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1.
Stroke ; 51(6): 1886-1890, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32404037

RESUMEN

Background and Purpose- Previous experimental studies found that the infusion of human purified nascent HDL (high-density lipoprotein) significantly reduced infarct volume and hemorrhagic transformation rate by decreasing neutrophil recruitment. ApoA1-M (apolipoprotein A1-Milano) is a natural variant of human ApoA1 that confers protection against atherosclerosis. Recombinant ApoA1-M has been formulated as a complex with phospholipids to mimic the properties of nascent HDL. The aim of this study was to assess the impact of intravenous ApoA1-M in a transient middle cerebral artery occlusion stroke model in rats. Methods- In a first experiment, rats were subjected to 120-minute transient middle cerebral artery occlusion and intravenous ApoA1-M was infused immediately or 4 hours after occlusion. In a second experiment, rats were subjected to 240-minute transient middle cerebral artery occlusion and intravenous ApoA1-M was infused with or without recombinant tPA (tissue-type plasminogen activator) immediately after recanalization. Primary outcome criteria were the infarct volume and hemorrhagic transformation rate measured at 24 hours. Platelets, coagulation, and neutrophil activation biomarkers were measured in brain homogenates and plasma. Additional in vitro experiments studied the effects of ApoA1-M on platelet aggregation and platelet-neutrophil interactions. Results- The infusion of ApoA1-M immediately or 4 hours after 120-minute transient middle cerebral artery occlusion significantly reduced the infarct volume compared with saline (P=0.034 and P=0.036, respectively). Compared with tPA alone, co-administration of ApoA1-M and tPA showed similar rates of hemorrhagic transformation. ApoA1-M had no significant inhibition effect on neutrophil activation biomarkers. Platelet activation was slightly decreased in rats treated with ApoA1-M compared with saline. In vitro, the incubation of human and rat platelet-rich plasma with ApoA1-M significantly reduced ADP-induced platelet aggregation (P=0.001 and P=0.02, respectively). Conclusions- ApoA1-Milano significantly decreased the infarct volume through an inhibition of platelet aggregation but did not reduce hemorrhagic transformation and neutrophils activation as expected after previous experimental studies with nascent HDL. Visual Overview- An online visual overview is available for this article.


Asunto(s)
Apolipoproteína A-I/farmacología , Aterosclerosis/prevención & control , Infarto de la Arteria Cerebral Media/prevención & control , Animales , Aterosclerosis/sangre , Aterosclerosis/patología , Biomarcadores/sangre , Plaquetas/metabolismo , Plaquetas/patología , Modelos Animales de Enfermedad , Humanos , Infarto de la Arteria Cerebral Media/sangre , Infarto de la Arteria Cerebral Media/patología , Masculino , Activación Neutrófila/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/patología , Agregación Plaquetaria/efectos de los fármacos , Ratas , Proteínas Recombinantes/farmacología
2.
Prostaglandins Other Lipid Mediat ; 121(Pt A): 4-16, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26463849

RESUMEN

The prostanoid E2 (PGE2) is known to modulate the aggregative response of platelets to their conventional agonists such as ADP, TXA2, thrombin or collagen. Through the activation of its receptor EP3, PGE2 sensitizes platelets to their agonists but also inhibits them through its two other receptors, EP2 and EP4. In mice, the net result of these opposed actions is the EP3-mediated potentiation of platelet aggregation and the in vivo aggravation of murine atherothrombosis. Since the pathway PGE2/EP3 is not involved in murine hemostasis, we propose a "platelet EP3 paradigm" to describe this apparently paradoxical association between the facilitating impact on atherothrombosis and the unaltered hemostasis. Consistent with this paradigm, a drug blocking EP3 dramatically decreased atherothrombosis without inducing bleeding in mice. In humans, several studies did not agree on the effect of PGE2 on platelets. Reinterpreting these data with the notion of "potentiation window" and taking the platelet initial cAMP level into account reconciled these inconsistent results. Thereby, the in vitro potentiating effect of PGE2 on human platelets becomes clear. In addition, the EP3 blocking drug DG-041 abrogated the potentiating effect of PGE2 in whole human blood but did not prolong bleeding times in volunteers. Thus, the murine "platelet EP3 paradigm" would apply to humans if the aggravating role of PGE2 on atherothrombosis is shown in patients. Therefore, testing an EP3 blocker in a phase III trial would be of high interest to fulfill the unmet medical need which is to control atherothrombosis without impacting hemostasis and thus to improve the prevention of myocardial infarction.


Asunto(s)
Aterosclerosis/complicaciones , Dinoprostona/metabolismo , Hemorragia/inducido químicamente , Terapia Molecular Dirigida/métodos , Subtipo EP3 de Receptores de Prostaglandina E/metabolismo , Trombosis/complicaciones , Trombosis/prevención & control , Animales , Dinoprostona/biosíntesis , Humanos , Subtipo EP3 de Receptores de Prostaglandina E/antagonistas & inhibidores , Trombosis/metabolismo , Trombosis/fisiopatología
3.
J Immunother Cancer ; 12(4)2024 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-38580334

RESUMEN

BACKGROUND: Checkpoint inhibitor-induced hepatitis (CPI-hepatitis) is an emerging problem with the widening use of CPIs in cancer immunotherapy. Here, we developed a mouse model to characterize the mechanism of CPI-hepatitis and to therapeutically target key pathways driving this pathology. METHODS: C57BL/6 wild-type (WT) mice were dosed with toll-like receptor (TLR)9 agonist (TLR9-L) for hepatic priming combined with anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) plus anti-programmed cell death 1 (PD-1) ("CPI") or phosphate buffered saline (PBS) control for up to 7 days. Flow cytometry, histology/immunofluorescence and messenger RNA sequencing were used to characterize liver myeloid/lymphoid subsets and inflammation. Hepatocyte damage was assessed by plasma alanine transaminase (ALT) and cytokeratin-18 (CK-18) measurements. In vivo investigations of CPI-hepatitis were carried out in Rag2-/- and Ccr2rfp/rfp transgenic mice, as well as following anti-CD4, anti-CD8 or cenicriviroc (CVC; CCR2/CCR5 antagonist) treatment. RESULTS: Co-administration of combination CPIs with TLR9-L induced liver pathology closely resembling human disease, with increased infiltration and clustering of granzyme B+perforin+CD8+ T cells and CCR2+ monocytes, 7 days post treatment. This was accompanied by apoptotic hepatocytes surrounding these clusters and elevated ALT and CK-18 plasma levels. Liver RNA sequencing identified key signaling pathways (JAK-STAT, NF-ΚB) and cytokine/chemokine networks (Ifnγ, Cxcl9, Ccl2/Ccr2) as drivers of CPI-hepatitis. Using this model, we show that CD8+ T cells mediate hepatocyte damage in experimental CPI-hepatitis. However, their liver recruitment, clustering, and cytotoxic activity is dependent on the presence of CCR2+ monocytes. The absence of hepatic monocyte recruitment in Ccr2rfp/rfp mice and CCR2 inhibition by CVC treatment in WT mice was able to prevent the development and reverse established experimental CPI-hepatitis. CONCLUSION: This newly established mouse model provides a platform for in vivo mechanistic studies of CPI-hepatitis. Using this model, we demonstrate the central role of liver infiltrating CCR2+ monocyte interaction with tissue-destructive CD8+ T cells in the pathogenesis of CPI-hepatitis and highlight CCR2 inhibition as a novel therapeutic target.


Asunto(s)
Hepatitis , Monocitos , Humanos , Ratones , Animales , Linfocitos T CD8-positivos , Receptor Toll-Like 9 , Ratones Endogámicos C57BL , Hepatitis/tratamiento farmacológico , Hepatitis/etiología
4.
J Exp Clin Cancer Res ; 43(1): 84, 2024 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-38493157

RESUMEN

BACKGROUND: How platelets interact with and influence the tumor microenvironment (TME) remains poorly characterized. METHODS: We compared the presence and participation of platelets in the TME of two tumors characterized by highly different TME, PyMT AT-3 mammary tumors and B16F1 melanoma. RESULTS: We show that whereas firmly adherent platelets continuously line tumor vessels of both AT-3 and B16F1 tumors, abundant extravascular stromal clusters of platelets from thrombopoietin-independent origin were present only in AT-3 mammary tumors. We further show that platelets influence the angiogenic and inflammatory profiles of AT-3 and B16F1 tumors, though with very different outcomes according to tumor type. Whereas thrombocytopenia increased bleeding in both tumor types, it further caused severe endothelial degeneration associated with massive vascular leakage, tumor swelling, and increased infiltration of cytotoxic cells, only in AT-3 tumors. CONCLUSIONS: These results indicate that while platelets are integral components of solid tumors, their localization and origin in the TME, as well as their impact on its shaping, are tumor type-dependent.


Asunto(s)
Neoplasias Mamarias Animales , Microambiente Tumoral , Animales , Humanos
5.
Arthritis Rheumatol ; 75(1): 84-97, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36428281

RESUMEN

OBJECTIVE: Syk is a cytoplasmic protein tyrosine kinase that plays a role in signaling via B cell and Fc receptors (FcR). FcR engagement and signaling via Syk is thought to be important in antineutrophil cytoplasm antibody (ANCA) IgG-mediated neutrophil activation. This study was undertaken to investigate the role of Syk in ANCA-induced myeloid cell activation and vasculitis pathogenesis. METHODS: Phosphorylation of Syk in myeloid cells from healthy controls and ANCA-associated vasculitis (AAV) patients was analyzed using flow cytometry. The effect of Syk inhibition on myeloperoxidase (MPO)-ANCA IgG activation of cells was investigated using functional assays (interleukin-8 and reactive oxygen species production) and targeted gene analysis with NanoString. Total and phosphorylated Syk at sites of tissue inflammation in patients with AAV was assessed using immunohistochemistry and RNAscope in situ hybridization. RESULTS: We identified increased phosphorylated Syk at critical activatory tyrosine residues in blood neutrophils and monocytes from patients with active AAV compared to patients with disease in remission or healthy controls. Syk was phosphorylated in vitro following MPO-ANCA IgG stimulation, and Syk inhibition was able to prevent ANCA-mediated cellular responses. Using targeted gene expression analysis, we identified up-regulation of FcR- and Syk-dependent signaling pathways following MPO-ANCA IgG stimulation. Finally, we showed that Syk is expressed and phosphorylated in tissue leukocytes at sites of organ inflammation in AAV. CONCLUSION: These findings indicate that Syk plays a critical role in MPO-ANCA IgG-induced myeloid cell responses and that Syk is activated in circulating immune cells and tissue immune cells in AAV; therefore, Syk inhibition may be a potential therapeutic option.


Asunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos , Anticuerpos Anticitoplasma de Neutrófilos , Humanos , Inflamación , Receptores Fc , Inmunoglobulina G , Inmunidad Innata , Peroxidasa , Quinasa Syk
6.
J Clin Invest ; 131(4)2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33320839

RESUMEN

Patients with acute liver failure (ALF) have systemic innate immune suppression and increased susceptibility to infections. Programmed cell death 1 (PD-1) expression by macrophages has been associated with immune suppression during sepsis and cancer. We therefore examined the role of the programmed cell death 1/programmed death ligand 1 (PD-1/PD-L1) pathway in regulating Kupffer cell (KC) inflammatory and antimicrobial responses in acetaminophen-induced (APAP-induced) acute liver injury. Using intravital imaging and flow cytometry, we found impaired KC bacterial clearance and systemic bacterial dissemination in mice with liver injury. We detected increased PD-1 and PD-L1 expression in KCs and lymphocyte subsets, respectively, during injury resolution. Gene expression profiling of PD-1+ KCs revealed an immune-suppressive profile and reduced pathogen responses. Compared with WT mice, PD-1-deficient mice and anti-PD-1-treated mice with liver injury showed improved KC bacterial clearance, a reduced tissue bacterial load, and protection from sepsis. Blood samples from patients with ALF revealed enhanced PD-1 and PD-L1 expression by monocytes and lymphocytes, respectively, and that soluble PD-L1 plasma levels could predict outcomes and sepsis. PD-1 in vitro blockade restored monocyte functionality. Our study describes a role for the PD-1/PD-L1 axis in suppressing KC and monocyte antimicrobial responses after liver injury and identifies anti-PD-1 immunotherapy as a strategy to reduce infection susceptibility in ALF.


Asunto(s)
Bacterias/inmunología , Infecciones Bacterianas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Macrófagos del Hígado/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Acetaminofén/efectos adversos , Acetaminofén/uso terapéutico , Adulto , Animales , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/microbiología , Femenino , Humanos , Macrófagos del Hígado/microbiología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología
7.
Elife ; 102021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33704068

RESUMEN

End-stage kidney disease (ESKD) patients are at high risk of severe COVID-19. We measured 436 circulating proteins in serial blood samples from hospitalised and non-hospitalised ESKD patients with COVID-19 (n = 256 samples from 55 patients). Comparison to 51 non-infected patients revealed 221 differentially expressed proteins, with consistent results in a separate subcohort of 46 COVID-19 patients. Two hundred and three proteins were associated with clinical severity, including IL6, markers of monocyte recruitment (e.g. CCL2, CCL7), neutrophil activation (e.g. proteinase-3), and epithelial injury (e.g. KRT19). Machine-learning identified predictors of severity including IL18BP, CTSD, GDF15, and KRT19. Survival analysis with joint models revealed 69 predictors of death. Longitudinal modelling with linear mixed models uncovered 32 proteins displaying different temporal profiles in severe versus non-severe disease, including integrins and adhesion molecules. These data implicate epithelial damage, innate immune activation, and leucocyte-endothelial interactions in the pathology of severe COVID-19 and provide a resource for identifying drug targets.


COVID-19 varies from a mild illness in some people to fatal disease in others. Patients with severe disease tend to be older and have underlying medical problems. People with kidney failure have a particularly high risk of developing severe or fatal COVID-19. Patients with severe COVID-19 have high levels of inflammation, causing damage to tissues around the body. Many drugs that target inflammation have already been developed for other diseases. Therefore, to repurpose existing drugs or design new treatments, it is important to determine which proteins drive inflammation in COVID-19. Here, Gisby, Clarke, Medjeral-Thomas et al. measured 436 proteins in the blood of patients with kidney failure and compared the levels between patients who had COVID-19 to those who did not. This revealed that patients with COVID-19 had increased levels of hundreds of proteins involved in inflammation and tissue injury. Using a combination of statistical and machine learning analyses, Gisby et al. probed the data for proteins that might predict a more severe disease progression. In total, over 200 proteins were linked to disease severity, and 69 with increased risk of death. Tracking how levels of blood proteins changed over time revealed further differences between mild and severe disease. Comparing this data with a similar study of COVID-19 in people without kidney failure showed many similarities. This suggests that the findings may apply to COVID-19 patients more generally. Identifying the proteins that are a cause of severe COVID-19 ­ rather than just correlated with it ­ is an important next step that could help to select new drugs for severe COVID-19.


Asunto(s)
COVID-19/sangre , Fallo Renal Crónico/sangre , Fallo Renal Crónico/virología , Diálisis Renal/métodos , Anciano , Biomarcadores/sangre , COVID-19/mortalidad , COVID-19/virología , Femenino , Predicción , Hospitalización , Humanos , Fallo Renal Crónico/mortalidad , Fallo Renal Crónico/terapia , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Pronóstico , Proteómica/métodos , Diálisis Renal/mortalidad , SARS-CoV-2/aislamiento & purificación , Índice de Severidad de la Enfermedad
8.
Elife ; 92020 11 25.
Artículo en Inglés | MEDLINE | ID: mdl-33236983

RESUMEN

Neutrophils constitute the largest population of phagocytic granulocytes in the blood of mammals. The development and function of neutrophils and monocytes is primarily governed by the granulocyte colony-stimulating factor receptor family (CSF3R/CSF3) and macrophage colony-stimulating factor receptor family (CSF1R/IL34/CSF1) respectively. Using various techniques this study considered how the emergence of receptor:ligand pairings shaped the distribution of blood myeloid cell populations. Comparative gene analysis supported the ancestral pairings of CSF1R/IL34 and CSF3R/CSF3, and the emergence of CSF1 later in lineages after the advent of Jawed/Jawless fish. Further analysis suggested that the emergence of CSF3 lead to reorganisation of granulocyte distribution between amphibian and early reptiles. However, the advent of endothermy likely contributed to the dominance of the neutrophil/heterophil in modern-day mammals and birds. In summary, we show that the emergence of CSF3R/CSF3 was a key factor in the subsequent evolution of the modern-day mammalian neutrophil.


Asunto(s)
Evolución Biológica , Cordados/fisiología , Factores Estimulantes de Colonias/farmacología , Simulación por Computador , Células Mieloides/fisiología , Neutrófilos/fisiología , Animales , Cordados/genética , Regulación de la Expresión Génica/fisiología , Especificidad de la Especie
9.
Res Pract Thromb Haemost ; 2(2): 228-239, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30046725

RESUMEN

Glycoprotein VI (GPVI), the main platelet receptor for collagen, has been shown to play a central role in various models of thrombosis, and to be a minor actor of hemostasis at sites of trauma. These observations have made of GPVI a novel target for antithrombotic therapy, as its inhibition would ideally combine efficacy with safety. Nevertheless, recent studies have indicated that GPVI could play an important role in preventing bleeding caused by neutrophils in the inflamed skin and lungs. Remarkably, there is evidence that the GPVI-dependent hemostatic function of platelets at the acute phase of inflammation in these organs does not involve aggregation. From a therapeutic perspective, the vasculoprotective action of GPVI in inflammation suggests that blocking of GPVI might bear some risks of bleeding at sites of neutrophil infiltration. In this review, we summarize recent findings on GPVI functions in inflammation and discuss their possible clinical implications and applications.

10.
Cardiovasc Res ; 114(12): 1656-1666, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-29800147

RESUMEN

Aims: Both leukotrienes and neutrophils have been linked to plaque destabilization. Despite being evoked, the role of leukotriene B4 (LTB4) in neutrophil recruitment to plaques and the concomitant effects of these two actors on plaque stability remain to be proven. Since both actors are elicited during endotoxaemia, a condition associated with the risk of cardiovascular events, we investigated whether endotoxaemia promotes LTB4-mediated neutrophil infiltration in plaques and explored the roles of LTB4 and neutrophils in plaque destabilization. Methods and results: Endotoxaemia induced by repeated peritoneal endotoxin injections at a non-lethal dose (1.5 mg/kg, 5 days) in chow-fed aged Apoe-/- mice (over 45 weeks old) resulted in neutrophil infiltration and activation in plaques. Subsequently to neutrophil invasion, plaques exhibited increased features of vulnerability: reduced collagen content, expanded necrotic cores, and thinned fibrous caps. These plaque features were reproduced by direct deposition of isolated neutrophils onto murine atheromatous carotid arteries in an in vivo assay. In endotoxemic mice, plaques produced increased amounts of LTB4. Genomic or pharmacological impairments of this production reduced neutrophil infiltration, collagenolysis, and apoptosis of smooth muscle cells in plaques of endotoxemic mice. Furthermore, conditioned media of human culprit plaques (CPs) contained more LTB4 than non-CPs and levels of LTB4 correlated to both neutrophil activation markers and endotoxin releases in CPs. Conclusion: These results show that the increased neutrophil recruitment elicited by LTB4 contributes to increase features of plaque destabilization in endotoxemic contexts and point out LTB4 as a potential therapeutic target in atherosclerosis.


Asunto(s)
Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Endotoxemia/metabolismo , Leucotrieno B4/metabolismo , Activación Neutrófila , Infiltración Neutrófila , Neutrófilos/metabolismo , Placa Aterosclerótica , Animales , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Modelos Animales de Enfermedad , Endotoxemia/inducido químicamente , Endotoxemia/patología , Femenino , Fibrosis , Humanos , Lipopolisacáridos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Necrosis , Comunicación Paracrina , Transducción de Señal , Técnicas de Cultivo de Tejidos
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