Perturbations of neurovascular integrity and breakdown of the blood–brain barrier may lead to neu... more Perturbations of neurovascular integrity and breakdown of the blood–brain barrier may lead to neuronal hypersynchronization and epileptiform activity. However, the mechanisms underlying these processes are still unclear. Furthermore, inflammatory processes related to the activation of brain vessel endothelia and/or BBB leakage have been reported in epilepsy, but no explanations have yet been provided for the relevant mechanisms of action. Our main goal is to adopt an integrated and multidisciplinary approach to this issue, taking into account different factors in the in vivo study of alterations of the ‘whole brain’ in epilepsy. Toward that end, we combine electrophysiological, neuropathological, and neuroimmunological data, in order to consider the multiple players involved in vivo in the pathogenesis of epilepsy. We report that vascular alterations and BBB leakage induced by protracted seizures are not merely epiphenomena detected in animal models of chemically induced epilepsy, but may represent crucial steps of a cascade of events responsible for epileptogenic mechanisms. Furthermore, we suggest that such events can implicate leukocyte recruitment and cytokine and chemokine release.
The regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the surv... more The regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton’s tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7–mediated apoptosis in CLL cells in a manner quantit...
Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neuro... more Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a−/− mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a−/− basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a−/− Lyn−/− showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a−/− hematological and neurological phenotypes, improving autophagy and prevent...
The blood–brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) represent two com... more The blood–brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) represent two complex structures protecting the central nervous system (CNS) against potentially harmful agents and circulating immune cells. The immunosurveillance of the CNS is governed by immune cells that constantly patrol the BCSFB, whereas during neuroinflammatory disorders, both BBB and BCSFB undergo morphological and functional alterations, promoting leukocyte intravascular adhesion and transmigration from the blood circulation into the CNS. Multiple sclerosis (MS) is the prototype of neuroinflammatory disorders in which peripheral T helper (Th) lymphocytes, particularly Th1 and Th17 cells, infiltrate the CNS and contribute to demyelination and neurodegeneration. Th1 and Th17 cells are considered key players in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis. They can actively interact with CNS borders by complex adhesion mechanisms and secretion of a variet...
The first events of leukocyte recruitment into the tissues are leukocyte tethering (capture) and ... more The first events of leukocyte recruitment into the tissues are leukocyte tethering (capture) and rolling along the vessel wall, which are mediated primarily by selectins. P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-type glycoprotein ligand expressed by all leukocytes and is a ligand for E-, P- and L-selectin. Quantitative and qualitative differences in the expression, affinity binding to the selectins and glycosylation of PSGL-1 between leukocyte subtypes suggest that potential PSGL-1 blockade should not have broad negative consequences in physiology and host defense. PSGL-1 has a well-documented role in organ targeting during inflammation in animal models, and inhibition of PSGL-1, though challenging, represents an attractive basis for antiinflammatory strategies. The consistent number of studies accumulated in the last 10 years since PSGL-1 was first characterized should induce us to ask whether we should put more effort into developing new drugs aimed to target more effectively PSGL-1 function in human diseases
Leukocyte migration into the central nervous system (CNS) represents a central process in the dev... more Leukocyte migration into the central nervous system (CNS) represents a central process in the development of neurological diseases with a detrimental inflammatory component. Infiltrating neutrophils have been detected inside the brain of patients with several neuroinflammatory disorders, including stroke, multiple sclerosis and Alzheimer’s disease. During inflammatory responses, these highly reactive innate immune cells can rapidly extravasate and release a plethora of pro-inflammatory and cytotoxic factors, potentially inducing significant collateral tissue damage. Indeed, several studies have shown that neutrophils promote blood-brain barrier damage and increased vascular permeability during neuroinflammatory diseases. Recent studies have shown that neutrophils migrate into the meninges and choroid plexus, suggesting these cells can also damage the blood-cerebrospinal fluid barrier (BCSFB). In this review, we discuss the emerging role of neutrophils in the dysfunction of brain bar...
Neurodegenerative diseases are closely related to inflammatory and autoimmune events, suggesting ... more Neurodegenerative diseases are closely related to inflammatory and autoimmune events, suggesting that the dysregulation of the immune system is a key pathological factor. Both multiple sclerosis (MS) and Alzheimer's disease (AD) are characterized by infiltrating immune cells, activated microglia, astrocyte proliferation, and neuronal damage. Moreover, MS and AD share a common pro-inflammatory signature, characterized by peripheral leukocyte activation and transmigration to the central nervous system (CNS). MS and AD are both characterized by the accumulation of activated neutrophils in the blood, leading to progressive impairment of the blood–brain barrier. Having migrated to the CNS during the early phases of MS and AD, neutrophils promote local inflammation that contributes to pathogenesis and clinical progression. The role of circulating T cells in MS is well-established, whereas the contribution of adaptive immunity to AD pathogenesis and progression is a more recent discove...
This article presents a stochastic model of lymphocyte recruitment in inflamed brain microvessels... more This article presents a stochastic model of lymphocyte recruitment in inflamed brain microvessels. Recent studies about the inflammatory process of the brain that leads to multiple sclerosis have revealed that lymphocyte extravasation is a sequence of dynamical states, mediated by partially overlapped interactions of different adhesion molecules and activation factors. This study’s model of lymphocyte recruitment is based on process algebras for mobile systems. The biochemical system is modelled as a set of concurrent processes of the biochemical stochastic π-calculus. Processes are driven by suitable probability distributions that quantitatively describe the rates and the times at which reactions to simulations occur. The results of the model reproduce, within the estimated experimental errors, the functional behavior of the data obtained from laboratory measurements.
The conventional notion that neurons are exclusively responsible for brain signaling is increasin... more The conventional notion that neurons are exclusively responsible for brain signaling is increasingly challenged by the idea that brain function in fact depends on a complex interplay between neurons, glial cells, vascular endothelium, and immune-related blood cells. Recent data demonstrates that neuronal activity is profoundly affected by an entire cellular and extracellular 'orchestra', the so-called neurovascular unit (NVU). Among the 'musical instruments' of this orchestra, there may be molecules long-known in biomedicine as important mediators of inflammatory and immune responses in the organism, as well as non-neuronal cells, e.g., leukocytes. We here review recent evidence on the structure and function of the NVU, both in the healthy brain and in pathological conditions, such as the abnormal NVU activation observed in epilepsy. We will argue that a better understanding of NVU function will require the addition of new players to the 'orchestra'.
Perturbations of neurovascular integrity and breakdown of the blood–brain barrier may lead to neu... more Perturbations of neurovascular integrity and breakdown of the blood–brain barrier may lead to neuronal hypersynchronization and epileptiform activity. However, the mechanisms underlying these processes are still unclear. Furthermore, inflammatory processes related to the activation of brain vessel endothelia and/or BBB leakage have been reported in epilepsy, but no explanations have yet been provided for the relevant mechanisms of action. Our main goal is to adopt an integrated and multidisciplinary approach to this issue, taking into account different factors in the in vivo study of alterations of the ‘whole brain’ in epilepsy. Toward that end, we combine electrophysiological, neuropathological, and neuroimmunological data, in order to consider the multiple players involved in vivo in the pathogenesis of epilepsy. We report that vascular alterations and BBB leakage induced by protracted seizures are not merely epiphenomena detected in animal models of chemically induced epilepsy, but may represent crucial steps of a cascade of events responsible for epileptogenic mechanisms. Furthermore, we suggest that such events can implicate leukocyte recruitment and cytokine and chemokine release.
The regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the surv... more The regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton’s tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7–mediated apoptosis in CLL cells in a manner quantit...
Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neuro... more Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a−/− mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a−/− basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a−/− Lyn−/− showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a−/− hematological and neurological phenotypes, improving autophagy and prevent...
The blood–brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) represent two com... more The blood–brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) represent two complex structures protecting the central nervous system (CNS) against potentially harmful agents and circulating immune cells. The immunosurveillance of the CNS is governed by immune cells that constantly patrol the BCSFB, whereas during neuroinflammatory disorders, both BBB and BCSFB undergo morphological and functional alterations, promoting leukocyte intravascular adhesion and transmigration from the blood circulation into the CNS. Multiple sclerosis (MS) is the prototype of neuroinflammatory disorders in which peripheral T helper (Th) lymphocytes, particularly Th1 and Th17 cells, infiltrate the CNS and contribute to demyelination and neurodegeneration. Th1 and Th17 cells are considered key players in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis. They can actively interact with CNS borders by complex adhesion mechanisms and secretion of a variet...
The first events of leukocyte recruitment into the tissues are leukocyte tethering (capture) and ... more The first events of leukocyte recruitment into the tissues are leukocyte tethering (capture) and rolling along the vessel wall, which are mediated primarily by selectins. P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-type glycoprotein ligand expressed by all leukocytes and is a ligand for E-, P- and L-selectin. Quantitative and qualitative differences in the expression, affinity binding to the selectins and glycosylation of PSGL-1 between leukocyte subtypes suggest that potential PSGL-1 blockade should not have broad negative consequences in physiology and host defense. PSGL-1 has a well-documented role in organ targeting during inflammation in animal models, and inhibition of PSGL-1, though challenging, represents an attractive basis for antiinflammatory strategies. The consistent number of studies accumulated in the last 10 years since PSGL-1 was first characterized should induce us to ask whether we should put more effort into developing new drugs aimed to target more effectively PSGL-1 function in human diseases
Leukocyte migration into the central nervous system (CNS) represents a central process in the dev... more Leukocyte migration into the central nervous system (CNS) represents a central process in the development of neurological diseases with a detrimental inflammatory component. Infiltrating neutrophils have been detected inside the brain of patients with several neuroinflammatory disorders, including stroke, multiple sclerosis and Alzheimer’s disease. During inflammatory responses, these highly reactive innate immune cells can rapidly extravasate and release a plethora of pro-inflammatory and cytotoxic factors, potentially inducing significant collateral tissue damage. Indeed, several studies have shown that neutrophils promote blood-brain barrier damage and increased vascular permeability during neuroinflammatory diseases. Recent studies have shown that neutrophils migrate into the meninges and choroid plexus, suggesting these cells can also damage the blood-cerebrospinal fluid barrier (BCSFB). In this review, we discuss the emerging role of neutrophils in the dysfunction of brain bar...
Neurodegenerative diseases are closely related to inflammatory and autoimmune events, suggesting ... more Neurodegenerative diseases are closely related to inflammatory and autoimmune events, suggesting that the dysregulation of the immune system is a key pathological factor. Both multiple sclerosis (MS) and Alzheimer's disease (AD) are characterized by infiltrating immune cells, activated microglia, astrocyte proliferation, and neuronal damage. Moreover, MS and AD share a common pro-inflammatory signature, characterized by peripheral leukocyte activation and transmigration to the central nervous system (CNS). MS and AD are both characterized by the accumulation of activated neutrophils in the blood, leading to progressive impairment of the blood–brain barrier. Having migrated to the CNS during the early phases of MS and AD, neutrophils promote local inflammation that contributes to pathogenesis and clinical progression. The role of circulating T cells in MS is well-established, whereas the contribution of adaptive immunity to AD pathogenesis and progression is a more recent discove...
This article presents a stochastic model of lymphocyte recruitment in inflamed brain microvessels... more This article presents a stochastic model of lymphocyte recruitment in inflamed brain microvessels. Recent studies about the inflammatory process of the brain that leads to multiple sclerosis have revealed that lymphocyte extravasation is a sequence of dynamical states, mediated by partially overlapped interactions of different adhesion molecules and activation factors. This study’s model of lymphocyte recruitment is based on process algebras for mobile systems. The biochemical system is modelled as a set of concurrent processes of the biochemical stochastic π-calculus. Processes are driven by suitable probability distributions that quantitatively describe the rates and the times at which reactions to simulations occur. The results of the model reproduce, within the estimated experimental errors, the functional behavior of the data obtained from laboratory measurements.
The conventional notion that neurons are exclusively responsible for brain signaling is increasin... more The conventional notion that neurons are exclusively responsible for brain signaling is increasingly challenged by the idea that brain function in fact depends on a complex interplay between neurons, glial cells, vascular endothelium, and immune-related blood cells. Recent data demonstrates that neuronal activity is profoundly affected by an entire cellular and extracellular 'orchestra', the so-called neurovascular unit (NVU). Among the 'musical instruments' of this orchestra, there may be molecules long-known in biomedicine as important mediators of inflammatory and immune responses in the organism, as well as non-neuronal cells, e.g., leukocytes. We here review recent evidence on the structure and function of the NVU, both in the healthy brain and in pathological conditions, such as the abnormal NVU activation observed in epilepsy. We will argue that a better understanding of NVU function will require the addition of new players to the 'orchestra'.
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Papers by Gabriela Constantin