Arteriosclerosis, Thrombosis, and Vascular Biology, 2014
Dendritic cells (DCs) play a key role in chronic inflammatory diseases such as atherosclerosis. M... more Dendritic cells (DCs) play a key role in chronic inflammatory diseases such as atherosclerosis. Myeloid cells with features of DCs are abundant in the normal arterial intima of mice, in regions that are predisposed to atherosclerosis. Upon induction of hypercholesterolemia, these intimal DCs rapidly engulf lipids and transform into the initial foam cells of nascent atherosclerotic lesions. The function of intimal DCs in normal mice remains unknown. We observed that systemic stimulation of toll-like receptors (TLRs), specifically TLR4 by LPS and TLR3 by Poly(I:C), induced a rapid reduction in the number of DCs in the normal aortic intima. Absence of TUNNEL staining and use of CD11c-hBcl2 transgenic mice suggested that apoptosis did not account for the loss of intimal DCs. Immuno-gold staining for CD11c coupled with scanning electron microscopy of the aortic surface and ex vivo experiments determined that reverse transmigration (RTM) through the intact endothelial monolayer accounts f...
Mononuclear phagocytes interact with vascular endothelium in a variety of pathological and physio... more Mononuclear phagocytes interact with vascular endothelium in a variety of pathological and physiological settings. Reversible adhesive interactions with blood-vessel walls lead to mar-gination of a substantial proportion of the circulating monocyte pool (1). Constitutive emigration into uninflamed tissues occurs through mechanisms that remain obscure. Increasing evidence suggests that local activation of vascular endothelium plays an important role in initial adhesion and subsequent emigration of monocytes at inflammatory sites, and that similar mechanisms promote monocyte accumulation in atherosclerotic plaques (2).
We screened three separate cohorts of healthcare workers for SARS-CoV-2 via nasopharyngeal swab P... more We screened three separate cohorts of healthcare workers for SARS-CoV-2 via nasopharyngeal swab PCR. A seroprevalence analysis using multiple assays was performed in a subgroup. The asymptomatic health care worker cohorts had a combined positivity rate of 29/5776 (0.50%, 95%CI 0.32-0.75) compared to the symptomatic cohort rate of 54/1597 (3.4%) (ratio of symptomatic to asymptomatic 6.8:1). Sequencing demonstrated several variants. The seroprevalence (n=996) was 1.4-3.4% depending on assay. Protein microarray analysis showed differing SARS-CoV-2 protein reactivities and helped define likely true positives vs. suspected false positives. Routine screening of asymptomatic health care workers helps identify a significant proportion of infections.
To generate new mouse models of human hematopoietic disease and increase our knowledge of the gen... more To generate new mouse models of human hematopoietic disease and increase our knowledge of the genetic networks that control hematopoiesis, we are performing dominant (generation 1 or G1) and pharmacologically-sensitized forward ethylnitrososurea (ENU) mutagenesis screens. Mice are phenotyped by saphenous vein peripheral blood analysis using an automated hematological analyzer. ENU is ideally suited to generating models of human disease and annotating gene function because the spectrum of mutations (point mutations generating leading to subtle amino acid substitutions, splicing errors, or premature termination) are similar to those often found in human disease. Furthermore, null mutations often do not represent the full extent of a gene’s function, requiring multiple alleles to fully define gene function. While dominant mutations can unequivocally cause some human diseases, often mutations in multiple genes interact and contribute to disease progression. Thus, we have developed sensi...
Macrophages promote both injury and repair after myocardial infarction, but discriminating functi... more Macrophages promote both injury and repair after myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping, parabiosis and single-cell transcriptomics to demonstrate that at steady state, TIMD4LYVE1MHC-IICCR2 resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4LYVE1MHC-IICCR2 macrophages and fully replaced TIMD4LYVE1MHC-IICCR2 macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4 and TIMD4 resident macrophage abundance, whereas CCR2 monocyte-derived macrophages adopted multiple cell fates within infarcted tissue, including those nearly indistinguishable from resident macrophages. Recruited macrophages did not express TIMD4, highlighting the ability of TIMD4 to track a subset of resident macrophages in the absence of fate mapping. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1-based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, revealing a nonredundant, cardioprotective role of resident cardiac macrophages.
Background: Reactive glucose-protein intermediates and advanced glycation endproducts (AGEs) are ... more Background: Reactive glucose-protein intermediates and advanced glycation endproducts (AGEs) are shown to colocalize with atheromatous lesions and to trigger complex chemical and biological responses through interaction with vessel wall elements. In diabetes and renal insufficiency, atherosclerosis is common, as are elevated levels of serum and vascular tissue AGEs. In the present study, AGEs supplied exogenously to nornal animals elicited vascular and renal pathology. Materials and Methods: Nondiabetic rabbits were injected intravenously with low doses of AGE-modified rabbit serum albumin (AGE-RSA, 16 mg/kg/day) for 4 months alone, or combined with a brief terminal period (2 weeks) of a cholesterol-rich diet (CRD) (2% cholesterol, 10% corn oil). AGE-RSA associated expression of vascular cell adhesion molecules and the development of atheromatous changes within the aorta were determined by immunohistology. Results: The AGE content of aortic tissue increased by 2.2-fold in AGE-treated and by 3.2-fold in AGE + CRDtreated rabbits compared with normal saline-treated control rabbits (p < 0.025 and 0.001, respectively). Serum AGE levels in AGE groups rose up to 3-fold above the controls (p < 0.025 and p < 0.01). Ascending aortic sections from AGE-treated rabbits showed significant focal intimal proliferation, enhanced endothelial cell adhesion with infrequent intirnal macrophages, oil-redO staining lipid deposits and positive focal expression of vascular cell adhesion molecule-I (VCAM-1), and intercellular adhesion molecule-I (ICAM-1), a pattern not observed in controls. These AGE-induced changes were markedly enhanced in animals cotreated with AGEs and a brief period of CRD. Lesions consisted of multifocal atheromas, containing foam cells, massive lipid droplets, and strong endothelial expression of VCAM-1 and ICAM-1 restricted to the affected areas. Conclusions: This study provides in vivo evidence for a causal relationship between chronic AGE accumulation and atherosclerosis independent of diabetic hyperglycemia, and suggests the utility of this animal model for the study of diabetic vascular disease in relation to glycation.
While the preponderance of morbidity and mortality in medulloblastoma patients are due to metasta... more While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate in vivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma in vivo is sufficient to drive leptomeningeal dissemination. Hematogenous disseminat...
Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, ye... more Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, yet their identities and functions remain poorly defined. We utilized a combination of genetic fate mapping, parabiotic, transcriptional, and functional analyses and demonstrated that the heart contained two major conventional dendritic cell (cDC) subsets, CD103+ and CD11b+, which differentially relied on local proliferation and precursor recruitment to maintain their tissue residency. Following viral infection of the myocardium, cDCs accumulated in the heart coincident with monocyte infiltration and loss of resident reparative embryonic-derived cardiac macrophages. cDC depletion abrogated antigen-specific CD8+ T cell proliferative expansion, transforming subclinical cardiac injury to overt heart failure. These effects were mediated by CD103+ cDCs, which are dependent on the transcription factor BATF3 for their development. Collectively, our findings identified resident cardiac cDC subsets...
Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identifie... more Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor κ light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)-derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. To define the role of endogenous miR-146a during atherogenesis. Paradoxically, Ldlr(-/-) (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr(-/-);miR-146a(-/-) mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr(-/-) mice receiving ...
Leukocyte crawling and transendothelial migration (TEM) are potentiated by shear stress caused by... more Leukocyte crawling and transendothelial migration (TEM) are potentiated by shear stress caused by blood flow. The mechanism that couples shear stress to migration has not been fully elucidated. We found that mice lacking GEF-H1 (GEF-H1(-/-)), a RhoA-specific guanine nucleotide exchange factor (GEF), displayed limited migration and recruitment of neutrophils into inflamed tissues. GEF-H1(-/-) leukocytes were deficient in in vivo crawling and TEM in the postcapillary venules. We demonstrated that although GEF-H1 deficiency had little impact on the migratory properties of neutrophils under static conditions, shear stress triggered GEF-H1-dependent spreading and crawling of neutrophils and relocalization of GEF-H1 to flotillin-2-rich uropods. Our results identify GEF-H1 as a component of the shear stress response machinery in neutrophils required for a fully competent immune response to bacterial infection.
In late April, the laboratory of immunologist Cheolho Cheong, Assistant Professor at the IRCM in ... more In late April, the laboratory of immunologist Cheolho Cheong, Assistant Professor at the IRCM in Montreal, was celebrating that their work was scheduled for publication in the May 2016 issue of Cell Metabolism. The article, ''Indoleamine 2,3-Dioxygenase-Expressing Aortic Plasmacytoid Dendritic Cells Protect against Atherosclerosis by Induction of Regulatory T Cells,'' was the first senior author publication from the nascent independent laboratory of Dr. Cheong. Cheolho was so excited about the work, exclaiming, ''Now I can show my friends I am still alive, still in science!'' With profound sadness and shock, we report that, on April 24, days after celebrating his 41 st birthday and returning the page proofs, Cheolho collapsed at home, passing from this life suddenly and unexpectedly. For all of us, this news seemed incomprehensible, as Cheolho was counted universally among us as one of the most energetic, full-of-life colleagues we knew. We cannot imagine the sorrow of his wife and two small children. As his friends and colleagues, we can easily recall his voice, once in our phones and our hallways, but now only in our minds, greeting us with a hearty ''hello'' or ''bonjour'' and an inquiry as to how we were doing and whether we had a moment to talk about science. Indeed, many of us had
Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyt... more Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However, the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum, blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus, RTM protects the normal arterial intima, and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atheros...
The majority of integrins expressed on circulating leukocytes are quiescent and do not mediate ad... more The majority of integrins expressed on circulating leukocytes are quiescent and do not mediate adhesion. In the process of emigrating from blood into tissues, leukocytes are exposed to a variety of stimuli, including chemokines, which rapidly activate integrin function through changes in affinity and/or avidity. High affinity is brought about by a change in integrin conformation and results in formation of a stable bond with ligand. This can be measured in soluble ligand-binding assays. Integrin conformation changes also result in exposure of previously masked epitopes that can be detected with monoclonal antibodies. In this report, methods used to detect high affinity integrins are reviewed. Four different approaches are discussed, including detection of masked epitopes with monoclonal antibodies, and binding of soluble ligands, ligand-coated beads, and ligandmimetic peptides. Improved techniques to measure rapid changes in integrin affinity in real time may be valuable both to study mechanisms of inside-out integrin activation and as a tool to screen for effective inhibitors of integrin activation in inflammatory disease models.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2014
Dendritic cells (DCs) play a key role in chronic inflammatory diseases such as atherosclerosis. M... more Dendritic cells (DCs) play a key role in chronic inflammatory diseases such as atherosclerosis. Myeloid cells with features of DCs are abundant in the normal arterial intima of mice, in regions that are predisposed to atherosclerosis. Upon induction of hypercholesterolemia, these intimal DCs rapidly engulf lipids and transform into the initial foam cells of nascent atherosclerotic lesions. The function of intimal DCs in normal mice remains unknown. We observed that systemic stimulation of toll-like receptors (TLRs), specifically TLR4 by LPS and TLR3 by Poly(I:C), induced a rapid reduction in the number of DCs in the normal aortic intima. Absence of TUNNEL staining and use of CD11c-hBcl2 transgenic mice suggested that apoptosis did not account for the loss of intimal DCs. Immuno-gold staining for CD11c coupled with scanning electron microscopy of the aortic surface and ex vivo experiments determined that reverse transmigration (RTM) through the intact endothelial monolayer accounts f...
Mononuclear phagocytes interact with vascular endothelium in a variety of pathological and physio... more Mononuclear phagocytes interact with vascular endothelium in a variety of pathological and physiological settings. Reversible adhesive interactions with blood-vessel walls lead to mar-gination of a substantial proportion of the circulating monocyte pool (1). Constitutive emigration into uninflamed tissues occurs through mechanisms that remain obscure. Increasing evidence suggests that local activation of vascular endothelium plays an important role in initial adhesion and subsequent emigration of monocytes at inflammatory sites, and that similar mechanisms promote monocyte accumulation in atherosclerotic plaques (2).
We screened three separate cohorts of healthcare workers for SARS-CoV-2 via nasopharyngeal swab P... more We screened three separate cohorts of healthcare workers for SARS-CoV-2 via nasopharyngeal swab PCR. A seroprevalence analysis using multiple assays was performed in a subgroup. The asymptomatic health care worker cohorts had a combined positivity rate of 29/5776 (0.50%, 95%CI 0.32-0.75) compared to the symptomatic cohort rate of 54/1597 (3.4%) (ratio of symptomatic to asymptomatic 6.8:1). Sequencing demonstrated several variants. The seroprevalence (n=996) was 1.4-3.4% depending on assay. Protein microarray analysis showed differing SARS-CoV-2 protein reactivities and helped define likely true positives vs. suspected false positives. Routine screening of asymptomatic health care workers helps identify a significant proportion of infections.
To generate new mouse models of human hematopoietic disease and increase our knowledge of the gen... more To generate new mouse models of human hematopoietic disease and increase our knowledge of the genetic networks that control hematopoiesis, we are performing dominant (generation 1 or G1) and pharmacologically-sensitized forward ethylnitrososurea (ENU) mutagenesis screens. Mice are phenotyped by saphenous vein peripheral blood analysis using an automated hematological analyzer. ENU is ideally suited to generating models of human disease and annotating gene function because the spectrum of mutations (point mutations generating leading to subtle amino acid substitutions, splicing errors, or premature termination) are similar to those often found in human disease. Furthermore, null mutations often do not represent the full extent of a gene’s function, requiring multiple alleles to fully define gene function. While dominant mutations can unequivocally cause some human diseases, often mutations in multiple genes interact and contribute to disease progression. Thus, we have developed sensi...
Macrophages promote both injury and repair after myocardial infarction, but discriminating functi... more Macrophages promote both injury and repair after myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping, parabiosis and single-cell transcriptomics to demonstrate that at steady state, TIMD4LYVE1MHC-IICCR2 resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4LYVE1MHC-IICCR2 macrophages and fully replaced TIMD4LYVE1MHC-IICCR2 macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4 and TIMD4 resident macrophage abundance, whereas CCR2 monocyte-derived macrophages adopted multiple cell fates within infarcted tissue, including those nearly indistinguishable from resident macrophages. Recruited macrophages did not express TIMD4, highlighting the ability of TIMD4 to track a subset of resident macrophages in the absence of fate mapping. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1-based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, revealing a nonredundant, cardioprotective role of resident cardiac macrophages.
Background: Reactive glucose-protein intermediates and advanced glycation endproducts (AGEs) are ... more Background: Reactive glucose-protein intermediates and advanced glycation endproducts (AGEs) are shown to colocalize with atheromatous lesions and to trigger complex chemical and biological responses through interaction with vessel wall elements. In diabetes and renal insufficiency, atherosclerosis is common, as are elevated levels of serum and vascular tissue AGEs. In the present study, AGEs supplied exogenously to nornal animals elicited vascular and renal pathology. Materials and Methods: Nondiabetic rabbits were injected intravenously with low doses of AGE-modified rabbit serum albumin (AGE-RSA, 16 mg/kg/day) for 4 months alone, or combined with a brief terminal period (2 weeks) of a cholesterol-rich diet (CRD) (2% cholesterol, 10% corn oil). AGE-RSA associated expression of vascular cell adhesion molecules and the development of atheromatous changes within the aorta were determined by immunohistology. Results: The AGE content of aortic tissue increased by 2.2-fold in AGE-treated and by 3.2-fold in AGE + CRDtreated rabbits compared with normal saline-treated control rabbits (p < 0.025 and 0.001, respectively). Serum AGE levels in AGE groups rose up to 3-fold above the controls (p < 0.025 and p < 0.01). Ascending aortic sections from AGE-treated rabbits showed significant focal intimal proliferation, enhanced endothelial cell adhesion with infrequent intirnal macrophages, oil-redO staining lipid deposits and positive focal expression of vascular cell adhesion molecule-I (VCAM-1), and intercellular adhesion molecule-I (ICAM-1), a pattern not observed in controls. These AGE-induced changes were markedly enhanced in animals cotreated with AGEs and a brief period of CRD. Lesions consisted of multifocal atheromas, containing foam cells, massive lipid droplets, and strong endothelial expression of VCAM-1 and ICAM-1 restricted to the affected areas. Conclusions: This study provides in vivo evidence for a causal relationship between chronic AGE accumulation and atherosclerosis independent of diabetic hyperglycemia, and suggests the utility of this animal model for the study of diabetic vascular disease in relation to glycation.
While the preponderance of morbidity and mortality in medulloblastoma patients are due to metasta... more While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate in vivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma in vivo is sufficient to drive leptomeningeal dissemination. Hematogenous disseminat...
Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, ye... more Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, yet their identities and functions remain poorly defined. We utilized a combination of genetic fate mapping, parabiotic, transcriptional, and functional analyses and demonstrated that the heart contained two major conventional dendritic cell (cDC) subsets, CD103+ and CD11b+, which differentially relied on local proliferation and precursor recruitment to maintain their tissue residency. Following viral infection of the myocardium, cDCs accumulated in the heart coincident with monocyte infiltration and loss of resident reparative embryonic-derived cardiac macrophages. cDC depletion abrogated antigen-specific CD8+ T cell proliferative expansion, transforming subclinical cardiac injury to overt heart failure. These effects were mediated by CD103+ cDCs, which are dependent on the transcription factor BATF3 for their development. Collectively, our findings identified resident cardiac cDC subsets...
Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identifie... more Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor κ light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)-derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. To define the role of endogenous miR-146a during atherogenesis. Paradoxically, Ldlr(-/-) (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr(-/-);miR-146a(-/-) mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr(-/-) mice receiving ...
Leukocyte crawling and transendothelial migration (TEM) are potentiated by shear stress caused by... more Leukocyte crawling and transendothelial migration (TEM) are potentiated by shear stress caused by blood flow. The mechanism that couples shear stress to migration has not been fully elucidated. We found that mice lacking GEF-H1 (GEF-H1(-/-)), a RhoA-specific guanine nucleotide exchange factor (GEF), displayed limited migration and recruitment of neutrophils into inflamed tissues. GEF-H1(-/-) leukocytes were deficient in in vivo crawling and TEM in the postcapillary venules. We demonstrated that although GEF-H1 deficiency had little impact on the migratory properties of neutrophils under static conditions, shear stress triggered GEF-H1-dependent spreading and crawling of neutrophils and relocalization of GEF-H1 to flotillin-2-rich uropods. Our results identify GEF-H1 as a component of the shear stress response machinery in neutrophils required for a fully competent immune response to bacterial infection.
In late April, the laboratory of immunologist Cheolho Cheong, Assistant Professor at the IRCM in ... more In late April, the laboratory of immunologist Cheolho Cheong, Assistant Professor at the IRCM in Montreal, was celebrating that their work was scheduled for publication in the May 2016 issue of Cell Metabolism. The article, ''Indoleamine 2,3-Dioxygenase-Expressing Aortic Plasmacytoid Dendritic Cells Protect against Atherosclerosis by Induction of Regulatory T Cells,'' was the first senior author publication from the nascent independent laboratory of Dr. Cheong. Cheolho was so excited about the work, exclaiming, ''Now I can show my friends I am still alive, still in science!'' With profound sadness and shock, we report that, on April 24, days after celebrating his 41 st birthday and returning the page proofs, Cheolho collapsed at home, passing from this life suddenly and unexpectedly. For all of us, this news seemed incomprehensible, as Cheolho was counted universally among us as one of the most energetic, full-of-life colleagues we knew. We cannot imagine the sorrow of his wife and two small children. As his friends and colleagues, we can easily recall his voice, once in our phones and our hallways, but now only in our minds, greeting us with a hearty ''hello'' or ''bonjour'' and an inquiry as to how we were doing and whether we had a moment to talk about science. Indeed, many of us had
Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyt... more Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However, the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum, blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus, RTM protects the normal arterial intima, and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atheros...
The majority of integrins expressed on circulating leukocytes are quiescent and do not mediate ad... more The majority of integrins expressed on circulating leukocytes are quiescent and do not mediate adhesion. In the process of emigrating from blood into tissues, leukocytes are exposed to a variety of stimuli, including chemokines, which rapidly activate integrin function through changes in affinity and/or avidity. High affinity is brought about by a change in integrin conformation and results in formation of a stable bond with ligand. This can be measured in soluble ligand-binding assays. Integrin conformation changes also result in exposure of previously masked epitopes that can be detected with monoclonal antibodies. In this report, methods used to detect high affinity integrins are reviewed. Four different approaches are discussed, including detection of masked epitopes with monoclonal antibodies, and binding of soluble ligands, ligand-coated beads, and ligandmimetic peptides. Improved techniques to measure rapid changes in integrin affinity in real time may be valuable both to study mechanisms of inside-out integrin activation and as a tool to screen for effective inhibitors of integrin activation in inflammatory disease models.
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Papers by Myron Cybulsky