LMP student seminars: 8 October
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
Mary-Jo Hatem
- Title: Investigating the effects of a smooth muscle cell-specific DDR1 knockout on atherosclerosis development
- Supervisor: Dr. Michelle Bendeck
Jasmeen Kaur Monga
- Title: B cells are involved in pathogenesis of angiotensin-II induced hypertension in mice
- Supervisor: Dr. Mansoor Husain
Group 5: Infectious Diseases, Inflammation and Immunology
Location: MSB 4279
Philip Sherif Samaan
- Title: Neuropsychiatric Long-COVID Patients Exhibit Elevated SARS-CoV-2-Specific GzmB and TNFα T-cell Responses with Diminished EBV Anti-VCA IgG Antibody Levels at 3-6 Months Post-Diagnosis
- Supervisor: Dr. Mario Ostrowski
Abstracts
Mary-Jo Hatem: Investigating the effects of a smooth muscle cell-specific DDR1 knockout on atherosclerosis development
Atherosclerosis is the stiffening and narrowing of major blood vessels as a result of high levels of low-density lipoproteins. The molecular mechanism of disease development has yet to be fully characterized due to the variety of contributing factors and cell types. One factor that has been determined to influence plaque development and calcification in atherosclerosis is a receptor tyrosine kinase and mechanosensor known as Discoidin Domain Receptor 1 (DDR1). DDR1 binds to collagen in the extracellular matrix, responds to stiffness changes and triggers downstream signaling. The global knockout of Ddr1 in atherosclerotic mice affects matrix protein accumulation, cell composition, and size of the plaques. However, with the global knockout affecting a variety of cell types and processes, like inflammation, it is difficult to understand the extent to which DDR1 in vSMCs influences disease progression.
Therefore, we have developed a SMC-specific knockout of Ddr1 using a Myh11-CreERT2 mouse model with accelerated atherosclerosis. We hypothesize that a SMC-specific knockout of DDR1 will change plaque size and composition. Using ultrasound imaging, western blotting, and immunohistochemistry, we have seen significant differences in plaque progression in SMC-specific knockout mice. SMC-specific DDR1 knockout suppresses vessel narrowing, increases collagen levels, decreases elastin levels, and alters cell composition within advanced plaques. Further research will be done to investigate differences in early-stage plaque development.
Additionally, vSMCs cultured from our mice models have shown differing levels of proteins such as β-catenin and uPAR as well as matrix metalloproteinases indicating potential pathways associated with results seen in vivo. Determining the changes in plaque stability and size within our SMC-specific DDR1 KO mice can lead us to better understand DDR1’s role in atherosclerosis. We can use these findings to determine mechanisms to prevent plaque rupture, decrease plaque size, as well as prevent advance plaque development.
Jasmeen Kaur Monga: B cells are involved in pathogenesis of angiotensin-II induced hypertension in mice
Background: Hypertension [HTN] is a leading modifiable risk factor for cardiovascular disease [CVD], affecting nearly 30% of adults globally1. Despite advances in treatment, resistant HTN – uncontrolled blood pressure [BP] despite using three antihypertensive medications – remains a significant challenge2. While traditional HTN management focuses on renin-angiotensin-aldosterone system modulation, emerging evidence suggests novel pathways involving immune regulation in HTN pathogenesis3. B cells, the sole cells of the body that secrete immunoglobulins, have been implicated in HTN susceptibility with both IgG and IgA – two major classes of immunoglobulins – positively correlated with BP in various human studies4,5.While IgG has been extensively studied in HTN development for its known pro-hypertensive role, IgA represents an emerging area of interest due to its potential pro-inflammatory involvement in gut dysbiosis, kidney damage, and neuroinflammation – all of which are associated with HTN6,7. This highlights a critical gap in understanding the combined role of B cells and their IgA production in angiotensin II [Ang II] induced HTN.
Aim 1: Investigate the causal role of B cells in experimental hypertension and characterizing how different B cell subsets contribute to experimental hypertension.
Aim 2: Elucidate the mechanisms by which B cells influence the development and progression of hypertension.
Methods & Results: We have used B-cell specific [CD19-Cre-mediated] expression of diphtheria-toxin [DT] receptor [ROSA26i-DTR] and showed through protocol optimization that low-dose (25ng/g body weight) DT once weekly effectively depletes all B cell subsets, including plasma cells, by 99%, a level not achievable with other anti-CD20 depletion methods. Using this model in experimental HTN, we also show that chronic B cell depletion via weekly DT injections for 4-wk led to blunted Ang-II-induced HTN (500ng/kg/min of Ang-II for 28-d) in CD19Cre/R26iDTR mice.
To investigate B-cell dynamics in during Ang-II-induced HTN, wild-type [WT] C57BL/6J male mice aged 10-12 wk were implanted with osmotic minipumps delivering Ang-II (500 ng/kg/min x 28-d) or vs. saline for underwent FC of all B-cell subsets in aorta, spleen, and bone marrow. CD19+ B cells in aorta increased 3.6-fold with Ang-II vs. saline (5.4±2.4E3 vs. 1.5±0.8E3 cells/aorta; N=5/group; p<0.01). In spleen, CD138+ plasma cells increased 2.3-fold with Ang-II vs. saline (7.6±3.8E5 vs 3.2±1.4E5 cells/spleen; N=5/group; p<0.001). By contrast, plasma cells were decreased 3-fold in bone marrow after Ang-II vs. saline (0.7±0.7E6 vs. 2.06±3.8E6 cells/bone marrow; N=5/group; p<0.05). Flow cytometric analysis of IgA+ plasma cells [PCs] in the small intestine lamina propria [SILP] and mesenteric lymph nodes [MLNs] revealed a 3.9-fold increase in IgA+ PCs in the SILP compared to saline controls (3.5±1.5E5 vs. 8.8±0.6E4 cells; N=5–6/group; p<0.05) and 2.6-fold decrease in PCs in the MLNs with Ang II treatment (2.2±0.2E4 vs. 5.9±0.2E4 cells; N=4-5/group; p<0.05), suggesting possible trafficking of IgA+ PCs from MLNs to the SILP during Ang II-induced HTN.
To investigate the role of IgA in HTN, IgA-/- and C57Bl6/J mice aged 10-12 wk were implanted with osmotic minipumps delivering Ang-II (500 ng/kg/min x 28-d) or vs. saline. We showed that IgA deficiency attenuates Ang II-induced HTN in mice, as evidenced by blunted BP responses in IgA-/- mice vs. C57Bl6/J controls. Co-housing experiments, where passive IgA transfer through coprophagy restored hypertensive responses in IgA-/- mice, solidified our hypothesis that IgA contributes to HTN pathogenesis. IgA-/- mice were protected even when offspring were separated by genotype post-weaning: crossing IgA-/- dams with IgA+/- sires, removing passive IgA transfer. Together, these data solidify IgA’s contribution to HTN pathogenesis and suggest its effects are mediated through mechanisms beyond mucosal immunity, potentially involving systemic inflammation.
Conclusion: These findings implicate a role of B cells and IgA in development of HTN, emphasizing the need for further elucidation of the exact mechanisms involved. Modulating B-cell numbers emerges as a novel therapeutic target for hypertension.
Philip Sherif Samaan: Neuropsychiatric Long-COVID Patients Exhibit Elevated SARS-CoV-2-Specific GzmB and TNFα T-cell Responses with Diminished EBV Anti-VCA IgG Antibody Levels at 3-6 Months Post-Diagnosis
Background: Infection with SARS-CoV-2 can lead to long-COVID, a chronic multisystemic condition estimated to affect over 70 million people worldwide. Although underlying mechanisms remain elusive, aberrant ongoing inflammation due to Epstein-Barr virus (EBV) reactivation and persistent SARS-CoV-2 viral reservoirs has been hypothesized. Here, cellular and humoral immune responses to SARS-CoV-2 and EBV were compared between neuropsychiatric long-COVID patients and recovered controls.
Methods: PBMC and serum were collected from 30 long-COVID patients and 27 matched controls at 3-6 months post-diagnosis (PD). FluoroSpot assays quantified IFN-γ, IL-2, TNF⍺, and granzyme B (GzmB) T-cell responses to SARS-CoV-2, EBV, and human cytomegalovirus (HCMV). Live viral neutralization assays assessed serological neutralizing capacity against SARS-CoV-2 and EBV. ELISA assays measured levels of anti-Early Antigen-Diffuse (EA-D) IgG, anti-viral capsid antigen (VCA) IgG, and anti-HCMV IgG to confirm EBV reactivation and seropositivity for EBV and HCMV, respectively.
Results: Neuropsychiatric long-COVID patients mounted significantly greater GzmB T-cell responses against SARS-CoV-spike and secreted significantly greater quantities of TNFα per T-cell against nucleoprotein. Long-COVID patients did not differ from controls in their capacity to neutralize SARS-CoV-2 or EBV at 3-6 months PD and EBV reactivation was detected in only one long-COVID patient. However, reduced EBV neutralization at 3-6 months correlated with having 2 or more neuropsychiatric symptoms at 12 months PD. Anti-VCA IgG levels also strongly correlated with EBV neutralization and were significantly diminished in long-COVID patients.
Conclusion: These findings overall indicate elevated SARS-CoV-2-specifc cytotoxic and pro-inflammatory T-cell immunity with diminished EBV-specific humoral immunity in neuropsychiatric long-COVID patients at 3-6 months PD.
Contact
No need to register.
Contact lmp.grad@utoronto.ca with any questions.