Publications by authors named "Melanie Demers"

31 Publications

Solid peripheral tumor leads to systemic inflammation, astrocyte activation and signs of behavioral despair in mice.

PLoS One 2018 15;13(11):e0207241. Epub 2018 Nov 15.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, United States of America.

Prevalence of depression is higher in patients with cancer than in the general population. Sustained systemic inflammation has been associated with depressive behavior and it has been reported that depressed patients commonly display alterations in their immune system. We previously showed that cancer in mice induces a systemic environment that promotes neutrophil activation and leukocytosis. We thus hypothesized that the peripheral systemic response to a solid tumor leads to endothelial activation, which may promote inflammatory changes in the brain with behavioral consequences. Using the Lewis lung carcinoma (LLC) model, we show that tumor growth induces a progressive increase in peripheral inflammation as observed by elevated interleukin-6 (IL-6). In behavioral studies, tumor-bearing mice showed no sign of motor, coordination or short term working memory deficits as assessed by rotarod, balance-beam, and novel object recognition tests. However, there was an impairment in the grip strength test and interestingly, an anxious and despair-like phenotype in the elevated plus-maze, and tail suspension tests, respectively. Immunostaining of perfused brains revealed fibrin accumulation in the vasculature with some leakage into the parenchyma, a process known to activate endothelial cells. Taken together, our results suggest that the inflamed and prothrombotic systemic environment created by the growth of a peripherally-located solid tumor induces endothelial activation, accumulation of fibrin in the brain and astrocyte activation, perhaps leading to depressive-like behavior.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207241PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237350PMC
April 2019

Thalamic and hippocampal volume associated with memory functions in multiple sclerosis.

Brain Cogn 2018 08 8;125:61-68. Epub 2018 Jun 8.

Department of Psychology, Université du Québec à Montréal, CP 8888, succ. Centre-ville, Montreal H3C 3P8, Canada; Centre de Recherche du Centre Hospitalier de l'Université de Montréal, 900 Rue Saint-Denis, Montréal, QC H2X 3H8, Canada. Electronic address:

Objectives: Although multiple sclerosis (MS) has long been considered to primarily affect white matter, it is now recognized that cognitive deficits in MS are also related to neocortical, thalamic and hippocampal damage. However, the association between damage to these structures and memory deficits in MS is unclear. This study examines whether MS patients with cognitive impairment have a reduction of hippocampal and/or thalamic volumes compared to cognitively intact patients, and whether these volume reductions correlate with various aspects of memory function.

Methodology: Volumetric MRI measures of thalamus and hippocampus of forty-one patients with MS were performed. The patients were divided in two groups depending on the presence or absence of cognitive impairment, based on their neuropsychological tests scores.

Results: Right hippocampal volume was found to be associated with learning, and the left thalamic volume was found to predict performance in verbal memory. Cognitively impaired patients had a tendency to have a reduced left thalamic volume compared to cognitively intact patients.

Conclusions: This study does not support a direct relationship between hippocampal atrophy and verbal memory. These results add to the growing evidence of the involvement of thalamus in cognitive impairment in MS and its association with verbal memory deficits.
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http://dx.doi.org/10.1016/j.bandc.2018.05.013DOI Listing
August 2018

ADAMTS13 Deficiency Worsens Colitis and Exogenous ADAMTS13 Administration Decreases Colitis Severity in Mice.

TH Open 2017 Jun;1(1):e11-e23

Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, United States.

Background: Inflammatory bowel disease (IBD) affects 1.6 million people in the United States. IBD is associated with an increased risk of thrombosis, which rises with disease activity. The pathogenesis of IBD and its increased thrombotic risk is not completely understood. Ultra large von Willebrand factor (ULVWF) multimers are secreted from activated endothelium, leading to recruitment of platelets and leukocytes. A disintegrin and metalloproteinase with thrombospondin type I repeats motif 13 (ADAMTS13) cleaves highly adhesive ULVWF into smaller, less bioactive, multimers, releasing them into circulation. Mice deficient in ADAMTS13 (ADAMTS13) have heightened inflammatory and thrombotic responses.

Objectives: We hypothesized that upon colitis induction, ADAMTS13 mice would have more severe symptoms compared with wild-type (WT) mice, and rhADAMTS13 administration to mice with colitis would improve their condition.

Results: Dextran sodium sulfate-induced colitis was worse in ADAMTS13 mice than WT. ADAMTS13 showed increased weight loss, worse anemia, and increased clinical and histologic colitis severity, compared with WT mice. ADAMTS13 mice had increased VWF release, with accumulation at inflamed colonic sites. Also, the majority of mice showed one or more submucosal colonic thrombi. ADAMTS13 deficiency worsened colitis and propagated intestinal inflammation, most likely through increased platelet-leukocyte recruitment by VWF. Treatment of WT mice with rhA-DAMTS13 decreased colitis severity without worsening anemia. Additionally, several immune-mediated chronic murine colitis models, and inflamed colon tissue specimens from IBD patients, showed increased VWF release at inflamed sites, suggesting a generalizability of our findings.

Conclusion: Measuring VWF/ADAMTS13 levels could have clinical utility. When applicable, the administration of ADAMTS13, in addition to primary treatment, may improve outcomes for IBD patients.
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http://dx.doi.org/10.1055/s-0037-1603927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5782810PMC
June 2017

Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer.

PLoS One 2018 11;13(1):e0191231. Epub 2018 Jan 11.

Department of Clinical Sciences, Danderyd Hospital, Division of Internal Medicine, Karolinska Institutet, Stockholm, Sweden.

Citrullinated histone H3 (H3Cit) is a central player in the neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs). NETs have been shown to elicit harmful effects on the host, and were recently proposed to promote tumor progression and spread. Here we report significant elevations of plasma H3Cit in patients with advanced cancer compared with age-matched healthy individuals. These elevations were specific to cancer patients as no increase was observed in severely ill and hospitalized patients with a higher non-malignant comorbidity. The analysis of neutrophils from cancer patients showed a higher proportion of neutrophils positive for intracellular H3Cit compared to severely ill patients. Moreover, the presence of plasma H3Cit in cancer patients strongly correlated with neutrophil activation markers neutrophil elastase (NE) and myeloperoxidase (MPO), and the inflammatory cytokines interleukin-6 and -8, known to induce NETosis. In addition, we show that high levels of circulating H3Cit strongly predicted poor clinical outcome in our cohort of cancer patients with a 2-fold increased risk for short-term mortality. Our results also corroborate the association of NE, interleukin-6 and -8 with poor clinical outcome. Taken together, our results are the first to unveil H3Cit as a potential diagnostic and prognostic blood marker associated with an exacerbated inflammatory response in patients with advanced cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0191231PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764486PMC
February 2018

Prospective memory impairment in multiple sclerosis: a review.

Clin Neuropsychol 2018 07 4;32(5):922-936. Epub 2017 Aug 4.

b Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM) , Montreal , Canada.

Objective: Multiple sclerosis (MS) is a progressive disease of the central nervous system affecting information processing speed, episodic memory, attention, and executive functions. MS patients also often report prospective memory (PM) failures that directly impact their functional autonomy, including professional and social life. The purpose of this paper was to review the literature concerning the assessment and remediation of PM deficits in MS.

Method: The literature pertaining to PM impairment in MS was carefully reviewed using PubMed, PsyINFO, and Google Scholar, as well as cross-references from the articles published on this topic. Since PM rehabilitation in MS patients is still in its infancy, this review mainly focuses on studies that have directly assessed PM through various measures including questionnaires, standardized clinical tests, and experimental procedures.

Conclusion: This literature review confirms the presence of PM deficits in MS patients, even in the early stages of the disease. A further need for controlled studies on PM assessment and PM interventions in patients with MS is stressed.
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http://dx.doi.org/10.1080/13854046.2017.1361473DOI Listing
July 2018

Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma.

Immunol Res 2017 06;65(3):706-712

Department of Clinical Sciences, Danderyd Hospital, Division of Internal Medicine, Karolinska Institutet, Stockholm, Sweden.

There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.
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http://dx.doi.org/10.1007/s12026-017-8905-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440486PMC
June 2017

Prospective memory in multiple sclerosis: The impact of cue distinctiveness and executive functioning.

Brain Cogn 2016 11 17;109:66-74. Epub 2016 Sep 17.

Centre hospitalier de l'Université de Montréal CHUM, Canada.

Objective: Prospective memory (PM), the ability to remember to do something at the appropriate time in the future, is crucial in everyday life. One way to improve PM performance is to increase the salience of a cue announcing that it is time to act. Multiple sclerosis (MS) patients often report PM failures and there is growing evidence of PM deficits among this population. However, such deficits are poorly characterized and their relation to cognitive status remains unclear. To better understand PM deficits in MS patients, this study investigated the impact of cue salience on PM, and its relation to retrospective memory (RM) and executive deficits.

Methods: Thirty-nine (39) MS patients were compared to 18 healthy controls on a PM task modulating cue salience during an ongoing general knowledge test.

Results: MS patients performed worse than controls on the PM task, regardless of cue salience. MS patients' executive functions contributed significantly to the variance in PM performance, whereas age, education and RM did not. Interestingly, low- and high-executive patients' performance differed when the cue was not salient, but not when it was, suggesting that low-executive MS patients benefited more from cue salience.

Conclusions: These findings add to the growing evidence of PM deficits in MS and highlight the contribution of executive functions to certain aspects of PM. In low-executive MS patients, high cue salience improves PM performance by reducing the detection threshold and need for environmental monitoring.
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http://dx.doi.org/10.1016/j.bandc.2016.07.011DOI Listing
November 2016

Priming of neutrophils toward NETosis promotes tumor growth.

Oncoimmunology 2016 May 18;5(5):e1134073. Epub 2016 Feb 18.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA; Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.

Neutrophils play a major role in cancer biology and both pro- and antitumoral functions of tumor-infiltrating neutrophils have been described. We have shown that tumors, by releasing G-CSF into the bloodstream, prime circulating neutrophils to form neutrophil extracellular traps (NETs) and we have detected the presence of NETs within the tumor microenvironment. Here, we report, using PAD4-deficient mice with a defect in neutrophil chromatin decondensation and NET formation, that the priming of neutrophils toward NETosis favors tumor growth. Interestingly, in a tumor model that does not release G-CSF and in which neutrophils are not primed for NETosis, PAD4-deficiency did not reduce tumor growth. However, supplying exogenous G-CSF to the wild-type (WT) host promoted intratumoral NETosis and tumor growth. Taken together, our results suggest that the priming of neutrophils for NETosis by the tumor or its environment leads to the accumulation of intratumoral NETs and a growth advantage to the tumor. Our work unveiled a pro-tumoral role for NETs which strengthens their potential as a new target in the fight against cancer.
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http://dx.doi.org/10.1080/2162402X.2015.1134073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910712PMC
May 2016

NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation.

Thromb Res 2016 Mar 12;139:56-64. Epub 2016 Jan 12.

Department of Clinical Sciences, Danderyd Hospital, Division of Cardiovascular Medicine, Karolinska Institutet, Stockholm, Sweden.

Introduction: Large elevations of high sensitive Troponin T (hsTnT) in ischemic stroke patients is associated with a poor outcome. In a pilot study we found a high prevalence of malignancies among these patients. Since neutrophil extracellular traps (NETs) have been linked to cancer-associated thrombosis, we hypothesized that the concomitant cerebral and myocardial ischemia could be the result of a NET-induced hypercoagulable state.

Materials And Methods: Clinical assessments, plasma analyses and autopsies with histopathology (in cases of in-hospital mortality) were performed on ischemic stroke patients with high elevations of hsTnT (N=12) and normal hsTnT (N=19).

Results: Patients with hsTnT elevation had an unexpectedly higher prevalence of cancer (p=0.002), half of which were diagnosed post-mortem. Autopsies of these patients revealed widespread myocardial, cerebral and pulmonary microthrombosis with H3Cit in thrombi. A pro-coagulant state and an increase of the NET specific marker citrullinated histone H3 (H3Cit) was found in plasma of patients with elevated hsTnT compared to patients with normal levels (p<0.001). Plasma analyses in cancer patients showed even higher H3Cit levels (p<0.001), and an increase in granulocyte colony-stimulating factor, known to prime neutrophils towards NETosis. H3Cit correlated positively with thrombin-antithrombin complex (p=0.004) and soluble P-selectin (p<0.001), further linking NETosis to the pro-thrombotic state.

Conclusions: The high prevalence of known or occult cancer in our study suggests that cancer-associated arterial microthrombosis may be underestimated. By linking the thrombosis to NETs, we suggest markers of NETosis that could aid in revealing cancer in arterial microthrombosis as well as arterial microthrombosis in cancer.
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http://dx.doi.org/10.1016/j.thromres.2016.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4769435PMC
March 2016

Role of executive functions in prospective memory in multiple sclerosis: Impact of the strength of cue-action association.

J Clin Exp Neuropsychol 2016 20;38(1):127-40. Epub 2015 Nov 20.

c Neurology Service , Hôpital Notre-Dame du CHUM , Montreal , QC , Canada.

Objectives: Patients diagnosed with multiple sclerosis (MS) often report prospective memory (PM) deficits. Although PM is important for daily functioning, it is not formally assessed in clinical practice. The aim of this study was to examine the role of executive functions in MS patients' PM revealed by the effect of strength of cue-action association on PM performance.

Method: Thirty-nine MS patients were compared to 18 healthy controls matched for age, gender, and education on a PM task modulating the strength of association between the cue and the intended action.

Results: Deficits in MS patients affecting both prospective and retrospective components of PM were confirmed using 2 × 2 × 2 mixed analyses of variance (ANOVAs). Among patients, multiple regression analyses revealed that the impairment was modulated by the efficiency of executive functions, whereas retrospective memory seemed to have little impact on PM performance, contrary to expectation. More specifically, results of 2 × 2 × 2 mixed-model analyses of covariance (ANCOVAs) showed that low-executive patients had more difficulty detecting and, especially, retrieving the appropriate action when the cue and the action were unrelated, whereas high-executive patients' performance seemed to be virtually unaffected by the cue-action association.

Conclusions: Using an objective measure, these findings confirm the presence of PM deficits in MS. They also suggest that such deficits depend on executive functioning and can be reduced when automatic PM processes are engaged through semantic cue-action association. They underscore the importance of assessing PM in clinical settings through a cognitive evaluation and offer an interesting avenue for rehabilitation.
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http://dx.doi.org/10.1080/13803395.2015.1091063DOI Listing
September 2016

Diabetes primes neutrophils to undergo NETosis, which impairs wound healing.

Nat Med 2015 Jul 15;21(7):815-9. Epub 2015 Jun 15.

1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [2] Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA. [3] Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.

Wound healing is impaired in diabetes, resulting in significant morbidity and mortality. Neutrophils are the main leukocytes involved in the early phase of healing. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs) by releasing decondensed chromatin lined with cytotoxic proteins. NETs, however, can also induce tissue damage. Here we show that neutrophils isolated from type 1 and type 2 diabetic humans and mice were primed to produce NETs (a process termed NETosis). Expression of peptidylarginine deiminase 4 (PAD4, encoded by Padi4 in mice), an enzyme important in chromatin decondensation, was elevated in neutrophils from individuals with diabetes. When subjected to excisional skin wounds, wild-type (WT) mice produced large quantities of NETs in wounds, but this was not observed in Padi4(-/-) mice. In diabetic mice, higher levels of citrullinated histone H3 (H3Cit, a NET marker) were found in their wounds than in normoglycemic mice and healing was delayed. Wound healing was accelerated in Padi4(-/-) mice as compared to WT mice, and it was not compromised by diabetes. DNase 1, which disrupts NETs, accelerated wound healing in diabetic and normoglycemic WT mice. Thus, NETs impair wound healing, particularly in diabetes, in which neutrophils are more susceptible to NETosis. Inhibiting NETosis or cleaving NETs may improve wound healing and reduce NET-driven chronic inflammation in diabetes.
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http://dx.doi.org/10.1038/nm.3887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631120PMC
July 2015

ADAMTS13 Endopeptidase Protects against Vascular Endothelial Growth Factor Inhibitor-Induced Thrombotic Microangiopathy.

J Am Soc Nephrol 2016 Jan 2;27(1):120-31. Epub 2015 Jun 2.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts; Department of Pediatrics, Harvard Medical School, Boston, Massachusetts; Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts

Thrombotic microangiopathy (TMA) is a life-threatening condition that affects some, but not all, recipients of vascular endothelial growth factor (VEGF) inhibitors given as part of chemotherapy. TMA is also a complication of preeclampsia, a disease characterized by excess production of the VEGF-scavenging soluble VEGF receptor 1 (soluble fms-like tyrosine kinase 1; sFlt-1). Risk factors for VEGF inhibitor-related TMA remain unknown. We hypothesized that deficiency of the VWF-cleaving ADAMTS13 endopeptidase contributes to the development of VEGF inhibitor-related TMA. ADAMTS13(-/-) mice overexpressing sFlt-1 presented all hallmarks of TMA, including thrombocytopenia, schistocytosis, anemia, and VWF-positive microthrombi in multiple organs. Similar to VEGF inhibitor-related TMA in humans, these mice exhibited severely impaired kidney function and hypertension. In contrast, wild-type mice overexpressing sFlt-1 developed modest hypertension but no other features of TMA. Recombinant ADAMTS13 therapy ameliorated all symptoms of TMA in ADAMTS13(-/-) mice overexpressing sFlt-1 and normalized BP in wild-type mice. ADAMTS13 activity may thus be a critical determinant for the development of TMA secondary to VEGF inhibition. Administration of recombinant ADAMTS13 may serve as a therapeutic approach to treat or prevent thrombotic complications of VEGF inhibition.
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http://dx.doi.org/10.1681/ASN.2014121165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696577PMC
January 2016

PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock.

Blood 2015 Mar 26;125(12):1948-56. Epub 2015 Jan 26.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA; Department of Pediatrics, Harvard Medical School, Boston, MA; Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA.

Neutrophil extracellular traps (NETs), consisting of nuclear DNA with histones and microbicidal proteins, are expelled from activated neutrophils during sepsis. NETs were shown to trap microbes, but they also fuel cardiovascular, thrombotic, and autoimmune disease. The role of NETs in sepsis, particularly the balance between their antimicrobial and cytotoxic actions, remains unclear. Neutrophils from peptidylarginine deiminase 4-(PAD4(-/-)) deficient mice, which lack the enzyme allowing for chromatin decondensation and NET formation, were evaluated. We found that neutrophil functions involved in bacterial killing, other than NETosis, remained intact. Therefore, we hypothesized that prevention of NET formation might not have devastating consequences in sepsis. To test this, we subjected the PAD4(-/-) mice to mild and severe polymicrobial sepsis produced by cecal ligation and puncture. Surprisingly, under septic conditions, PAD4(-/-) mice did not fare worse than wild-type mice and had comparable survival. In the presence of antibiotics, PAD4-deficiency resulted in slightly accelerated mortality but bacteremia was unaffected. PAD4(-/-) mice were partially protected from lipopolysaccharide-induced shock, suggesting that PAD4/NETs may contribute to the toxic inflammatory and procoagulant host response to endotoxin. We propose that preventing NET formation by PAD4 inhibition in inflammatory or thrombotic diseases is not likely to increase host vulnerability to bacterial infections.
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http://dx.doi.org/10.1182/blood-2014-07-587709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366625PMC
March 2015

NETosis: a new factor in tumor progression and cancer-associated thrombosis.

Semin Thromb Hemost 2014 Apr 3;40(3):277-83. Epub 2014 Mar 3.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts.

Neutrophils have long been known as innate immune cells that phagocytose and kill pathogens and mount inflammatory responses protecting the host from infection. In the past decades, new aspects of neutrophils have emerged unmasking their importance not only in inflammation but also in many pathological conditions including thrombosis and cancer. The 2004 discovery that neutrophils, upon strong activation, release decondensed chromatin to form neutrophil extracellular traps (NETs), has unveiled new avenues of research. Here, we review current knowledge regarding NETs in thrombosis, with a special focus on cancer-associated thrombosis as well as their potential role in cancer growth and metastasis. We discuss the prospective use of NET-specific biomarkers, such as citrullinated histone H3 and NET inhibitors, as tools to anticipate and fight cancer-associated thrombosis. We propose that the rapid developments in the field of NETosis may provide new targets to combat the thrombotic consequences of cancer and perhaps even help to contain the disease itself.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112728PMC
http://dx.doi.org/10.1055/s-0034-1370765DOI Listing
April 2014

Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice.

Proc Natl Acad Sci U S A 2013 May 6;110(21):8674-9. Epub 2013 May 6.

Immunology Graduate Program, Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA.

Deep vein thrombosis and pulmonary embolism are major health problems associated with high mortality. Recently, DNA-based neutrophil extracellular traps (NETs) resulting from the release of decondensed chromatin, were found to be part of the thrombus scaffold and to promote coagulation. However, the significance of nuclear decondensation and NET generation in thrombosis is largely unknown. To address this, we adopted a stenosis model of deep vein thrombosis and analyzed venous thrombi in peptidylarginine deiminase 4 (PAD4)-deficient mice that cannot citrullinate histones, a process required for chromatin decondensation and NET formation. Intriguingly, less than 10% of PAD4(-/-) mice produced a thrombus 48 h after inferior vena cava stenosis whereas 90% of wild-type mice did. Neutrophils were abundantly present in thrombi formed in both groups, whereas extracellular citrullinated histones were seen only in thrombi from wild-type mice. Bone marrow chimera experiments indicated that PAD4 in hematopoietic cells was the source of the prothrombotic effect in deep vein thrombosis. Thrombosis could be rescued by infusion of wild-type neutrophils, suggesting that neutrophil PAD4 was important and sufficient. Endothelial activation and platelet aggregation were normal in PAD4(-/-) mice, as was hemostatic potential determined by bleeding time and platelet plug formation after venous injury. Our results show that PAD4-mediated chromatin decondensation in the neutrophil is crucial for pathological venous thrombosis and present neutrophil activation and PAD4 as potential drug targets for deep vein thrombosis.
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http://dx.doi.org/10.1073/pnas.1301059110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3666755PMC
May 2013

Value of the MoCA test as a screening instrument in multiple sclerosis.

Can J Neurol Sci 2013 May;40(3):410-5

Department of Psychology, Université du Québec à Montréal.

Objective: Since a large proportion of multiple sclerosis (MS) patients exhibit cognitive deficits, it is important to have reliable and cost-effective screening measures that can be used to follow patients effectively. the objective of this study was to evaluate the clinical value of the Montreal Cognitive Assessment (MoCA) test in detecting cognitive deficits in MS patients.

Methods: Forty-one (70.1% women, mean age 44.51 ±7.43) mildly impaired (EDSS: 2.26 ±1.87) MS patients were recruited for this study. In addition to the MoCA, they were administered the MSNQ-P (patient version) and the MSNQ-I (informant version), the bDI-FS and a comprehensive neuropsychological test battery.

Results: there were significant correlations between the MoCA test and the three factors derived from the neuropsychological evaluation (Executive/speed of processing, Learning, Delayed recall). the MoCA test was correlated with the MSNQ-I but only marginally with the MSNQ-P. In addition, there was no significant correlation between the MSNQ-P and the neuropsychological factors, whereas significant correlations were found between two of those factors (Learning and Delayed recall) and the MSNQ-I, suggesting that the informant version is more reliable than the patient version for the presence of cognitive deficits.

Conclusion: the results obtained in the present study support the value of the MoCA test as a screening tool for the presence of cognitive dysfunction in MS patients, even in patients with mild functional disability (EDSS).
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http://dx.doi.org/10.1017/s0317167100014384DOI Listing
May 2013

Neutrophil extracellular traps: A new link to cancer-associated thrombosis and potential implications for tumor progression.

Oncoimmunology 2013 Feb;2(2):e22946

Program in Cellular and Molecular Medicine; Boston Children's Hospital; Boston, MA USA ; Department of Pediatrics; Harvard Medical School; Boston, MA USA.

Cancers prime neutrophils to release extracellular DNA traps through the systemic release of granulocyte colony-stimulating factor (G-CSF). We recently showed that these circulating neutrophil extracellular traps (NETs) promote the establishment of a pro-thrombotic state. The role of NETs in cancer biology and tumor progression may prove much more than an unfortunate side effect of cancer.
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http://dx.doi.org/10.4161/onci.22946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601165PMC
February 2013

Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice.

Blood 2013 Feb 12;121(6):1008-15. Epub 2012 Dec 12.

Immune Disease Institute, Boston, MA 02115, USA.

The majority of peripheral serotonin is stored in platelets, which secrete it on activation. Serotonin releases Weibel-Palade bodies (WPBs) and we asked whether absence of platelet serotonin affects neutrophil recruitment in inflammatory responses. Tryptophan hydroxylase (Tph)1–deficient mice, lacking non-neuronal serotonin, showed mild leukocytosis compared with wild-type (WT), primarily driven by an elevated neutrophil count. Despite this, 50% fewer leukocytes rolled on unstimulated mesenteric venous endothelium of Tph1(-/-) mice. The velocity of rolling leukocytes was higher in Tph1(-/-) mice, indicating fewer selectin-mediated interactions with endothelium. Stimulation of endothelium with histamine, a secretagogue of WPBs, or injection of serotonin normalized the rolling in Tph1(-/-) mice. Diminished rolling in Tph1(-/-) mice resulted in reduced firm adhesion of leukocytes after lipopolysaccharide treatment. Blocking platelet serotonin uptake with fluoxetine in WT mice reduced serum serotonin by > 80% and similarly reduced leukocyte rolling and adhesion. Four hours after inflammatory stimulation, neutrophil extravasation into lung, peritoneum, and skin wounds was reduced in Tph1(-/-) mice, whereas in vitro neutrophil chemotaxis was independent of serotonin. Survival of lipopolysaccharide-induced endotoxic shock was improved in Tph1(-/-) mice. In conclusion, platelet serotonin promotes the recruitment of neutrophils in acute inflammation, supporting an important role for platelet serotonin in innate immunity.
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http://dx.doi.org/10.1182/blood-2012-06-437392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567335PMC
February 2013

Isoflurane inhibits neutrophil recruitment in the cutaneous Arthus reaction model.

J Anesth 2013 Apr 25;27(2):261-8. Epub 2012 Oct 25.

Immune Disease Institute, Boston, MA, USA.

Purpose: Neutrophil recruitment to the inflammatory sites is regulated by a variety of adhesion molecules including β2 integrins. The dependency of neutrophil recruitment on β2 integrins is variable in different tissues, but has not yet been verified in the cutaneous passive reverse Arthus reaction. We examined this question and also evaluated the impact of isoflurane on neutrophil recruitment to the skin because we previously showed in vitro that isoflurane binds and inhibits β2 integrins.

Methods: The dependency on β2 integrins in neutrophil recruitment to the skin in the Arthus reaction was examined using αL, αM and β2 knockout mice. Then, we evaluated the effect of isoflurane on neutrophil recruitment to the skin. In addition, the effects of isoflurane on neutrophil binding to intercellular adhesion molecule-1 (ICAM-1), one of the β2 integrin ligands, were studied in vitro using cell adhesion assays.

Results: Neutrophil recruitment to the skin in the Arthus reaction model was totally dependent on β2 integrins, as β2 knockout mice completely abolished it. However, the defect of only one of the β2 integrins was not sufficient to abolish neutrophil recruitment. Isoflurane reduced neutrophil recruitment to the skin by approximately 90 %. Also, isoflurane inhibited neutrophil adhesion to β2 integrin ligand ICAM-1.

Conclusions: We demonstrated that (1) neutrophil recruitment to the skin was totally dependent on β2 integrins, and (2) isoflurane significantly impaired neutrophil recruitment. Based on the previous studies on the contribution of other adhesion molecules in neutrophil recruitment, it is likely that isoflurane at least partially affects on β2 integrins in this model.
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http://dx.doi.org/10.1007/s00540-012-1508-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3568683PMC
April 2013

Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis.

Proc Natl Acad Sci U S A 2012 Aug 23;109(32):13076-81. Epub 2012 Jul 23.

Immune Disease Institute, Boston, MA 02115, USA.

Cancer-associated thrombosis often lacks a clear etiology. However, it is linked to a poor prognosis and represents the second-leading cause of death in cancer patients. Recent studies have shown that chromatin released into blood, through the generation of neutrophil extracellular traps (NETs), is procoagulant and prothrombotic. Using a murine model of chronic myelogenous leukemia, we show that malignant and nonmalignant neutrophils are more prone to NET formation. This increased sensitivity toward NET generation is also observed in mammary and lung carcinoma models, suggesting that cancers, through a systemic effect on the host, can induce an increase in peripheral blood neutrophils, which are predisposed to NET formation. In addition, in the late stages of the breast carcinoma model, NETosis occurs concomitant with the appearance of venous thrombi in the lung. Moreover, simulation of a minor systemic infection in tumor-bearing, but not control, mice results in the release of large quantities of chromatin and a prothrombotic state. The increase in neutrophil count and their priming is mediated by granulocyte colony-stimulating factor (G-CSF), which accumulates in the blood of tumor-bearing mice. The prothrombotic state in cancer can be reproduced by treating mice with G-CSF combined with low-dose LPS and leads to thrombocytopenia and microthrombosis. Taken together, our results identify extracellular chromatin released through NET formation as a cause for cancer-associated thrombosis and unveil a target in the effort to decrease the incidence of thrombosis in cancer patients.
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http://dx.doi.org/10.1073/pnas.1200419109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420209PMC
August 2012

ADAMTS13 exerts a thrombolytic effect in microcirculation.

Thromb Haemost 2012 Sep 10;108(3):527-32. Epub 2012 Jul 10.

Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital Boston, Boston, MA 02115, USA.

Recombinant tissue plasminogen activator (r-tPA) is the drug of choice for thrombolysis, but it is associated with a significant risk of bleeding and is not always successful. By cleaving von Willebrand factor (VWF), the metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) down-regulates thrombus formation in injured vessels. We investigated whether recombinant ADAMTS13 (r-ADAMTS13) induces thrombolysis in vivo in mice. Thrombosis was produced by ferric chloride-induced (FeCl(3)) injury in the venules of a dorsal skinfold chamber. Phosphate-buffered saline (PBS, vehicle), r-tPA or r-ADAMTS13, supplemented with hirudin (to stop on-going thrombin generation), was directly applied onto the occluded vessel, and thrombus dissolution was evaluated by intravital microscopy. The incidence of blood flow restoration significantly increased 30 minutes (min) after r-ADAMTS13 vs. PBS treatment (60% vs. 0%, p<0.05) and 60 min after r-tPA treatment (75% vs. 17%, p<0.05). Both r-tPA and r-ADAMTS13 significantly reduced thrombus size 60 min after their superfusion (53.2% and 62.3% of the initial thrombus size, p<0.05 and p<0.01, respectively). Bleeding occurred in all r-tPA-treated chambers, while it was absent in mice treated with r-ADAMTS13 or PBS. We observed that, similar to r-tPA, r-ADAMTS13 can dissolve occlusive thrombi induced by FeCl(3) injury in venules. In contrast to r-tPA, the in vivo thrombolytic effect of ADAMTS13 was not associated with any signs of haemorrhage. ADAMTS13 could represent a new therapeutic option for thrombolysis.
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http://dx.doi.org/10.1160/TH12-01-0046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121884PMC
September 2012

Targeting platelet function to improve drug delivery.

Oncoimmunology 2012 Jan;1(1):100-102

Immune Disease Institute; Program in Cellular and Molecular Medicine; Children's Hospital Boston; Department of Pediatrics; Harvard Medical School; Boston, MA USA.

Thrombocytopenia-induced tumor hemorrhage improves drug delivery to tumors. This phenomenon presents a new way to increase drug efficacy with minimal side effects. Combining anti-platelet treatment with therapeutic drugs may help us in the search for more effective ways to fight cancer.
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http://dx.doi.org/10.4161/onci.1.1.17962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376975PMC
January 2012

Increased efficacy of breast cancer chemotherapy in thrombocytopenic mice.

Cancer Res 2011 Mar 6;71(5):1540-9. Epub 2011 Jan 6.

The Immune Disease Institute, Boston, Massachusetts 02115, USA.

Platelets contribute to homeostasis of the tumor vasculature by helping prevent hemorrhage. Thus, we hypothesized that inducing thrombocytopenia would increase tumor vascular leakiness and facilitate the effective delivery of chemotherapeutic agents to tumors. In a mammary carcinoma murine model, platelet depletion induced bleeding specifically at the tumor site, favoring the accumulation of fluorescently labeled microspheres only in the tumor. Moreover, induction of thrombocytopenia in tumor-bearing mice before injection of paclitaxel increased its intratumoral accumulation and reduced growth of both slow- and fast-growing tumors, compared with mice with normal platelet counts that were treated only with paclitaxel. Histologic analysis confirmed the expectation of an increase in tumor apoptosis and a reduction in tumor proliferation in thrombocytopenic mice receiving chemotherapy. No increased toxicity was seen in other organs or blood cells. Taken together, our results indicate that low platelet count selectively induces leakiness of tumor vessels and favors the delivery of chemotherapy to tumor sites, enhancing its tumoricidal effects.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-2038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078642PMC
March 2011

Overexpression of galectin-7, a myoepithelial cell marker, enhances spontaneous metastasis of breast cancer cells.

Am J Pathol 2010 Jun 9;176(6):3023-31. Epub 2010 Apr 9.

Institut national de la recherche scientifique-Institut Armand-Frappier, Laval, Québec, Canada, H7V 1B7.

Galectins are members of a family of beta-galactosides-binding proteins that have recently emerged as novel modulators in different aspects of cancer. The expression of galectins in tumors and/or the tissue surrounding them has been well documented. Since galectin-7 expression has been associated with epithelial tissues and varies significantly in various types of cancer, we have investigated for the first time its role in breast cancer. Using two preclinical mouse models, high levels of galectin-7 expression in breast cancer cells drastically increased their ability to metastasize to lungs and bones. Significant increases in the number of pulmonary metastases and osteolytic lesions were induced by overexpression of galectin-7 compared with control cells. In human tissues, galectin-7 was specifically found in myoepithelial cells of normal human breast tissue, but not in luminal cells. Its expression was severely altered in breast carcinoma, many samples showing greater than 70% of galectin-7 positive cells. High expression levels of galectin-7 were restricted to high-grade breast carcinomas, including HER2 overexpressing and basal-like groups. In HER2 overexpressing cases, galectin-7 expression was associated with lymph node axillary metastasis. Taken together, our results indicate that galectin-7 may represent a potential target for both specific detection and therapeutic inhibition of metastatic breast cancer.
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http://dx.doi.org/10.2353/ajpath.2010.090876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877862PMC
June 2010

Innate immune cells induce hemorrhage in tumors during thrombocytopenia.

Am J Pathol 2009 Oct 3;175(4):1699-708. Epub 2009 Sep 3.

Immune Disease Institute, Boston, MA 02115, USA.

Platelets are crucial regulators of tumor vascular homeostasis and continuously prevent tumor hemorrhage through secretion of their granules. However, the reason for tumor bleeding in the absence of platelets remains unknown. Tumors are associated with inflammation, a cause of hemorrhage in thrombocytopenia. Here, we investigated the role of the inflamed tumor microenvironment in the induction of tumor vessel injury in thrombocytopenic mice. Using s.c. injections of vascular endothelial growth factor or tumor necrosis factor-alpha combined with depletion of neutrophils, we demonstrate that enhancing the opening of endothelial cell junctions was not sufficient to cause bleeding in the absence of platelets; instead, induction of tissue hemorrhage in thrombocytopenia required recruitment of leukocytes. Immunohistology revealed that thrombocytopenia-induced tumor hemorrhage occurs at sites of macrophage and neutrophil accumulation. Mice deficient in beta2 or beta3 integrins, which have decreased neutrophil and/or macrophage infiltration in their tumor stroma, were protected from thrombocytopenia-induced tumor hemorrhage, indicating that, in the absence of platelets, stroma-infiltrating leukocytes induced tumor vessel injury. This injury was independent of reactive oxygen species generation and of complement activation, as suggested by the persistence of tumor hemorrhage in C3- and nicotinamide adenine dinucleotide phosphate oxidase-deficient thrombocytopenic mice. Our results show that platelets counteract tumor-associated inflammation and that the absence of this platelet function elicits vascular injuries by tumor-infiltrating innate immune cells.
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http://dx.doi.org/10.2353/ajpath.2009.090460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2751565PMC
October 2009

Increased galectin-7 gene expression in lymphoma cells is under the control of DNA methylation.

Biochem Biophys Res Commun 2009 Sep 9;387(3):425-9. Epub 2009 Jul 9.

INRS-Institut Armand-Frappier, Laval, Quebec, Canada.

Recent studies have reported that elevated levels of galectin-7 in different types of cancer. The mechanisms underlying its abnormal regulation in cancer cells remain, however, unknown. Here, we have examined the relationship between galectin-7 and p53, a gene previously associated with upregulation of galectin-7. While RNA and protein analyses revealed a consistent and irreversible upregulation of galectin-7 throughout progression of lymphoma, no correlation with p53 was found. In fact, most of the lymphoma cell lines expressing high levels of galectin-7 did not express any detectable level of p53, although expressed p21(Waf1/Cip1) gene following doxorubicin treatment, indicating that p53 was functional in these cells. Methylation-specific polymerase chain reaction (MS-PCR) analyses rather suggested that galectin-7 expression was associated with changes in DNA methylation. This conclusion was supported by data using demethylating agent 5-aza-dC. Furthermore, disruption of the DNA methylases dnmt1 and dnmt3a induced galectin-7. Collectively, our data suggest that abnormal expression of galectin-7 in lymphoma cells is not dependent on p53, but is rather associated with DNA hypomethylation.
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http://dx.doi.org/10.1016/j.bbrc.2009.07.015DOI Listing
September 2009

Galectin-7 in lymphoma: elevated expression in human lymphoid malignancies and decreased lymphoma dissemination by antisense strategies in experimental model.

Cancer Res 2007 Mar;67(6):2824-9

INRS-Institut Armand-Frappier, University of Québec, Laval, Québec, Canada.

Galectin-7 is found mainly in stratified squamous epithelia as well as in various other types of cancer cells. As with other members of the galectin family, the expression of galectin-7 has been shown to negatively regulate the development of some tumors while correlating with the progression of other tumor types. For example, up-regulation of galectin-7 is associated with rat mammary carcinomas and with progression to T-cell malignancy. Here, we provide evidence indicating that galectin-7 functions as an important molecule in the dissemination of lymphoma cells in vivo. We found that stable transfection of lymphoma cells with a plasmid encoding antisense galectin-7 cDNA significantly inhibited the dissemination and invasion of lymphoma cells to peripheral organs, thereby increasing the survival of mice. We also found that inhibition of galectin-7 in aggressive lymphoma cells correlated with a decreased invasion of tumor cells in target organs and a reduced expression of matrix metalloproteinase-9, a gene associated with a poor prognosis in non-Hodgkin's lymphoma. We finally examined the expression of galectin-7 in 50 specimens of different mature B-cell neoplasms and found high galectin-7 expression levels in a significant proportion of mature B-cell neoplasms but not in normal B cells. Taken together, these findings suggest that galectin-7 is a potential therapeutic target in the treatment of lymphoid malignancies.
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http://dx.doi.org/10.1158/0008-5472.CAN-06-3891DOI Listing
March 2007

The role of DNA hypomethylation in the control of stromelysin gene expression.

Biochem Biophys Res Commun 2006 Apr 21;342(4):1233-9. Epub 2006 Feb 21.

Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, University of Quebec, Laval, Que., Canada.

Genome-wide DNA hypomethylation is a critical mechanism underlying neoplastic transformation. Thus, genes that are suppressed in normal tissues or in specific cell types may become aberrantly expressed in neoplasia. To determine whether DNA methylation can modulate matrix metalloproteinase (mmp) gene expression, we have used a genetically engineered cell line in which both key DNA methyltransferase genes, Dnmt-1 and Dnmt-3b, were removed by homologous recombination. We found that cells bearing a dual knock-out of both Dnmt-1 and Dnmt-3b genes induced de novo expression of mmp-3 gene, but not that of mmp-1 and mmp-2. Furthermore, treatment of the wild-type cells with DNA methylase inhibitors 5-aza-dC and zebularine also induced mmp-3 gene expression. On the other hand, in vitro methylation of the mmp-3 promoter suppressed its transcriptional activity. Finally, we found that induction of mmp-3 and mmp-10 gene expression by hypomethylation was cell-specific, suggesting that epigenetic changes may predispose cells to express stromelysin genes.
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http://dx.doi.org/10.1016/j.bbrc.2006.02.068DOI Listing
April 2006

New roles for matrix metalloproteinases in metastasis.

Crit Rev Immunol 2005 ;25(6):493-523

INRS-Institut Armand-Frappier, University of Québec, Laval, Québec, Canada.

To form tumors successfully at sites remote from the primary tumor, metastatic cells must be endowed with particular properties. They must detach from the primary tumor and enter the blood circulation, where they must resist hemodynamic shearstress, "home" to the target organ, successfully extravasate, and then migrate through dense stroma to a site favorable for tumor growth. Recent results with genetically engineered mouse models have generated data which clearly challenge the classic dogma stating that matrix metalloproteinases (MMPs) promote metastasis solely by modulating the remodeling of extracellular matrix (ECM). Instead, it is becoming clear that MMPs and their natural inhibitors have multiple biological functions that not only challenge our view on how MMPs promote metastasis, but also raise for the first time the idea that secretion of MMPs by the host could protect it from tumor growth, at least in some types of cancer or at specific stages of tumor progression.
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http://dx.doi.org/10.1615/critrevimmunol.v25.i6.30DOI Listing
February 2006

[Galectin-7: a novel gene associated with metastasis].

Med Sci (Paris) 2005 Oct;21(10):790-2

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http://dx.doi.org/10.1051/medsci/20052110790DOI Listing
October 2005