RESUMO
Preservation of normal joint function in patients with haemophilia is a goal of modern therapy. Regular injections of anti-haemophilic factor concentrate reduce the risk of joint bleeding, the optimal regimen for which remains under investigation. The goals of the experiment described here are: (i) to assess the capacity of a murine model of severe haemophilic arthropathy to predict the likelihood of success of a test product to prevent joint bleeding and the complications that follow and (ii) to compare the effectiveness of recombinant human activated factor VII (rFVIIa) to recombinant human factor VIII (rFVIII) to prevent acute joint bleeding in the mouse model of haemarthrosis. Mice lacking expression of FVIII received a single intravenous injection of human rFVIII (280 U kg(-1)), rFVIIa (10 mg kg(-1)) or vehicle prior to blunt trauma injury to the knee joint. Mice receiving rFVIII and rFVIIa developed less injury-induced joint bleeding, swelling and loss of range of motion compared to mice pretreated with vehicle. Despite the reduction in clinical symptoms, synovial hyperplasia was evident in all groups after 7 days although less pronounced in mice receiving rFVIII and rFVIIa. The data under these experimental conditions demonstrate: (i) that this model can be used to evaluate novel therapies designed to prevent joint bleeding (prophylaxis) and (ii) both rFVIII and rFVIIa reduced acute haemarthrosis but did not completely prevent synovitis, the sequelae of blood induced joint injury.
Assuntos
Modelos Animais de Doenças , Hemartrose/prevenção & controle , Doença Aguda , Animais , Avaliação de Medicamentos/métodos , Fator VIII/uso terapêutico , Fator VIIa/uso terapêutico , Hemartrose/etiologia , Hemartrose/patologia , Hiperplasia/patologia , Hiperplasia/prevenção & controle , Traumatismos do Joelho/complicações , Traumatismos do Joelho/patologia , Camundongos , Camundongos Knockout , Proteínas Recombinantes/uso terapêutico , Membrana Sinovial/patologia , Sinovite/patologia , Sinovite/prevenção & controle , Ferimentos não Penetrantes/complicações , Ferimentos não Penetrantes/patologiaRESUMO
A poor response to the infusion of anti-haemophilic factor (AHF) concentrates used to control acute bleeding or prevent haemorrhage during surgery may have many causes, including the lack of functional activity of the infused protein concentrate. It is generally recommended to follow the manufacturers' instructions when administering the factor intravenously. For convenience, infusion sets with 23 gauge butterfly needles are packaged with the major brands of AHF concentrate. In hospitals, blunt connectors for i.v. infusion are becoming more prevalent to reduce the risk of needle stick injuries to both patients and hospital staff. The integrity of AHF infused using such devices has not been examined. We examined the possibility that passage of complex proteins such as AHF through such devices may result in alteration of the protein, rendering it inactive. The results presented here suggest that this is not the case; neither the structure nor the function of AHF protein products was adversely affected by the use of the needleless transfer device (NTD) or the needle types and sizes commonly used in clinical practice. Samples of factor VIII and factor IX AHF concentrate were passed through the B-D Blunt Plastic Cannula or 23, 27 and 30 gauge needles. No significant changes in electrophoretic mobility or coagulation activity were detected. Samples generally showed slightly increased coagulation activity when compared with the control in which the sample was passed directly out of the syringe with no device attached. These data indicate that the NTD and various needle gauges are acceptable for the infusion of AHF concentrate in a clinical setting. Furthermore, the use of small gauge needles has advantages that may improve adherence to rigorous factor replacement programmes.
Assuntos
Fatores de Coagulação Sanguínea/administração & dosagem , Hemofilia A/tratamento farmacológico , Hemofilia B/tratamento farmacológico , Agulhas , Coagulação Sanguínea/efeitos dos fármacos , Fatores de Coagulação Sanguínea/farmacocinética , Eletroforese em Gel de Poliacrilamida/métodos , Desenho de Equipamento , Fator IX/administração & dosagem , Fator IX/farmacocinética , Fator VIII/administração & dosagem , Fator VIII/farmacocinética , Humanos , Infusões Intravenosas/instrumentação , Peso Molecular , Peptídeos/farmacocinéticaRESUMO
Recurrent joint bleeding is the most common musculoskeletal manifestation of haemophilia and leads to a target joint and synovitis. The pathobiology of haemophilic synovitis (HS) is not well understood. Here the histopathological changes that occur following haemarthrosis were examined in an animal model for human HS. After two haemarthrosis, there was soft tissue and joint swelling and histological changes of acute synovitis included infiltration of the sub-synovial layer by mononuclear cells and neutrophils, thickening of the synovial membrane with villus formation, and hyperplasia of blood vessels. Subacute changes were evident after three haemarthrosis; muscle atrophy was present and an intense mononuclear cell infiltrate filled the sub-synovial space. There was destruction of articular surfaces and loss of cartilage. Seventeen months after three haemarthrosis, chronic joint changes included gross deformity and loss of congruence due to dense fibrotic tissue filling the joint space. The mononuclear inflammatory cell infiltrate and thickened synovial membrane persisted. Pits and erosions of articular surfaces and sub-chondral cysts were present. There was fibro-cartilage and new bone formation. This model of human HS should be useful to fully evaluate the biochemical and molecular changes that occur following joint bleeding and to test novel therapeutics to prevent HS.
Assuntos
Hemofilia A/patologia , Sinovite/patologia , Doença Aguda , Animais , Doença Crônica , Modelos Animais de Doenças , Hemartrose/etiologia , Hemartrose/patologia , Hemofilia A/complicações , Membro Posterior , Humanos , Deformidades Articulares Adquiridas/etiologia , Deformidades Articulares Adquiridas/patologia , Articulações/patologia , Camundongos , Camundongos Endogâmicos , Sinovite/etiologiaRESUMO
Transfusion-transmitted virus (TTV) is a potential cause of post-transfusion hepatitis in patients with haemophilia. Plasma-derived clotting factor concentrates currently undergo processes that are effective in removal and inactivation of viruses such as HIV, hepatitis B and C; however, their effectiveness with respect to TTV is unknown. To determine if TTV DNA is present in plasma-derived concentrates of factor IX, we tested 14 lots of Mononine and compared the results with BeneFix. Nucleic acid isolation, followed by a two-round polymerase chain reaction (PCR) and agarose gel analysis indicated that all 17 lots were negative for TTV. Although TTV may be considered an emerging pathogen, no evidence of the virus was detected in the commercially available plasma-derived concentrate of FIX most commonly used to treat haemophilia B.
Assuntos
Infecções por Circoviridae/transmissão , Fator IX/uso terapêutico , Hepatite B/terapia , Torque teno virus , DNA Viral/análise , Humanos , Reação em Cadeia da Polimerase/métodos , Reação TransfusionalRESUMO
Haemophilia is a genetic disease as a result of the deficiency of blood coagulation factor VIII or IX. Bleeding is common, especially into joints where an inflammatory, proliferative synovitis develops resulting in a debilitating arthritis, haemophilic arthropathy. The pathogenesis of blood-induced haemophilic synovitis (HS) is poorly understood. The gross, microscopic and ultrastructural changes that occur in the synovial membrane following human and experimental hemarthrosis have been described. Repeated episodes of bleeding induce synoviocyte hypertrophy and hyperplasia, an intense neovascular response and inflammation of the synovial membrane. The component(s) in blood that initiates these changes is(are) not known, although iron is often proposed as one possibility. Here, we describe a novel murine model of human haemophilia A, which facilitates the examination of large number of animals and tissue specimens. The effects of hemarthrosis on the physical, gross and microscopic changes evoked following joint bleeding are described. Controlled, blunt trauma to the knee joint consistently resulted in joint swelling because of a combination of bleeding and inflammation. Hemosiderin was found in the synovial membrane. Similar to hemarthrosis in human haemophilia, joint bleeding resulted in acute morbidity evidenced by inactivity, weight loss and immobility. With time the animals recovered. The model of experimental murine HS described here has utility in the study of the pathogenesis of HS. This is the first of a series of articles, which will discuss the pathophysiology and characterize the model, with comparison of his model to others which have been published previously. It should provide a useful model to test potential therapeutic interventions.
Assuntos
Hemartrose/etiologia , Hemofilia A/complicações , Sinovite/etiologia , Animais , Índice de Massa Corporal , Hemartrose/patologia , Hemofilia A/patologia , Camundongos , Camundongos Knockout , Modelos Animais , Atividade Motora , Sinovite/patologia , Redução de Peso , Ferimentos não Penetrantes/complicações , Ferimentos não Penetrantes/patologiaRESUMO
Gangliosides, sialic acid-containing glycosphingolipids, accumulate in atherosclerotic vessels. Their role in the pathogenesis of atherosclerosis is unknown. Gangliosides isolated from tumor cells promote collagen-stimulated platelet aggregation and ATP secretion and enhance platelet adhesion to immobilized collagen. These activities are all mediated by ganglioside effects on the platelet integrin collagen receptor alpha2beta1. Therefore, we hypothesized that gangliosides isolated from atherosclerotic plaques would enhance platelet adhesion to immobilized collagen, a major component of the subendothelial matrix of blood vessels. Furthermore, we questioned whether this effect of atherosclerotic gangliosides might play a role in the pathogenesis of atherosclerosis. To test this hypothesis, we isolated the gangliosides from postmortem aortas of patients with extensive atherosclerotic disease and examined their effects on platelet adhesion. Samples of aortic tissue taken from areas involved with atherosclerotic plaque demonstrated accumulation of gangliosides (64.9+/-6.5 nmol/g wet weight) compared with gangliosides isolated from control normal aortic tissue taken from children who died of noncardiac causes (NAGs; 21.1+/-6.4 nmol/g wet weight). Interestingly, samples of tissue taken from diseased aortas but from areas not involved with gross plaque formation also demonstrated ganglioside accumulation (47.6+/-12.8 nmol/g wet weight). Next, the activity of each of these gangliosides on platelet adhesion to immobilized type I collagen was studied. Atherosclerotic aortic gangliosides (AAGs) as well as those isolated from grossly unaffected areas of the same aorta (UAGs) both increased platelet adhesion compared with control NAGs (OD570, 0. 37+/-0.11 and 0.29+/-0.14 versus 0.16+/-0.07, respectively; P<0.01 and P<0.05, respectively). These OD570 values corresponded to 9x10(5), 8x10(4), and 6x10(3) platelets per well after preincubation with 5 micromol/L AAG, UAG, and NAG, respectively. Increased adhesion was observed after preincubation with as little as 0.5 micromol/L AAG, and maximal adhesion was seen at 2.5 micromol/L, with a plateau extending to the highest concentration tested, 10 micromol/L. The effect of AAGs on platelet adhesion to collagen was abrogated by incubation of treated platelets with F-17 anti-alpha2 monoclonal antibody (OD570, 0.13+/-0.02). Finally, the effects of the major individual gangliosides isolated from atherosclerotic tissues, GM3 and GD3, were tested. GM3 increased adhesion to collagen (OD570, 0.415+/-0.06) as did GD3 (0.31+/-0.08). Similar to that of AAGs, the effect of both molecules was blocked by F-17 (0. 09+/-0.04 and 0.13+/-0.06, respectively). These experiments demonstrate that accumulated atherosclerotic gangliosides promote platelet adhesion to collagen, the major component of the subendothelial matrix. Furthermore, this activity is mediated by an effect of the gangliosides on the collagen-binding integrin alpha2beta1. This activity may provide a mechanism for the development of platelet thrombi at sites where atherosclerotic gangliosides accumulate and help to explain the role of platelets in the process of atherosclerotic disease progression.
Assuntos
Doenças da Aorta/metabolismo , Arteriosclerose/metabolismo , Gangliosídeo G(M3)/metabolismo , Integrinas/metabolismo , Adesividade Plaquetária/fisiologia , Idoso , Aorta/química , Aorta/metabolismo , Plaquetas/química , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Colágeno/metabolismo , Colágeno/farmacologia , Feminino , Gangliosídeo G(M3)/análise , Gangliosídeos/análise , Gangliosídeos/metabolismo , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Receptores de ColágenoRESUMO
Gangliosides, sialic acid-containing glycosphingolipids, enhance tumor formation in experimental animals and are associated with tumor progression and metastasis in humans. The mechanism(s) for this activity is (are) unknown. One possibility is enhanced platelet activation, since the interaction of platelets with tumor cells contributes to tumor cell arrest in the vascular compartment. We have previously shown that neuroblastoma tumor gangliosides (NBTG) enhance platelet adenosine triphosphate (ATP) secretion, aggregation, and adhesion. We determined that these NBTG effects are specific for collagen and are mediated through an alpha2 beta1 integrin-dependent mechanism. This report describes the effects of NBTG on a physiologically relevant model of collagen-alpha2 betal interaction. Platelet adhesion to immobilized native collagen fibers similar to those found in the extracellular matrix of blood vessels was determined. Platelet adhesion is enhanced by NBTG in a concentration-dependent manner. Incubation with concentrations of 1 and 10 microM NBTG increased platelet adhesion by 9% and 52%, respectively, compared to less than 1% in controls not incubated with gangliosides (P = 0.001 and P < 0.0001, respectively). In addition to increasing the number of adherent platelets, NBTG promoted more rapid attachment. In NBTG-incubated platelets, platelet adhesion began after a 5-min lag phase and was maximal at 30 min compared to a 20-min lag phase and maximal adhesion at 60 min for control platelets. At 30 min this difference was significant (P = 0.017); however, by 120 min there was no difference between NBTG and controls (P = 0.259). NBTG also induces platelet adhesion at collagen concentrations (0.1 microg) that failed to support adhesion of control platelets. These effects of NBTG require Mg2+ or Mn2+ ions but are not supported by Zn2+ or Ca2+ ions. Furthermore, preincubation of platelets with a blocking antibody (6F1) to the integrin collagen receptor alpha2 beta1 abrogates all of the effects of NBTG. These results indicate that tumor gangliosides enhance platelet adhesion to extracellular matrix collagen and promote rapid stabilization of the collagen-alpha2 beta1 interaction, the initial steps in platelet activation.
Assuntos
Colágeno/metabolismo , Gangliosídeos/farmacologia , Neuroblastoma/química , Adesividade Plaquetária/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Gangliosídeos/isolamento & purificação , Humanos , Integrinas/fisiologia , Neuroblastoma/patologia , Receptores de Colágeno , Células Tumorais CultivadasRESUMO
N- and C-terminal amino acids of proteolipid proteins from the whole brain and some other organs were investigated. N-terminal amino acids were identified by the dansylation procedure. C-terminal amino acids were determined after the enzymatic hydrolysis with carboxy peptidases A and B with the following dansylation. Phenyl alanine and lysine were identified as C-terminal amino acids of the proteolipids from the whole brain and only lysine--as the C-terminal amino acid of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle. The corresponding N-terminal amino acids of the proteolipids from the whole brain were aspartic acid and glycine and of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle--only aspartic acid. A comparison of the data obtained with the previous ones has shown that in the brain there exist only two types of proteolipids--one characteristic of myelin, another-- of mitochondria, and in other organs--only one characteristic of mitochondria.