Jimmy D. Dikeakos's research while affiliated with The University of Western Ontario and other places
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Publications (90)
Background: Despite widespread study of dendritic cell (DC)-based cancer immunotherapies, the in vivo post-injection fate of DC remains largely unknown. Due in part to a lack of quantifiable imaging modalities, this is troubling as the amount of DC migration to secondary lymphoid organs correlates with therapeutic efficacy. Preliminary studies have...
The Severe Acute Respiratory Syndrome Coronavirus 2 (CoV-2) pandemic has affected millions globally. A significant complication of CoV-2 infection is secondary bacterial co-infection, as seen in approximately 25% of severe cases. The most common organism isolated during co-infection is Staphylococcus aureus. Here, we describe the development of an...
[This corrects the article DOI: 10.1371/journal.ppat.1010092.].
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) binds to angiotensin-converting enzyme 2 (ACE2) to mediate membrane fusion via two distinct pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In this study, we found that SARS-CoV-2 S has a wider protease...
Phosphofurin acidic cluster sorting protein 2 (PACS-2) is a multifunctional cytosolic membrane trafficking protein with distinct roles in maintaining cellular homeostasis. Recent clinical reports have described 28 individuals possessing a de novo PACS-2 E209K mutation that present with epileptic seizures and cerebellar dysgenesis. As the PACS-2 E20...
The emergence and spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) and the associated Coronavirus disease (COVID-19) pandemic have affected millions globally. Like other respiratory viruses, a significant complication of COVID-19 infection is secondary bacterial co-infection, which is seen in approximately 25% of severe cases....
Purpose:
The purpose of this study was to evaluate magnetic particle imaging (MPI) as a method for the in vivo tracking of dendritic cells (DC). DC are used in cancer immunotherapy and must migrate from the site of implantation to lymph nodes to be effective. The magnitude of the ensuing T cell response is proportional to the number of lymph node-...
Examining protein-protein interactions provides critical insight into numerous human diseases and infections. Here we describe a protocol for bimolecular fluorescence complementation, which can be used to directly visualize and characterize intracellular protein-protein interactions and ascertain their localization using fluorescence microscopy.
To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent path...
Antiretroviral therapy can control human immunodeficiency virus type 1 (HIV-1) replication in people living with HIV; however, these treatments are not curative and no practical approach for an HIV-1 cure has yet shown success in clinical trials. Counteracting the multiple barriers HIV-1 presents against a practical cure is a direct means to functi...
The development of safe and effective vaccines to prevent SARS-CoV-2 infections remains an urgent priority worldwide. We have used a recombinant vesicular stomatitis virus (rVSV)-based prime-boost immunization strategy to develop an effective COVID-19 vaccine candidate. We have constructed VSV genomes carrying exogenous genes resulting in the produ...
Phosphofurin acidic cluster sorting protein 1 (PACS‐1) is canonically a cytosolic trafficking protein, yet recent reports have described nuclear roles for PACS‐1. Herein, we sought to define the nuclear transport mechanism of PACS‐1. We demonstrate that PACS‐1 nucleocytoplasmic trafficking is dependent on its interaction with the nuclear transport...
Immunotherapies, such as dendritic cell- (DC-)based therapies, are useful for treating cancer as an alternative to or in combination with traditional therapies. Cells must migrate to lymphoid organs to be effective and the magnitude of the ensuing T cell response is proportional to the number of lymph node-migrated DC. With less than 10% of cells e...
Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real time imagi...
Prolonged immune activation drives the upregulation of multiple checkpoint receptors on the surface of virus-specific T cells, inducing their exhaustion. Reversing HIV-1-induced T cell exhaustion is imperative for efficient virus clearance; however, viral mediators of checkpoint receptor upregulation remain largely unknown. The enrichment of checkp...
Serine Incorporator 5 (SERINC5) reduces the infectivity of progeny HIV-1 virions by incorporating into the outer host-derived viral membrane during egress. To counter SERINC5, the HIV-1 accessory protein Nef triggers SERINC5 internalization by engaging the Adaptor Protein 2 (AP-2) complex using the [D/E]xxxL[L/I] 167 Nef dileucine motif. Nef also e...
Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We visualized seque...
Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We visualized seque...
Graphical Abstract Highlights d IgM has a major role in the capacity of convalescent plasma to neutralize SARS-CoV-2 d IgM represents only about 5% of total antibodies in plasma d
Characterization of the humoral response to SARS-CoV-2, the etiological agent of COVID-19, is essential to help control the infection. The neutralization activity of plasma from COVID-19 patients decreases rapidly during the first weeks after recovery. However, the specific role of each immunoglobulin isotype in the overall neutralizing capacity is...
Nutrient sequestration is an essential facet of host innate immunity. Macrophages play a critical role in controlling iron availability through expression of the iron transport protein ferroportin (FPN), which extrudes iron from the cytoplasm to the extracellular milieu. During phagocytosis, the limiting phagosomal membrane, which derives from the...
The loss of efferocytosis—the phagocytic clearance of apoptotic cells—is an initiating event in atherosclerotic plaque formation. While the loss of macrophage efferocytosis is a prerequisite for advanced plaque formation, the transcriptional and cellular events in the pre-lesion site that drive these defects are poorly defined. Transcriptomic analy...
Characterization of the humoral response to SARS-CoV-2, the etiological agent of Covid-19, is essential to help control the infection. In this regard, we and others recently reported that the neutralization activity of plasma from COVID-19 patients decreases rapidly during the first weeks after recovery. However, the specific role of each immunoglo...
Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We emplo...
Atherosclerosis is an inflammatory disease involving formation of lipid-rich lesions within the arteries. Macrophages clear apoptotic cells that accumulate within these lesions. Defective apoptotic cell clearance is a hallmark of advanced atherosclerotic disease, yet mechanisms that drive this defect are poorly understood. In this study, we identif...
Purpose
The purpose of this study was to test fluorine-19 (19F) cellular magnetic resonance (MRI) as a non-invasive imaging modality to track therapeutic cell migration as a surrogate marker of immunotherapeutic effectiveness.
Materials and methods
Human peripheral blood mononuclear cell- (PBMC)-derived antigen presenting cell (APC) were labeled w...
Along with other immune checkpoints, T cell i mmunoglobulin and m ucin domain-containing protein 3 (Tim-3) is expressed on exhausted CD4 ⁺ and CD8 ⁺ T cells and is upregulated on the surface of these cells upon infection by H uman I mmunodeficiency V irus Type 1 (HIV-1). Recent reports have suggested an antiviral role for Tim-3. However, the molecu...
Aberrant macrophage polarization is a major contributor to the onset and progression of atherosclerosis. Despite this, macrophage polarization during in early stages of human atherosclerotic disease is poorly understood. Using transcriptomic analysis of macrophages recovered from early-stage human atherosclerotic lesions, we have identified a uniqu...
The restriction factor BST-2 and the NK cell ligands NTB-A and PVR are among a growing list of membrane proteins found to be downregulated by HIV-1 Vpu. BST-2 antagonism enhances viral release, while NTB-A and PVR downmodulation contributes to NK cell evasion. However, it remains unclear how Vpu can target multiple cellular factors simultaneously....
Efferocytosis is essential for homeostasis and prevention of the inflammatory and autoimmune diseases resulting from apoptotic cell lysis. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis, with mutations in CD93 predisposing patients to efferocytosis‐associated diseases. CD93 is a cell surface protein which is proteolytic...
Background:
The HIV-1 accessory proteins Nef and Vpu alter cell surface levels of multiple host proteins to modify the immune response and increase viral persistence. Nef and Vpu can downregulate cell surface levels of the co-stimulatory molecule CD28, however the mechanism of this function has not been completely elucidated.
Results:
Here, we p...
Superantigens (SAgs) released by common Gram-positive bacterial pathogens have been reported to delete, anergize or activate mouse T cells. However, little is known about their effects on preexisting memory CD8 + T cell (TCD8) pools. Furthermore, whether SAgs manipulate human memory TCD8 responses to cognate antigens is unknown. We employed a human...
The regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in...
The regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in...
The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef, plays an essential role in disease progression and pathogenesis via hijacking the host cellular membrane-trafficking machinery. Interestingly, HIV-1 group-M subtypes display differences in the rate of disease progression. However, few reports investigated how the cellular behavi...
Footpad thickness in mice receiving low-dose mTOR inhibitors.
BALB/c mice were infected in footpad with L. major as in Fig 1A. Twenty-one days later, mice were treated daily with vehicle (n = 9), rapamycin (1.5 μg/dose, n = 9), GSK-2126458 (1.5 μg/dose, n = 9) or KU-0063794 (1.5 μg/dose, n = 9) via the i.p. route. Footpad swelling measurements befo...
The effect of treatment with low-dose mTOR inhibitors on the capacity of splenocytes to secrete cytokines.
Splenocytes that were prepared at the end of the experiment were stimulated with Leishmania lysate or anti-CD3 mAb, and culture supernatants were collected on days 1 and 3, in which IL-4 (A) and IFN-γ (B) levels were quantified. The IFN-γ/IL-4...
No differences in Leishmania copy number between vehicle-treated and low dose mTOR inhibitor-treated BALB/c mice.
Parasite burden was determined in the popliteal lymph nodes (pLN) of the infected footpad by PCR after 10 days of treatment with vehicle or daily low doses (1.5 μg/dose) of mTOR inhibitors (n = 9). Absolute copy numbers of Leishmania RV...
Leishmaniasis is a serious global health problem affecting many people worldwide. While patients with leishmaniasis can be treated with several agents, drug toxicicty and the emergence of resistant strains render available treatments ineffective in the long run. Inhibitors of the mammalian target of rapamycin (mTOR) have been demonstrated to exert...
A bstract
Efferocytosis – the phagocytic removal of apoptotic cells – is essential for the maintenance of homeostasis and prevention of the inflammatory and autoimmune diseases which can follow the lysis of uncleared apoptotic cells. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis and angiogenesis, and upon mutation, res...
The success of many current vaccines relies on a formulation that incorporates an immune activating adjuvant. This will hold true for the design of a successful therapeutic HIV vaccine targeted at controlling reactivated virus following cessation of combined antiretroviral therapy (cART). The HIV accessory protein Nef functions by interfering with...
Acquired Immune Deficiency Syndrome is characterized by a decline in CD4⁺ T cells. Here, we elucidated the mechanism underlying apoptosis in Human Immunodeficiency Virus-1 (HIV-1) infection by examining host apoptotic pathways hijacked by the HIV-1 Nef protein in the CD4⁺ T-cell line Sup-T1. Using a panel of Nef mutants unable to bind specific host...
TYRO3, AXL, and MERTK (TAM) receptors are a family of receptor tyrosine kinases that maintain homeostasis through the clearance of apoptotic cells, and when defective, contribute to chronic inflammatory and autoimmune diseases such as atherosclerosis, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and Crohn's disease. In ad...
For studies on vaccines and therapies for HIV disease, SIV-HIV chimeric viruses harboring the HIV-1 env gene (SHIVenv) remain the best virus in non-human primate models. However, there are still very few SHIVenv viruses that can cause AIDS in non-CD8-depleted animals. In the present study, a recently created CCR5-using SHIVenv_B3 virus with env gen...
A defining characteristic of HIV-1 infection is the ability of the virus to persist within the host. Specifically, MHC-I downregulation by the HIV-1 accessory protein Nef is of critical importance in preventing infected cells from cytotoxic T-cell mediated killing. Nef downregulates MHC-I by modulating the host membrane trafficking machinery, resul...
Extensive genetic diversity is a defining characteristic of human immunodeficiency virus type 1 (HIV-1) and poses a significant barrier to the development of an effective vaccine. To better understand the impact of this genetic diversity on the HIV-1 pathogenic factor Nef, we compiled a panel of reference strains from the NIH Los Alamos HIV Databas...
Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1) is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in...
Related to Fig 7: PKA is required for WT levels of HAdV-5 protein production.
A549 cells were treated with control siRNA or siRNA specific for PKA subunits and infected with WT HAdV-5 (dl309; MOI of 5). Cells were harvested at 12, 24, and 36 hr post-infection and viral protein production was assayed by western blot using antibodies against represen...
The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protei...
Related to Fig 6: HAdV-4 E1A does not relocalize PKA subunits.
A549 cells were transfected with EGFP-tagged constructs for full-length HAdV-5 or HAdV-4 E1A. Cells were fixed, permeabilized and stained with antibodies for PKA subunits and DAPI as indicated. Unlike HAdV-5 E1A, HAdV-4 E1A was unable to noticeably relocalize PKA, suggesting that an AKA...
Related to Fig 7: PKA is recruited to HAdV early gene promoters in an E1A-dependent manner.
A549 cells were infected with the indicated viruses at an MOI of 5 and harvested 20 hours post-infection. Chromatin immunoprecipitation (ChIP) was performed with antibodies specific for the indicated proteins. DNA was probed via qPCR for the presence of mult...
Related to Fig 6: Subcellular localization of RIIα and Cα during HAdV-5 infection and localization of all PKA subunits in HEK293 cells.
(A) A549 cells were infected with either WT HAdV-5 (dl309), ΔE1A virus (dl312) or a virus lacking PKA-binding (dl1101; Δ4–25). Cells were fixed, permeabilized and stained for confocal immunofluorescence. RIIα appea...
Related to Fig 3: HAdV-4 E1A is not predicted to form a helix that is capable of binding PKA in an equivalent manner as the HAdV-5 E1A AKAP-like sequence.
(A) The PSI-PRED protein sequence analysis workbench was used to predict the helical propensity of the N-terminal regions of HAdV-5 and HAdV-4 E1A. Although both sequences are predicted to form h...
List of primers used in this study.
(DOCX)
Related to Fig 6: Co-staining of PKA and viral replication centres during HAdV-5 infection.
A549 cells were infected with the indicated virus (MOI 5) and were subsequently fixed, permeabilized and stained with antibodies specific for the indicated PKA subunits or HAdV-5 DNA-binding protein (DBP) and DAPI as indicated. Images were acquired on a Niko...
List of antibodies used in this study.
(DOCX)
List of silencing RNAs used in this study.
(DOCX)
Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regu...
Derivation of the SAA, RDF, Ripley’s, DBSCAN and OPTICS algorithm.
Complete derivation of all algorithms used in the MIiSR toolbox.
(DOCX)
Operation of the DBSCAN and OPTICS algorithms.
(A) The DBSCAN algorithm requires the user to provide the minimum number of particles in a cluster (k) and the neighbourhood size in which a cluster would exist (ε). Assuming k = 5, molecule ‘a’ (green) would be considered directly density reachable, and thus a core molecule in a cluster, as more than...
Spatial association analysis–Operation and effect of over- and under-sampling.
(A) Spatial association analysis functions by assessing distances between the molecule of interest (blue circles) and the nearest-neighbour molecule in the other colour channel (red circles). The intermolecular separation distances between nearest neighbours is calculate...
MIiSR program.
This zip-compressed archive contains the MIiSRconvert and MIiSR GUI’s, as well as stand-alone function for scripts for converting common super-resolution microscopy position files to Matlab-compatible matrices (fileConv.m), for cropping and filtering super-resolution microscopy position files (LoadCrop.m), and to perform the SAA (SAA...
Evolution of RDF and Ripley’s H-function curves with increasing clustering of two initially unclustered populations.
Model of two randomly distributed populations of 5,000 molecules/population (red and blue dots, left panel) which coalesce into 50 co-clusters. Upper-right panel display’s cross-Ripley’s H function for each frame, while the lower-rig...
Radial distribution function–Operation and effect of over- and under-sampling.
(A) RDF (pair-correlation or G(r)) analysis operates by quantifying the number of molecules (blue points) in an expanding torus centered on each molecule, normalized to the average density of molecules in the sample. For an unclustered dataset, G will have a value of 1 a...
Ripley’s K-function–Operation and effect of over- and under-sampling.
(A) Ripley’s K function operates by quantifying the density of particles (blue points) in an expanding circular area around each point, compared to the number of points predicted given the average point density of the image. When K is plotted against radius, clustering appears as...
Evolution of RDF and Ripley’s H-function curves with increasing clustering of a random population with a pre-clustered population.
Model of one randomly distributed population of 5,000 molecules (blue dots) which coalesce into 50 pre-existing clusters of red molecules. Upper-right panel display’s cross-Ripley’s H function for each frame, while the...
Impact of partial clustering on RDF and Ripley’s H-function.
Model of two co-clustered molecules (5,000/color) with an increasing portion of unclustered molecules. Each frame 200 additional unclustered molecules are added to each population (left panel). Upper-right panel display’s cross-Ripley’s H function for each frame, while the lower-right pan...
The HIV-1 accessory protein Nef is essential for HIV disease progression and pathogenesis. A major Nef function is the downregulation of cell surface MHC-I, which contributes to the ability of HIV-1 to evade immune surveillance by cytotoxic T cells.1 This function is mediated by multiple Nef binding interfaces that interact with the cytosolic tail...
The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, the...
Background:
Many viral genomes encode a limited number of proteins, illustrating their innate efficiency in bypassing host immune surveillance. This concept of genomic efficiency is exemplified by the 9 kb RNA genome of human immunodeficiency virus 1 (HIV-1), encoding 15 proteins sub-divided according to function. The enzymatic group includes prot...
