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1.
Shock ; 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38813920

RESUMO

BACKGROUND: The association between neutrophil extracellular traps (NETs) and the requirement for vasopressor and inotropic support in vasoplegic shock is unclear. This study aimed to investigate the dynamics of plasma levels of NETs and cell-free DNA (cfDNA) up to 48 hours after the admission to the intensive care unit (ICU) for management of vasoplegic shock of infectious (SEPSIS) or non-infectious (following cardiac surgery, CARDIAC) origin. METHODS: Prospective, observational study of NETs and cfDNA plasma levels at 0H (admission) and then at 12H, 24H and 48H in SEPSIS and CARDIAC patients. The Vasopressor Inotropic Score (VIS), the Sequential Organ Failure Assessment (SOFA) score and time spent with invasive ventilation, in ICU and in hospital were recorded. Associations between NETs/cfDNA and VIS and SOFA were analysed by Spearman's correlation (rho), and between NETs/cfDNA and ventilation/ICU/hospitalisation times by generalised linear regression. RESULTS: Both NETs and cfDNA remained elevated over 48 hours in SEPSIS (n = 46) and CARDIAC (n = 30) patients, with time weighted average concentrations greatest in SEPSIS (NETs median difference 0.06 [0.02-0.11], p = 0.005; cfDNA median difference 0.48 [0.20-1.02], p < 0.001). The VIS correlated to NETs (rho = 0.3-0.60 in SEPSIS, p < 0.01, rho = 0.36-0.57 in CARDIAC, p ≤ 0.01) and cfDNA (rho = 0.40-0.56 in SEPSIS, p < 0.01, rho = 0.38-0.47 in CARDIAC, p < 0.05). NETs correlated with SOFA. Neither NETs nor cfDNA were independently associated with ventilator/ICU/hospitalisation times. CONCLUSION: Plasma levels of NETs and cfDNA correlated with the dose of vasopressors and inotropes administered over 48 hours in patients with vasoplegic shock from sepsis or following cardiac surgery. NETs levels also correlated with organ dysfunction. These findings suggest that similar mechanisms involving release of NETs are involved in the pathophysiology of vasoplegic shock irrespective of an infectious or non-infectious etiology.

2.
Acta Anaesthesiol Scand ; 68(6): 772-780, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38497568

RESUMO

BACKGROUND: Surgery on cardiopulmonary bypass (CPB) elicits a pleiomorphic systemic host response which, when severe, requires prolonged intensive care support. Given the substantial cross-talk between inflammation, coagulation, and fibrinolysis, the aim of this hypothesis-generating observational study was to document the kinetics of fibrinolysis recovery post-CPB using ClotPro® point-of-care viscoelastometry. Tissue plasminogen activator-induced clot lysis time (TPA LT, s) was correlated with surgical risk, disease severity, organ dysfunction and intensive care length of stay (ICU LOS). RESULTS: In 52 patients following CPB, TPA LT measured on the first post-operative day (D1) correlated with surgical risk (EuroScore II, Spearman's rho .39, p < .01), time on CPB (rho = .35, p = .04), disease severity (APACHE II, rho = .52, p < .001) and organ dysfunction (SOFA, rho = .51, p < .001) scores, duration of invasive ventilation (rho = .46, p < .01), and renal function (eGFR, rho = -.65, p < .001). In a generalized linear regression model containing TPA LT, CPB run time and markers of organ function, only TPA LT was independently associated with the ICU LOS (odds ratio 1.03 [95% CI 1.01-1.05], p = .01). In a latent variables analysis, the association between TPA LT and the ICU LOS was not mediated by renal function and thus, by inference, variation in the clearance of intraoperative tranexamic acid. CONCLUSIONS: This observational hypothesis-generating study in patients undergoing cardiac surgery with cardiopulmonary bypass demonstrated an association between the severity of fibrinolysis resistance, measured on the first post-operative day, and the need for extended postoperative ICU level support. Further examination of the role of persistent fibrinolysis resistance on the clinical outcomes in this patient cohort is warranted through large-scale, well-designed clinical studies.


Assuntos
Procedimentos Cirúrgicos Cardíacos , Ponte Cardiopulmonar , Fibrinólise , Tempo de Internação , Humanos , Ponte Cardiopulmonar/efeitos adversos , Masculino , Estudos Prospectivos , Fibrinólise/efeitos dos fármacos , Feminino , Idoso , Pessoa de Meia-Idade , Tempo de Internação/estatística & dados numéricos , Resultado do Tratamento , Ativador de Plasminogênio Tecidual/uso terapêutico , Complicações Pós-Operatórias/epidemiologia , Tempo de Lise do Coágulo de Fibrina
3.
Biomedicines ; 11(4)2023 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-37189750

RESUMO

The survival rate of patients with osteosarcoma (OS) has not improved over the last 30 years. Mutations in the genes TP53, RB1 and c-Myc frequently occur in OS and enhance RNA Polymerase I (Pol I) activity, thus supporting uncontrolled cancer cell proliferation. We therefore hypothesised that Pol I inhibition may be an effective therapeutic strategy for this aggressive cancer. The Pol I inhibitor CX-5461 has demonstrated therapeutic efficacy in different cancers in pre-clinical and phase I clinical trials; thus, the effects were determined on ten human OS cell lines. Following characterisation using genome profiling and Western blotting, RNA Pol I activity, cell proliferation and cell cycle progression were evaluated in vitro, and the growth of TP53 wild-type and mutant tumours was measured in a murine allograft model and in two human xenograft OS models. CX-5461 treatment resulted in reduced ribosomal DNA (rDNA) transcription and Growth 2 (G2)-phase cell cycle arrest in all OS cell lines. Additionally, tumour growth in all allograft and xenograft OS models was effectively suppressed without apparent toxicity. Our study demonstrates the efficacy of Pol I inhibition against OS with varying genetic alterations. This study provides pre-clinical evidence to support this novel therapeutic approach in OS.

4.
Transl Res ; 258: 60-71, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-36921796

RESUMO

DICER1 mutations predispose to increased risk for various cancers, particularly pleuropulmonary blastoma (PPB), the commonest lung malignancy of childhood. There is a paucity of directly actionable molecular targets as these tumors are driven by loss-of-function mutations of DICER1. Therapeutic development for PPB is further limited by a lack of biologically and physiologically-representative disease models. Given recent evidence of Dicer's role as a haploinsufficient tumor suppressor regulating RNA polymerase I (Pol I), Pol I inhibition could abrogate mutant Dicer-mediated accumulation of stalled polymerases to trigger apoptosis. Hence, we developed a novel subpleural orthotopic PPB patient-derived xenograft (PDX) model that retained both RNase IIIa and IIIb hotspot mutations and recapitulated the cardiorespiratory physiology of intra-thoracic disease, and with it evaluated the tolerability and efficacy of first-in-class Pol I inhibitor CX-5461. In PDX tumors, CX-5461 significantly reduced H3K9 di-methylation and increased nuclear p53 expression, within 24 hours' exposure. Following treatment at the maximum tolerated dosing regimen (12 doses, 30 mg/kg), tumors were smaller and less hemorrhagic than controls, with significantly decreased cellular proliferation, and increased apoptosis. As demonstrated in a novel intrathoracic tumor model of PPB, Pol I inhibition with CX-5461 could be a tolerable and clinically-feasible therapeutic strategy for mutant Dicer tumors, inducing antitumor effects by decreasing H3K9 methylation and enhancing p53-mediated apoptosis.


Assuntos
Blastoma Pulmonar , RNA Polimerase I , Humanos , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Proteína Supressora de Tumor p53/genética , Blastoma Pulmonar/genética , Blastoma Pulmonar/metabolismo , Blastoma Pulmonar/patologia , Carcinogênese , Ribonuclease III/genética , Ribonuclease III/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo
5.
Crit Care ; 27(1): 55, 2023 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-36765421

RESUMO

BACKGROUND: Fibrinolysisis is essential for vascular blood flow maintenance and is triggered by endothelial and platelet release of tissue plasminogen activator (t-PA). In certain critical conditions, e.g. sepsis, acute respiratory failure (ARF) and trauma, the fibrinolytic response is reduced and may lead to widespread thrombosis and multi-organ failure. The mechanisms underpinning fibrinolysis resistance include reduced t-PA expression and/or release, reduced t-PA and/or plasmin effect due to elevated inhibitor levels, increased consumption and/or clearance. This study in critically ill patients with fibrinolysis resistance aimed to evaluate the ability of t-PA and plasminogen supplementation to restore fibrinolysis with assessment using point-of-care ClotPro viscoelastic testing (VET). METHODS: In prospective, observational studies, whole-blood ClotPro VET evaluation was carried out in 105 critically ill patients. In 32 of 58 patients identified as fibrinolysis-resistant (clot lysis time > 300 s on the TPA-test: tissue factor activated coagulation with t-PA accelerated fibrinolysis), consecutive experimental whole-blood VET was carried out with repeat TPA-tests spiked with additional t-PA and/or plasminogen and the effect on lysis time determined. In an interventional study in a patient with ARF and fibrinolysis resistance, the impact of a 24 h intravenous low-dose alteplase infusion on coagulation and fibrinolysis was prospectively monitored using standard ClotPro VET. RESULTS: Distinct response groups emerged in the ex vivo experimental VET, with increased fibrinolysis observed following supplementation with (i) t-PA only or (ii) plasminogen and t-PA. A baseline TPA-test lysis time of > 1000 s was associated with the latter group. In the interventional study, a gradual reduction (25%) in serial TPA-test lysis times was observed during the 24 h low-dose alteplase infusion. CONCLUSIONS: ClotPro viscoelastic testing, the associated TPA-test and the novel experimental assays may be utilised to (i) investigate the potential mechanisms of fibrinolysis resistance, (ii) guide corrective treatment and (iii) monitor in real-time the treatment effect. Such a precision medicine and personalised treatment approach to the management of fibrinolysis resistance has the potential to increase treatment benefit, while minimising adverse events in critically ill patients. TRIAL REGISTRATION: VETtiPAT-ARF, a clinical trial evaluating ClotPro-guided t-PA (alteplase) administration in fibrinolysis-resistant patients with ARF, is ongoing (ClinicalTrials.gov NCT05540834 ; retrospectively registered September 15th 2022).


Assuntos
Fibrinólise , Ativador de Plasminogênio Tecidual , Humanos , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tecidual/uso terapêutico , Tempo de Lise do Coágulo de Fibrina , Sistemas Automatizados de Assistência Junto ao Leito , Estudos Prospectivos , Estudos de Viabilidade , Estado Terminal/terapia , Plasminogênio/farmacologia
6.
Blood Transfus ; 21(4): 289-295, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36346878

RESUMO

BACKGROUND: Platelet concentrates have a limited shelf life due to room temperature storage and therefore, are not kept in regional centres where turnover is low. Cryopreserved platelets have been proposed as an alternative to platelet transfusion in austere circumstances and fibrinogen concentrate has improved thromboelastometry parameters in thrombocytopenia. This study compared the ability of stored haemostatic products and platelets to correct thromboelastometry parameters in thrombocytopenia. MATERIALS AND METHODS: Blood from eight patients with severe thrombocytopenia was combined with platelet concentrates, cryoprecipitate, fibrinogen concentrate, factor VIII, factor XIII and cryopreserved platelets in ratios equivalent to transfusion. Tissue factor initiated thromboelastometry (EXTEM) was compared between the products. RESULTS: EXTEM amplitude at 20 minutes (A20) improved by 13.1 mm with platelets (p<0.01). The 5mm increase in A20 seen with cryoprecipitate (p=0.06) was not statistically different from platelets (p=0.19). No improvement in A20 was observed with cryopreserved platelets or factor concentrates. EXTEM clotting times (CT) improved with cryopreserved platelets (19.4 s, p=0.001) and cryoprecipitate (24.1 s, p<0.05), but not fibrinogen, and both were superior to platelets (9.9 s, p<0.05). Clotting concentrates did not improve EXTEM parameters although further studies suggested the improvement in A20 was largely driven by higher fibrinogen concentrations in cryoprecipitate. DISCUSSION: These results suggest that cryopreserved platelets enhance clot initiation but do not contribute to clot strength in thrombocytopenia. When platelets are not available for transfusion, cryoprecipitate may be of value, however this requires further clinical studies.


Assuntos
Anemia , Hemostáticos , Trombocitopenia , Humanos , Fibrinogênio/uso terapêutico , Hemostasia , Trombocitopenia/terapia , Coagulação Sanguínea , Tromboelastografia/métodos
7.
Macromol Biosci ; 22(10): e2200222, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35906813

RESUMO

Many materials have been engineered and commercialized as hemostatic agents. However, there is still a gap in the availability of hemostats that offer biocompatibility and biodegradability in combination with effective hemostatic properties. Cellulose nanofibers are investigated as hemostatic materials with most studies focusing on oxidized cellulose-derived hemostats. The recent studies demonstrate that by optimizing the morphological properties of nonoxidized cellulose nanofibers (CNFs) enhanced hemostasis is achieved. Herein, the hemostatic and wound-healing properties of CNFs with optimized morphology using two forms, gel, and sponge is investigated. In vitro thromboelastometry studies demonstrate that CNFs reduce clotting time by 68% (±SE 2%) and 88% (±SE 5%) in gel and sponge forms, respectively. In an in vivo murine liver injury model, CNFs significantly reduce blood loss by 38% (±SE 10%). The pH-neutral CNFs do not damage red blood cells, nor do they impede the proliferation of fibroblast or endothelial cells. Subcutaneously-implanted CNFs show a foreign body reaction resolving with the degradation of CNFs on histological examination and there is no scarring in the skin after 8 weeks. Demonstrating superior hemostatic performance in a variety of forms, as well as biocompatibility and biodegradability, CNFs hold significant potential for use in surgical and first-aid environments.


Assuntos
Celulose Oxidada , Hemostáticos , Nanofibras , Animais , Celulose/farmacologia , Celulose Oxidada/farmacologia , Células Endoteliais , Hemostasia , Hemostáticos/farmacologia , Camundongos
8.
EJHaem ; 3(1): 80-85, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35846213

RESUMO

Platelet transfusions are not always available for bleeding in severe thrombocytopenia, as storage outside of major centers is limited by their short shelf-life. Data are lacking to support alternative available blood products; however, additional fibrinogen has been shown to enhance clot formation in vitro. To test the hypothesis that cryoprecipitate supplementation could improve clot formation in severe thrombocytopenia, eight hematological malignancy patients with platelet counts under 10 × 109/L each had 10 units of apheresis cryoprecipitate transfused prior to planned prophylactic platelet transfusions. The primary endpoint of thromboelastometry amplitude at 20 min increased by a mean of 5.1 mm (p < 0.01) following cryoprecipitate transfusion despite persisting thrombocytopenia. Thromboelastometry clotting times reduced by a mean of 7.8 s (p < 0.05) and alpha angle increased by a mean of 10.6° (p < 0.01). These results are consistent with cryoprecipitate enhancing the strength of the fibrin/platelet meshwork within the forming thrombus. While platelet transfusion remains the standard of care, where platelet supplies are limited, these data provide a rationale for the use of cryoprecipitate to obtain hemostasis in bleeding thrombocytopenic patients.

9.
J Immunol ; 208(12): 2738-2748, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35649630

RESUMO

Liver-resident CD8+ T cells can play critical roles in the control of pathogens, including Plasmodium and hepatitis B virus. Paradoxically, it has also been proposed that the liver may act as the main place for the elimination of CD8+ T cells at the resolution of immune responses. We hypothesized that different adhesion processes may drive residence versus elimination of T cells in the liver. Specifically, we investigated whether the expression of asialo-glycoproteins (ASGPs) drives the localization and elimination of effector CD8+ T cells in the liver, while interactions with platelets facilitate liver residence and protective function. Using murine CD8+ T cells activated in vitro, or in vivo by immunization with Plasmodium berghei sporozoites, we found that, unexpectedly, inhibition of ASGP receptors did not inhibit the accumulation of effector cells in the liver, but instead prevented these cells from accumulating in the spleen. In addition, enforced expression of ASGP on effector CD8+ T cells using St3GalI-deficient cells lead to their loss from the spleen. We also found, using different mouse models of thrombocytopenia, that severe reduction in platelet concentration in circulation did not strongly influence the residence and protective function of CD8+ T cells in the liver. These data suggest that platelets play a marginal role in CD8+ T cell function in the liver. Furthermore, ASGP-expressing effector CD8+ T cells accumulate in the spleen, not the liver, prior to their destruction.


Assuntos
Linfócitos T CD8-Positivos , Malária , Animais , Receptor de Asialoglicoproteína , Fígado , Camundongos , Plasmodium berghei , Esporozoítos
10.
Invest New Drugs ; 40(3): 529-536, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35201535

RESUMO

BACKGROUND: Uterine leiomyosarcoma is a rare aggressive smooth muscle cancer with poor survival rates. RNA Polymerase I (Pol I) activity is elevated in many cancers supporting tumour growth and prior studies in uterine leiomyosarcoma revealed enlarged nucleoli and upregulated Pol I activity-related genes. This study aimed to investigate the anti-tumour potential of CX-5461, a Pol I transcription inhibitor currently being evaluated in clinical trials for several cancers, against the human uterine leiomyosarcoma cell line, SK-UT-1. METHODS: SK-UT-1 was characterised using genome profiling and western blotting. The anti-tumour effects of CX-5461 were investigated using cell proliferation assays, expression analysis using qRT-PCR, and BrdU/PI based cell cycle analysis. RESULTS: Genetic analysis of SK-UT-1 revealed mutations in TP53, RB1, PTEN, APC and TSC1 & 2, all potentially associated with increased Pol I activity. Protein expression analysis showed dysregulated p53, RB1 and c-Myc. CX-5461 treatment resulted in an anti-proliferation response, G2 phase cell-cycle arrest and on-target activity demonstrated by reduced ribosomal DNA transcription. CONCLUSIONS: SK-UT-1 was confirmed as a representative model of uterine leiomyosarcoma and CX-5461 has significant potential as a novel adjuvant for this rare cancer.


Assuntos
Benzotiazóis , Leiomiossarcoma , Naftiridinas , Neoplasias Uterinas , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Leiomiossarcoma/tratamento farmacológico , Leiomiossarcoma/genética , Leiomiossarcoma/metabolismo , Naftiridinas/farmacologia , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Uterinas/tratamento farmacológico , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo
11.
JCI Insight ; 7(2)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35076023

RESUMO

Platelet-neutrophil aggregates (PNAs) facilitate neutrophil activation and migration and could underpin the recruitment of neutrophils to the pancreas during type 1 diabetes (T1D) pathogenesis. PNAs, measured by flow cytometry, were significantly elevated in the circulation of autoantibody-positive (Aab+) children and new-onset T1D children, as well as in pre-T1D (at 4 weeks and 10-12 weeks) and T1D-onset NOD mice, compared with relevant controls, and PNAs were characterized by activated P-selectin+ platelets. PNAs were similarly increased in pre-T1D and T1D-onset NOD isolated islets/insulitis, and immunofluorescence staining revealed increased islet-associated neutrophil extracellular trap (NET) products (myeloperoxidase [MPO] and citrullinated histones [CitH3]) in NOD pancreata. In vitro, cell-free histones and NETs induced islet cell damage, which was prevented by the small polyanionic drug methyl cellobiose sulfate (mCBS) that binds to histones and neutralizes their pathological effects. Elevated circulating PNAs could, therefore, act as an innate immune and pathogenic biomarker of T1D autoimmunity. Platelet hyperreactivity within PNAs appears to represent a previously unrecognized hematological abnormality that precedes T1D onset. In summary, PNAs could contribute to the pathogenesis of T1D and potentially function as a pre-T1D diagnostic.


Assuntos
Plaquetas/imunologia , Agregação Celular/imunologia , Diabetes Mellitus Tipo 1 , Armadilhas Extracelulares , Neutrófilos/imunologia , Pâncreas , Animais , Autoanticorpos/sangue , Criança , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/imunologia , Diagnóstico Precoce , Armadilhas Extracelulares/diagnóstico por imagem , Armadilhas Extracelulares/imunologia , Feminino , Imunofluorescência/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Ativação de Neutrófilo/imunologia , Selectina-P/metabolismo , Pâncreas/imunologia , Pâncreas/patologia
12.
Carbohydr Polym ; 265: 118043, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33966826

RESUMO

Hemorrhage remains a significant cause of morbidity and mortality following trauma and during complex surgeries. A variety of nanomaterials, including oxidized cellulose nanofibers (OCNFs), have been studied to overcome the disadvantages of current commercial topical hemostats. However, the relationship between nano-structural characteristics and hemostatic efficacy of non-oxidized cellulose nanofibers (CNFs) has not been elucidated. Herein, we present the first report of the correlation between structure and hemostatic performance of CNFs. In vitro thromboelastometry studies on CNFs, synthesized by ball-milling, showed that there is an optimum balance point between the aspect ratio (AR) and specific surface area (SSA) of nanofibers in terms of their maximum contribution to platelet function and plasma coagulation. The optimized CNFs with high SSA (17 m2/g) and a high AR (166) shortened normal whole blood clotting time by 68 %, outperforming cellulose-based hemostats. Additionally, CNFs reduced clotting time in platelet-deficient blood (by 80 %) and heparinized blood (by 54 %).


Assuntos
Celulose/química , Hemostáticos/química , Nanofibras/química , Tromboelastografia/métodos , Celulose/farmacologia , Celulose Oxidada/química , Hemorragia/patologia , Hemorragia/terapia , Hemostáticos/farmacologia , Humanos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
13.
Platelets ; 32(5): 662-670, 2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-32664767

RESUMO

The ability of platelets to promote carcinoma and melanoma progression has been thoroughly studied and occurs in numerous ways. In contrast, the effect of platelets on sarcomas, tumors arising from mesenchymal cells, has received very little attention. This study was undertaken to simultaneously compare the effects of platelets on murine and human sarcomas and carcinomas. In contrast to their effect on carcinomas, platelets inhibited the invasion of some murine- and all human sarcomas tested in vitro. Further invasion studies with TGFß treatment only partially recapitulated the results seen with whole platelets. In a spontaneous tumor growth and lung metastasis model, platelets promoted 4T1 mammary carcinoma metastasis but not MCA-1 fibrosarcoma metastasis. Gene expression analysis of the platelet-promoted MDA-MB-231 breast carcinoma, and the platelet-inhibited HT1080 fibrosarcoma cell lines revealed that exposure of MDA-MB-231 to platelets, resulted in upregulation of oncogenes and EMT-associated genes whereas in HT1080 a tumor-suppressor gene was significantly upregulated. Thus, this study has revealed a potential diametrically opposing effect of platelets on mesenchymal and epithelial cancers, a finding that warrants further investigation.


Assuntos
Plaquetas/metabolismo , Carcinoma/sangue , Sarcoma/sangue , Animais , Movimento Celular , Proliferação de Células , Humanos , Camundongos , Voluntários
14.
Nat Commun ; 11(1): 6408, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33328478

RESUMO

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9-1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.


Assuntos
Armadilhas Extracelulares/efeitos dos fármacos , Histonas/metabolismo , Polímeros/farmacologia , Sepse/sangue , Sepse/tratamento farmacológico , Animais , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Feminino , Histonas/toxicidade , Humanos , Bicamadas Lipídicas , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/sangue , Ativação Plaquetária/efeitos dos fármacos , Polieletrólitos , Polímeros/química , Ratos Wistar , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/patologia , Sepse/patologia
15.
Blood Adv ; 4(12): 2623-2630, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32556282

RESUMO

Trauma-induced coagulopathy (TIC) is a complex, multifactorial failure of hemostasis that occurs in 25% of severely injured patients and results in a fourfold higher mortality. However, the role of platelets in this state remains poorly understood. We set out to identify molecular changes that may underpin platelet dysfunction after major injury and to determine how they relate to coagulopathy and outcome. We performed a range of hemostatic and platelet-specific studies in blood samples obtained from critically injured patients within 2 hours of injury and collected prospective data on patient characteristics and clinical outcomes. We observed that, although platelet counts were preserved above critical levels, circulating platelets sampled from trauma patients exhibited a profoundly reduced response to both collagen and the selective glycoprotein VI (GPVI) agonist collagen-related peptide, compared with those from healthy volunteers. These responses correlated closely with overall clot strength and mortality. Surface expression of the collagen receptors GPIbα and GPVI was reduced on circulating platelets in trauma patients, with increased levels of the shed ectodomain fragment of GPVI detectable in plasma. Levels of shed GPVI were highest in patients with more severe injuries and TIC. Collectively, these observations demonstrate that platelets experience a loss of GPVI and GPIbα after severe injury and translate into a reduction in the responsiveness of platelets during active hemorrhage. In turn, they are associated with reduced hemostatic competence and increased mortality. Targeting proteolytic shedding of platelet receptors is a potential therapeutic strategy for maintaining hemostatic competence in bleeding and improving the efficacy of platelet transfusions.


Assuntos
Plaquetas , Transfusão de Plaquetas , Hemorragia/etiologia , Hemostasia , Humanos , Estudos Prospectivos
16.
J Adolesc Young Adult Oncol ; 9(6): 667-671, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32397787

RESUMO

Two thirds of metastatic osteosarcoma patients die within 5 years of diagnosis. Improved experimental models of osteosarcoma metastasis will facilitate the development of more effective therapies. Intravenous cancer cell injection can produce lung metastases in nude mice, but this "experimental metastasis" technique has been predominantly applied to a single osteosarcoma cell line (143B) and required injection of 1-2 million cells. Using two human osteosarcoma cell lines, we discovered that transient Natural Killer cell depletion dramatically enhanced the efficiency of experimental pulmonary osteosarcoma metastasis. This technique for modeling osteosarcoma metastasis may enable the identification of better treatments for this aggressive cancer.


Assuntos
Células Matadoras Naturais/metabolismo , Neoplasias Pulmonares/secundário , Osteossarcoma/terapia , Administração Intravenosa , Animais , Feminino , Camundongos , Camundongos Nus
17.
J Thromb Haemost ; 18(6): 1447-1458, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32198957

RESUMO

BACKGROUND: Collagen and fibrin engagement and activation of glycoprotein (GP) VI induces proteolytic cleavage of the GPVI ectodomain generating shed soluble GPVI (sGPVI). Collagen-mediated GPVI shedding requires intracellular signalling to release the sGPVI, mediated by A Disintegrin And Metalloproteinase 10 (ADAM10); however, the precise mechanism by which fibrin induces GPVI shedding remains elusive. Plasma sGPVI levels are elevated in patients with coagulopathies, sepsis, or inflammation and can predict onset of sepsis and sepsis-related mortality; therefore, it is clinically important to understand the mechanisms of GPVI shedding under conditions of minimal collagen exposure. OBJECTIVES: Our aim was to characterize mechanisms by which fibrin-GPVI interactions trigger GPVI shedding. METHODS: Platelet aggregometry, sGPVI ELISA, and an ADAM10 fluorescence resonance energy transfer assay were used to measure fibrin-mediated platelet responses. RESULTS: Fibrin induced αIIbß3-independent washed platelet aggregate formation, GPVI shedding, and increased ADAM10 activity, all of which were insensitive to pre-treatment with inhibitors of Src family kinases but were divalent cation- and metalloproteinase-dependent. In contrast, treatment of washed platelets with other GPVI ligands, collagen, and collagen-related peptide caused αIIbß3-dependent platelet aggregation and GPVI release but did not increase constitutive ADAM10 activity. CONCLUSIONS: Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin-induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin-induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.


Assuntos
Fibrina , Glicoproteínas da Membrana de Plaquetas , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Plaquetas , Humanos , Proteínas de Membrana , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas
18.
Platelets ; 31(3): 315-321, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32054377

RESUMO

Diagnosis of immune thrombocytopenia (ITP) and prediction of response to therapy remain significant and constant challenges in hematology. In patients who present with ITP, the platelet count is frequently used as a surrogate marker for disease severity, and so often determines the need for therapy. Although there is a clear link between thrombocytopenia and hemostasis, a direct correlation between the extent of thrombocytopenia and bleeding symptoms, especially at lower platelet counts is lacking. Thus, bleeding in ITP is heterogeneous, unpredictable, and nearly always based on a multitude of risk factors, beyond the platelet count. The development of an evidence-based, validated risk stratification model for ITP treatment is a major goal in the ITP community and this review discusses new laboratory approaches to evaluate the various pathobiologies of ITP that may inform such a model.


Assuntos
Suscetibilidade a Doenças , Púrpura Trombocitopênica Idiopática/etiologia , Pesquisa/tendências , Animais , Biomarcadores , Plaquetas/imunologia , Plaquetas/metabolismo , Plaquetas/patologia , Suscetibilidade a Doenças/imunologia , Humanos , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Megacariócitos/imunologia , Megacariócitos/metabolismo , Megacariócitos/patologia , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/metabolismo , Púrpura Trombocitopênica Idiopática/terapia
19.
Methods Protoc ; 3(1)2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31979362

RESUMO

Bioluminescent tumor cell lines are used extensively in vivo to monitor tumor growth and metastasis but rarely used in vitro to follow tumor cell behavior. Tumor cell migration is frequently studied in vitro using transwell assays, however, current methods do not permit the co-incubation of tumor cells with different stromal cell types for analysis of the effects of intercellular cross-talk on tumor cell migration. We describe a novel migration assay using bioluminescent tumor cell lines that is rapid, accurate, and permits the study of the effects of tumor cell-stromal cell interactions on tumor cell migratory behavior.

20.
Pharmacol Res Perspect ; 7(6): e00526, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31624634

RESUMO

Dichloroacetate (DCA) is an investigational drug targeting the glycolytic hallmark of cancer by inhibiting pyruvate dehydrogenase kinases (PDK). It is metabolized by GSTZ1, which has common polymorphisms altering enzyme or promoter activity. GSTZ1 is also irreversibly inactivated by DCA. In the first clinical trial of DCA in a hematological malignancy, DiCAM (DiChloroAcetate in Myeloma), we have examined the relationship between DCA concentrations, GSTZ1 genotype, side effects, and patient response. DiCAM recruited seven myeloma patients in partial remission. DCA was administered orally for 3 months with a loading dose. Pharmacokinetics were performed on day 1 and 8. Trough and peak concentrations of DCA were measured monthly. GSTZ1 genotypes were correlated with drug concentrations, tolerability, and disease outcomes. One patient responded and two patients showed a partial response after one month of DCA treatment, which included the loading dose. The initial half-life of DCA was shorter in two patients, correlating with heterozygosity for GSTZ1*A genotype, a high enzyme activity variant. Over 3 months, one patient maintained DCA trough concentrations approximately threefold higher than other patients, which correlated with a low activity promoter genotype (-1002A, rs7160195) for GSTZ1. This patient displayed the strongest response, but also the strongest neuropathy. Overall, serum concentrations of DCA were sufficient to inhibit the constitutive target PDK2, but unlikely to inhibit targets induced in cancer. Promoter GSTZ1 polymorphisms may be important determinants of DCA concentrations and neuropathy during chronic treatment. Novel dosing regimens may be necessary to achieve effective DCA concentrations in most cancer patients while avoiding neuropathy.


Assuntos
Ácido Dicloroacético/farmacocinética , Resistencia a Medicamentos Antineoplásicos/genética , Glutationa Transferase/genética , Mieloma Múltiplo/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/genética , Administração Oral , Idoso , Ácido Dicloroacético/administração & dosagem , Ácido Dicloroacético/efeitos adversos , Drogas em Investigação/administração & dosagem , Drogas em Investigação/efeitos adversos , Drogas em Investigação/farmacocinética , Feminino , Genótipo , Glutationa Transferase/metabolismo , Meia-Vida , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/genética , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Estudos Prospectivos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo
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