RESUMO
Multipotent mesenchymal stromal cells (MSCs) are tested in numerous clinical trials. Questions have been raised concerning fate and function of these therapeutic cells after systemic infusion. We therefore asked whether culture-expanded human MSCs elicit an innate immune attack, termed instant blood-mediated inflammatory reaction (IBMIR), which has previously been shown to compromise the survival and function of systemically infused islet cells and hepatocytes. We found that MSCs expressed hemostatic regulators similar to those produced by endothelial cells but displayed higher amounts of prothrombotic tissue/stromal factors on their surface, which triggered the IBMIR after blood exposure, as characterized by formation of blood activation markers. This process was dependent on the cell dose, the choice of MSC donor, and particularly the cell-passage number. Short-term expanded MSCs triggered only weak blood responses in vitro, whereas extended culture and coculture with activated lymphocytes increased their prothrombotic properties. After systemic infusion to patients, we found increased formation of blood activation markers, but no formation of hyperfibrinolysis marker D-dimer or acute-phase reactants with the currently applied dose of 1.0-3.0 × 10(6) cells per kilogram. Culture-expanded MSCs trigger the IBMIR in vitro and in vivo. Induction of IBMIR is dose-dependent and increases after prolonged ex vivo expansion. Currently applied doses of low-passage clinical-grade MSCs elicit only minor systemic effects, but higher cell doses and particularly higher passage cells should be handled with care. This deleterious reaction can compromise the survival, engraftment, and function of these therapeutic cells.
Assuntos
Células-Tronco Mesenquimais/citologia , Terapia Baseada em Transplante de Células e Tecidos , Células Endoteliais/citologia , Humanos , Células-Tronco Mesenquimais/imunologia , Trombose/sangueRESUMO
Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood. Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time. We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation.
Assuntos
Plaquetas/fisiologia , Comunicação Celular , Fator XIIa/metabolismo , Ativação Plaquetária , Plasma Rico em Plaquetas/fisiologia , Serpinas/metabolismo , Antitrombinas/metabolismo , Coagulação Sanguínea , Plaquetas/enzimologia , Fator Xa/metabolismo , Citometria de Fluxo , Humanos , Calicreínas/metabolismo , Plasma Rico em Plaquetas/enzimologiaRESUMO
BACKGROUND: A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non-human primates. The detrimental instant blood-mediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss. This reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen. MATERIALS AND METHODS: Low molecular weight dextran sulfate (LMW-DS) and the complement inhibitor Compstatin were used in a combination of in vitro and in vivo studies designed to dissect the xenogeneic IBMIR in a non-human primate model of pancreatic islet transplantation. Adult porcine islets (10,000 IEQs/kg) were transplanted intraportally into three pairs of cynomolgus monkeys that had been treated with LMW-DS or heparin (control), and the effects on the IBMIR were characterized. Porcine islets were also incubated in human blood plasma in vitro to assess complement inhibition by LMW-DS and Compstatin. RESULTS: Morphological scoring and immunohistochemical staining revealed that the severe islet destruction and macrophage, neutrophilic granulocyte, and T-cell infiltration observed in the control (heparin-treated) animals were abrogated in the LMW-DS-treated monkeys. Both coagulation and complement activation were significantly reduced in monkeys treated with LMW-DS, but IgM and complement fragments were still found on the islet surface. This residual complement activation could be inhibited by Compstatin in vitro. CONCLUSIONS: The xenogeneic IBMIR in this non-human primate model is characterized by an immediate binding of antibodies that triggers deleterious complement activation and a subsequent clotting reaction that leads to further complement activation. The effectiveness of LMW-DS (in vivo and in vitro) and Compstatin (in vitro) in inhibiting this IBMIR provides the basis for a protocol that can be used to abrogate the IBMIR in pig-human clinical islet transplantation.
Assuntos
Inflamação/sangue , Inflamação/etiologia , Transplante das Ilhotas Pancreáticas/efeitos adversos , Transplante das Ilhotas Pancreáticas/imunologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Ativação do Complemento/efeitos dos fármacos , Citocinas/biossíntese , Sulfato de Dextrana/administração & dosagem , Heparina/administração & dosagem , Humanos , Técnicas In Vitro , Transplante das Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas/fisiologia , Macaca fascicularis , Peptídeos Cíclicos/administração & dosagem , Sus scrofa , Transplante HeterólogoRESUMO
Rapid destruction of hepatocytes after hepatocyte transplantation has hampered the application of this procedure clinically. The instant blood-mediated inflammatory reaction (IBMIR) is a plausible underlying cause for this cell loss. The present study was designed to evaluate the capacity of low molecular weight dextran sulfate (LMW-DS) to control these initial reactions from the innate immune system. Fresh and cryopreserved hepatocytes were tested in an in vitro whole-blood model using ABO-compatible blood. The ability to elicit IBMIR and the capacity of LMW-DS (100 µg/ml) to attenuate the degree of activation of the cascade systems were monitored. The effect was also compared to conventional anticoagulant therapy using unfractionated heparin (1 IU/ml). Both fresh and freeze-thawed hepatocytes elicited IBMIR to the same extent. LMW-DS reduced the platelet loss and maintained the cell counts at the same degree as unfractionated heparin, but controlled the coagulation and complement systems significantly more efficiently than heparin. LMW-DS also attenuated the IBMIR elicited by freeze-thawed cells. Therefore, LMW-DS inhibits the cascade systems and maintains the cell counts in blood triggered by both fresh and cryopreserved hepatocytes in direct contact with ABO-matched blood. LMW-DS at a previously used and clinically applicable concentration (100 µg/ml) inhibits IBMIR in vitro and is therefore a potential IBMIR inhibitor in hepatocyte transplantation.
Assuntos
Criopreservação/métodos , Sulfato de Dextrana/uso terapêutico , Heparina/uso terapêutico , Hepatócitos/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Imunidade Inata/efeitos dos fármacos , Imunoensaio , Inflamação/prevenção & controle , Fígado/citologia , Peso Molecular , Transplante Heterólogo/métodosRESUMO
There are strong indications that only a small fraction of grafts successfully engraft in clinical islet transplantation. One explanation may be the instant blood-mediated inflammatory reaction (IBMIR) elicited by tissue factor, which is produced by the endocrine cells. In the present study, we show that islets intended for islet transplantation produce tissue factor in both the transmembrane and the alternatively spliced form and that the membrane-bound form is released as microparticles often associated with both insulin and glucagon granules. A low-molecular mass factor VIIa (FVIIa) inhibitor that indirectly blocks both forms of tissue factor was shown in vitro to be a promising drug to eliminate the IBMIR. Thrombin-antithrombin complex (TAT) and FVIIa-antithrombin complex (FVIIa-AT) were measured in nine patients who together received 20 infusions of isolated human islets. Both the TAT and FVIIa-AT complexes increased rapidly within 15-60 min after infusion. When the initial TAT and FVIIa-AT levels were plotted against the increase in C-peptide concentration after 7 days, patients with an initially strong IBMIR showed no significant increase in insulin synthesis after 7 days. In conclusion, tissue factor present in both the islets and the culture medium and elicits IBMIR, which affects the function of the transplanted islets.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/fisiologia , Tromboplastina/fisiologia , Adulto , Processamento Alternativo/fisiologia , Antitrombina III/fisiologia , Fator VIIa/antagonistas & inibidores , Fator VIIa/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Inflamação/fisiopatologia , Ilhotas Pancreáticas/ultraestrutura , Masculino , Pessoa de Meia-Idade , Peptídeo Hidrolases/fisiologia , Fatores de TempoRESUMO
Human islets induce an injurious clotting reaction at the time of transplantation. A potential strategy to counteract this reaction would be to allow the islets to express hirudin, a protein with direct anticoagulative activity. Human islets were transduced with an adenoviral vector encoding hirudin, an empty corresponding vector, or left untreated. Islet culture supernatants were analyzed for hirudin using an ELISA, a chromogenic substrate assay based on the thrombin-binding properties of hirudin and in a whole blood viscosimetry assay. Immunohistochemical evaluation and determination of hirudin content revealed an abundant expression of hirudin after transduction. Hirudin content in transduced islets was in the range of the insulin content levels. A delay in human whole blood clotting time could be observed after addition of supernatants taken from islet cultures expressing hirudin. However, transduced islets showed an impaired glucose-stimulated insulin release, but could readily be retrieved 6 weeks after transplantation to athymic mice. A marked expression and secretion of hirudin with functional capacity can be induced in human islets using an adenoviral vector. The impairment in glucose-stimulated insulin release in hirudin-secreting islets, compared to controls, indicates that the additional protein synthesis affects the functional capacity of the islets.
Assuntos
Adenoviridae/genética , Anticoagulantes/metabolismo , Hirudinas/genética , Ilhotas Pancreáticas/metabolismo , Animais , DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/genética , Hirudinas/metabolismo , Humanos , Imuno-Histoquímica , Insulina/metabolismo , Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Transplante Heterólogo , Tempo de Coagulação do Sangue TotalRESUMO
A thrombotic/inflammatory reaction is elicited when isolated islets of Langerhans come in contact with ABO-compatible blood. The detrimental effects of this instant blood-mediated inflammatory reaction (IBMIR) provide a reasonable explanation for the observation that an unexpectedly high number of islets, from several donors, are needed to produce normoglycemia in transplant patients with type 1 diabetes. In this study, the hypothesis that a specific thrombin inhibitor, Melagatran, could reduce IBMIR in an in vitro model in which human islets are exposed to ABO-compatible blood was tested. The administration of Melagatran abrogated IBMIR dose-dependently. Islets exposed to blood, in the absence or presence of 0.4 micromol/l Melagatran, exhibited a loss of integrity and were found to be trapped in macroscopic clots containing platelets and CD11b(+) leukocytes. At concentrations from 1 to 10 micromol/l, Melagatran inhibited both coagulation and complement activation. Also, platelet and leukocyte activation and consumption were decreased. Islet morphology was maintained with almost no platelets adhering to the surface, and infiltration by CD11b(+) leukocytes was considerably reduced. In conclusion, Melagatran significantly reduced IBMIR in this model system. This protective effect indicates that thrombin plays a pivotal role in IBMIR and suggests that thrombin inhibition can improve the outcome of clinical islet transplantation.
Assuntos
Glicina/análogos & derivados , Glicina/uso terapêutico , Inflamação/sangue , Inflamação/prevenção & controle , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/fisiopatologia , Trombina/antagonistas & inibidores , Sistema ABO de Grupos Sanguíneos , Idoso , Idoso de 80 Anos ou mais , Anticoagulantes/uso terapêutico , Azetidinas , Benzilaminas , Plaquetas/patologia , Antígenos CD11/análise , Cadáver , Feminino , Fibrina/fisiologia , Humanos , Imuno-Histoquímica , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/patologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/análise , Trombose/sangue , Trombose/patologia , Trombose/prevenção & controleRESUMO
UNLABELLED: The aim of this study was to assess the combined effect of heparin coating of a stent graft and administration of abciximab, on platelet and coagulation activity in vitro. METHODS: Stent grafts with an expanded polytetrafluoroethylene (ePTFE) membrane interfoliated between two stents were deployed in tubings to form Chandler loops. Fresh human blood with a low concentration of heparin and various doses of abciximab was rotated for 1 h, then collected and used for measurements of platelets, thrombin-antithrombin complex (TAT), CD11b, complement and contact activation of coagulation. In the first set of experiments, all stent grafts were heparin coated. There were three study groups: Group 1a (no abciximab, n=5); Group1b (abciximab in a concentration of 3.3 microg/ml, n=5); Group 1c (abciximab in a concentration of 8.3 microg/ml, n=5). In the second set of experiments, the concentration of abciximab was 3.3 microg/ml. There were three study groups: Group 2a (untreated stent grafts, n=4); Group 2b (heparin-coated stent grafts, n=4); Group 2c (heparin-coated PVC tubings with no stent grafts, n=4). RESULTS: In the first set of experiments, there was a significant reduction in platelet count in Group 1c compared to Group 1a and Group 1b. There was a significant reduction in TAT in Group 1b and Group 1c as compared to Group 1a. In the second set of experiments, TAT was reduced in Group 2b and Group 2c compared to Group 2a. Contact activation was lowered in Group 1b and Group 1c as compared to Group 1a for both FXIa-AT (0.088 and 0.088 vs. 0.115) and FXIIa-AT (0.12 and 0.12 vs. 0.19). CONCLUSION: Heparin coating of a stent graft was shown to improve blood compatibility and this was further enhanced by addition of abciximab.
Assuntos
Anticorpos Monoclonais/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Heparina/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Stents , Abciximab , Anticoagulantes/farmacologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Ativação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Politetrafluoretileno/química , Politetrafluoretileno/farmacologiaRESUMO
BACKGROUND: One of the main obstacles to clinical islet xenotransplantation is the injurious instant blood-mediated inflammatory reaction (IBMIR) that causes rapid binding of platelets to the islet surface, activation of the coagulation and complement systems, and leukocyte infiltration of the islets when the islets are exposed to blood. METHODS: This study assesses the effect of low molecular weight dextran sulfate (LMW-DS) on IBMIR induced by porcine islets in an in vitro tubing loop assay using human blood and in an in vivo model using diabetic athymic mice. RESULTS: In vitro experiments demonstrated that platelet consumption, coagulation, and complement activation were already reduced in the presence of LMW-DS at 0.01 mg/mL, and that at 0.1 mg/mL, LMW-DS prevented IBMIR. Immunohistochemical investigation showed that the leukocyte infiltration was abrogated at the highest dose. In vivo experiments showed that the transplanted pig islets survived for a significantly longer period in recipients treated with LMW-DS, and morphologic examination of transplanted islets showed a reduction in IBMIR analogous to that demonstrated by in vitro studies. CONCLUSIONS: Given that LMW-DS has been used in clinical studies without serious adverse reactions, it has potential as a drug candidate that can control the strong innate immune response induced by pig islets when transplanted through the portal vein.
Assuntos
Sulfato de Dextrana/farmacologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/cirurgia , Tolerância Imunológica/efeitos dos fármacos , Transplante das Ilhotas Pancreáticas/imunologia , Substitutos do Plasma/farmacologia , Fatores Etários , Animais , Coagulação Sanguínea/efeitos dos fármacos , Ativação do Complemento/efeitos dos fármacos , Sulfato de Dextrana/química , Fibrinólise/efeitos dos fármacos , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Nus , Peso Molecular , Substitutos do Plasma/química , Suínos , Transplante HeterólogoRESUMO
OBJECTIVES: Heparin coating of the cardiopulmonary bypass circuit is shown to improve the biocompatibility of the surface. We have studied a new heparin surface, the Corline Heparin Surface, applied to a complete set of an extracorporeal device used during coronary artery bypass grafting in terms of activation of inflammation, coagulation, and fibrinolysis in patients and in shed mediastinal blood. METHODS: Sixty patients scheduled for coronary artery bypass grafting were randomized to one of 3 groups with heparin-coated devices receiving either a standard, high, or low dose of systemic heparin or to an uncoated but otherwise identical circuit receiving a standard dose of systemic heparin. Samples were drawn before, during, and after the operation from the pericardial cavity and in shed mediastinal blood. No autotransfusion of shed mediastinal blood was performed. RESULTS: The Corline Heparin Surface significantly reduced the activation of coagulation, fibrinolysis, platelets, and inflammation compared with that seen with the uncoated surface in combination with a standard dose of systemic heparin during cardiac surgery with cardiopulmonary bypass. Both a decrease and an increase of systemic heparin in combination with the coated heparin surface resulted in higher activation of these processes. A significantly higher expression of all studied parameters was found in the shed mediastinal blood compared with in systemic blood at the same time. CONCLUSIONS: The Corline Heparin Surface used in cardiopulmonary bypass proved to be more biocompatible than an uncoated surface when using a standard systemic heparin dose. The low dose of systemic heparin might not be sufficient to maintain the antithrombotic activity, and the high dose resulted in direct cell activation rather than a further anti-inflammatory and anticoagulatory effect.
Assuntos
Anticoagulantes/administração & dosagem , Perda Sanguínea Cirúrgica/prevenção & controle , Ponte de Artéria Coronária , Heparina/administração & dosagem , Idoso , Análise de Variância , Fatores de Coagulação Sanguínea/análise , Materiais Revestidos Biocompatíveis , Citocinas/sangue , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Citometria de Fluxo , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Estatísticas não Paramétricas , Resultado do TratamentoRESUMO
Contact between blood and a biomaterial surface takes place in many applications and is known to activate the coagulation and complement systems. Heparin surface coatings have been shown to reduce blood activation upon contact with artificial surfaces. To establish the optimal heparin surface concentration, blood was incubated in a tubing loop model at 37 degrees C. The tubing was coated with different surface concentrations of heparin and rotated at three different velocities. We demonstrate that the blood compatibility of a surface with regard to coagulation, complement, and platelet activation can be improved by increasing the heparin surface concentration in the 6-12 pmol antithrombin/cm2 concentration interval. The binding of factor H is not influenced by the increased heparin surface concentration, suggesting that this factor is not the primary regulator of complement on heparin surfaces. In addition, the heparin coating has no effect on the complement activation that occurs on gas surfaces in extracorporeal circuits.
Assuntos
Antitrombinas/metabolismo , Materiais Biocompatíveis/metabolismo , Sangue/metabolismo , Heparina/metabolismo , Células Sanguíneas/fisiologia , Adesão Celular/fisiologia , Fator H do Complemento/metabolismo , HumanosRESUMO
OBJECTIVE: Diabetes is associated with various vascular complications and is suggested to induce a prothrombotic state. In the current study, we characterized antiplasmin incorporation into fibrin in relation to other fibrinolytic compounds in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 236 patients with type 1 diabetes and 78 control subjects were investigated. The incorporation of antiplasmin into the fibrin network and the plasma levels of plasminogen activator inhibitor type 1 (PAI-1) activity, tissue plasminogen activator (tPA) activity, tPA/PAI-1 complex, plasmin-antiplasmin complex, antiplasmin, factor XIII, and d-dimer were measured. In addition, we used global assays to study fibrinolysis. RESULTS: The incorporation of antiplasmin into the fibrin network was significantly higher in patients with type 1 diabetes than in control subjects without diabetes (1.65 ± 0.25 vs. 1.35 ± 0.18 mg/L, respectively; P < 0.0001). The patients also had lower PAI-1 activity (2.19 units/mL [interquartile range 0.96-5.42] vs. 4.25 units/mL [1.95-9.0]; P = 0.0012) and antiplasmin level in plasma (78.5 ± 13.3 vs. 83.2 ± 15.4 mg/L; P < 0.05), resulting in a higher fibrinolytic capacity (shorter clot lysis time; P = 0.0090). We did not find any important sex differences regarding fibrinolysis in the patients or in the control subjects. CONCLUSIONS: Patients with type 1 diabetes incorporate more antiplasmin into the fibrin network than control subjects without diabetes do and have a reduced PAI-1 activity and a shorter clot lysis time. These results suggest that patients with type 1 diabetes produce a fibrin clot that is more resistant to fibrinolysis, which, however, may be counteracted by an increased fibrinolytic potential in plasma.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Fibrina/metabolismo , alfa 2-Antiplasmina/metabolismo , Adulto , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/fisiopatologia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinolisina/metabolismo , Fibrinólise/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/sangue , Ativador de Plasminogênio Tecidual/sangueRESUMO
OBJECTIVE: The antiphospholipid syndrome (APS) is defined by persistent antiphospholipid antibodies together with thrombosis and/or pregnancy morbidity. We investigated the tightness of fibrin clot and fibrinolytic function in plasma samples from APS patients compared with two control groups. MATERIAL AND METHODS: APS patients (n=49), healthy controls (HC) (n=19) and warfarin-treated nonAPS thrombosis controls (nonAPS-TC) (n=39) were investigated. Fibrin permeability was assessed as the permeability coefficient (Ks) by a flow measurement technique. Additionally, clot density and fibrinolytic function was analysed by a turbidimetric clotting and lysis assay. Fibrin structure was visualised using scanning electron microscopy. Finally, the number of cell-derived microparticles (MPs) in the samples were correlated to fibrin permeability RESULTS AND CONCLUSIONS: The Ks value was lower in samples from APS-patients compared to HC and nonAPS-TC (p<0.0001 for both) indicating a less permeable fibrin clot in APS patients. Scanning electron microscopy images confirmed compact fibrin with smaller intrinsic pores and thinner fibers in samples from APS patients as compared to HC. Prolonged fibrinolysis (clot lysis) times were present in the subgroup of APS patients with previous arterial thrombosis (n=15) as compared to HC and to nonAPS-TC (all p-values<0.05). In conclusion, tighter fibrin clots were formed in plasma from APS patients compared with healthy controls and warfarin treated patients with thrombosis of "nonAPS origin". This new observation presents a possible mechanism contributing to the thrombotic predisposition of APS patients. Impaired fibrinolysis, selectively present among APS patients with previous arterial thrombosis, may further aggravate the pro-thrombotic state in this APS subgroup.
Assuntos
Síndrome Antifosfolipídica/sangue , Fibrina/metabolismo , Fibrinólise/fisiologia , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Trombose/sangueRESUMO
BACKGROUND: Hepatocyte transplantation (HcTx) has proven to be a safe procedure, although the functional results have been unsatisfactory, probably due to insufficient engraftment or a loss of transplanted mass or function. In this study, we investigate whether hepatocytes in contact with blood induce an inflammatory reaction leading to, similar to what happens in clinical islet transplantation, an instant blood-mediated inflammatory reaction (IBMIR) resulting in an early loss of transplanted cells. METHODS: By using an experimental model that mimics the portal vein blood flow, we could study different parameters reflecting the effects on the innate immunity elicited by hepatocytes in contact with ABO-matched human blood. RESULTS: We report that all aspects of the IBMIR such as platelet and granulocyte consumption, coagulation, and complement activation were demonstrated. Addition of various specific inhibitors of coagulation allowed us to clearly delineate the various stages of the hepatocyte-triggered IBMIR and show that the reaction was triggered by tissue factor. Analysis of a case of clinical HcTx showed that hepatocyte-induced IBMIR also occurs in vivo. Both the inflammatory and the coagulation aspects were controlled by low-molecular-weight dextran sulfate. CONCLUSION: Isolated hepatocytes in contact with blood induce the IBMIR in vitro, and there are indications that these events are also relevant in vivo. According to these findings, HcTx would benefit from controlling a wider range of signals from the innate immune system.
Assuntos
Hepatócitos/imunologia , Hepatócitos/transplante , Inflamação/etiologia , Complicações Pós-Operatórias/etiologia , Sistema ABO de Grupos Sanguíneos , Coagulação Sanguínea , Células Cultivadas , Sulfato de Dextrana/farmacologia , Humanos , Imuno-Histoquímica , Inflamação/prevenção & controle , Tromboplastina/fisiologiaRESUMO
Infusion of human third-party mesenchymal stromal cells (MSCs) appears to be a promising therapy for acute graft-versus-host disease (aGvHD). To date, little is known about how MSCs interact with the body's innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46) and DAF (CD55), but were protected from complement lysis via expression of protectin (CD59). Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.
Assuntos
Proteínas do Sistema Complemento/metabolismo , Imunidade Inata , Mesoderma/citologia , Receptores de Complemento/metabolismo , Animais , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Humanos , Células Estromais/imunologia , Células Estromais/metabolismoRESUMO
BACKGROUND: Small intestine neuroendocrine tumors (SI-NETs) belong to a rare group of cancers. Most patients have developed metastatic disease at the time of diagnosis, for which there is currently no cure. The delay in diagnosis is a major issue in the clinical management of the patients and new markers are urgently needed. We have previously identified paraneoplastic antigen Ma2 (PNMA2) as a novel SI-NET tissue biomarker. Therefore, we evaluated whether Ma2 autoantibodies detection in the blood stream is useful for the clinical diagnosis and recurrence of SI-NETs. METHODOLOGY/PRINCIPAL FINDINGS: A novel indirect ELISA was set up to detect Ma2 autoantibodies in blood samples of patients with SI-NET at different stages of disease. The analysis was extended to include typical and atypical lung carcinoids (TLC and ALC), to evaluate whether Ma2 autoantibodies in the blood stream become a general biomarker for NETs. In total, 124 blood samples of SI-NET patients at different stages of disease were included in the study. The novel Ma2 autoantibody ELISA showed high sensitivity, specificity and accuracy with ROC curve analysis underlying an area between 0.734 and 0.816. Ma2 autoantibodies in the blood from SI-NET patients were verified by western blot and sequential immunoprecipitation. Serum antibodies of patients stain Ma2 in the tumor tissue and neurons. We observed that SI-NET patients expressing Ma2 autoantibody levels below the cutoff had a longer progression and recurrence-free survival compared to those with higher titer. We also detected higher levels of Ma2 autoantibodies in blood samples from TLC and ALC patients than from healthy controls, as previously shown in small cell lung carcinoma samples. CONCLUSION: Here we show that high Ma2 autoantibody titer in the blood of SI-NET patients is a sensitive and specific biomarker, superior to chromogranin A (CgA) for the risk of recurrence after radical operation of these tumors.
Assuntos
Antígenos de Neoplasias/química , Autoanticorpos/química , Biomarcadores Tumorais/sangue , Neoplasias Intestinais/sangue , Neoplasias Intestinais/diagnóstico , Intestino Delgado/patologia , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Proteínas do Tecido Nervoso/química , Tumores Neuroendócrinos/sangue , Tumores Neuroendócrinos/diagnóstico , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Carcinoma de Pequenas Células do Pulmão/patologia , Adulto , Idoso , Cromogranina A/química , Intervalo Livre de Doença , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunoprecipitação , Masculino , Pessoa de Meia-Idade , Recidiva , Sensibilidade e EspecificidadeAssuntos
Materiais Biocompatíveis/efeitos adversos , Coagulação Sanguínea/efeitos dos fármacos , Teste de Materiais/métodos , Materiais Biocompatíveis/normas , Testes de Coagulação Sanguínea , Materiais Revestidos Biocompatíveis/efeitos adversos , Materiais Revestidos Biocompatíveis/normas , Fator XII/biossíntese , Fibrina/metabolismo , Vidro , Heparina/farmacologia , Humanos , Cinética , Teste de Materiais/instrumentação , Polietilenotereftalatos/farmacologia , Politetrafluoretileno/farmacologia , Análise EspectralRESUMO
Activated human plate lets trigger FXII-mediated contact activation, which leads to the generation of FXIIa-antithrombin (AT) and FXIa-AT complexes. This suggests that contact activation takes place at different sites, on activated platelets and material surfaces, during therapeutic procedures involving biomaterials in contact with blood and is differentially regulated. Here we show that activation in platelet-poor plasma, platelet-rich plasma (PRP), and whole blood induced by glass, kaolin, and polyphosphate elicited high levels of FXIIa-C1-inhibitor (C1INH), low levels of FXIa-C1INH and KK-C1INH, and almost no AT complexes. Platelet activation, in both PRP and blood, led to the formation of FXIIa-AT, FXIa-AT, and kallikrein (KK)-AT but almost no C1INH complexes. In severe trauma patients, FXIIa-AT and FXIa-AT were correlated with the release of thrombospondin-1 (TSP-1) from activated platelets. In contrast, FXIIa-C1INH complexes were detected when the FXIIa-AT levels were low. No correlations were found between FXIIa-C1INH and FXIIa-AT or TSP-1. Inhibition of FXIIa on material surfaces was also shown to affect the function of aggregating platelets. In conclusion, formation of FXIIa-AT and FXIIa-C1INH complexes can help to distinguish between contact activation triggered by biomaterial surfaces and by activated platelets. Platelet aggregation studies also demonstrated that platelet function is influenced by material surface-mediated contact activation and that generation of FXIIa-AT complexes may serve as a new biomarker for thrombotic reactions during therapeutic procedures employing biomaterial devices.
Assuntos
Antitrombinas/fisiologia , Materiais Biocompatíveis , Plaquetas/citologia , Proteínas Inativadoras do Complemento 1/fisiologia , Ativação Plaquetária , Plaquetas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Trombospondina 1/metabolismoRESUMO
Interactions of gene therapy vectors with human blood components upon intravenous administration have a significant effect on vector efficacy and patient safety. Here we describe methods to evaluate these interactions and their effects in whole human blood, using baculovirus vectors as a model. Opsonisation of baculovirus particles by binding of IgM and C3b was demonstrated, which is likely to be the cause of the significant blood cell-associated virus that was detected. Preventing formation of the complement C5b-9 (membrane attack) complex maintained infectivity of baculovirus particles as shown by studying the effects of two specific complement inhibitors, Compstatin and a C5a receptor antagonist. Formation of macroscopic blood clots after 4h was prevented by both complement inhibitors. Pro- and anti-inflammatory cytokines Il-1beta, IL-6, IL-8 and TNF-alpha were produced at variable levels between volunteers and complement inhibitors showed patient-specific effects on cytokine levels. Whilst both complement inhibitors could play a role in protecting patients from aggressive inflammatory reactions, only Compstatin maintained virus infectivity. We conclude that this ex vivo model, used here for the first time with infectious agents, is a valuable tool in evaluating human innate immune responses to gene therapy vectors or to predict the response of individual patients as part of a clinical trial or treatment. The use of complement inhibitors for therapeutic viruses should be considered on a patient-specific basis.