca with any questions.