Cisplatin and progesterone separate and combined action on rat brain nuclear phospholipids content.

The antitumor agent cisplatin and steroid hormone progesterone separate and combined action on content of total phospholipids and their individual classes in nuclei from rat brain cells were investigated. Cisplatin and progesterone exhibit their own characteristic properties, when used separately. Cisplatin reduces, and progesterone, on the contrary, increases the content of total phospholipids. When used together, the effects of these drugs are summed up. Cisplatin reduces the content of all 7 individual phospholipids found in rat brain nuclear preparations. Progesterone, on the other hand, increases the content of 5 classes of phospholipids. The combined use of cisplatin and progesterone restores 5 classes of nuclear phospholipids to the baseline level, and increases the quantity of 2 classes. The obtained results are discussed in terms of antagonistic effects of studied drugs, which can help in reducing undesirable side effects of cisplatin in case of combined use of antitumor drug and steroid.

Haemarthrosis model in mice: BSS - Bleeding Severity Score assessment system.

INTRODUCTION: The prophylactic administration of clotting factor concentrate is currently the most effective strategy for the prevention of joint bleeding. As new agents with different mechanisms of action and administration schedules are developed, it will be important to study them in relevant preclinical models. AIM: The aim of this study was the standardization of a mouse haemarthrosis model in a haemophilia mouse and the development and validation of a comprehensive bleeding assessment system, the Bleeding Severity Score (BSS). METHODS AND RESULTS: Four outcome measurements were assessed, two of which, the extra-articular bleeding score and intra-articular bleeding score, were determined to be the most reliable and were summarized into a BSS which was validated using a mouse haemarthrosis variability model. CONCLUSION: Using this model, the haemostatic effect of prospective drugs can be assessed in a clinically relevant joint bleeding model and will significantly increase the value of preclinical studies.

The biological efficacy profile of BAX 855, a PEGylated recombinant factor VIII molecule.

Prophylaxis prevents joint and other bleeding episodes in patients with haemophilia A. Development of new factor concentrates with longer circulating half-lives may encourage patients to start, continue or resume prophylaxis. The aim of this study was to compare the pharmacodynamic effect of a PEGylated full-length recombinant factor VIII (rFVIII) concentrate with that of an unmodified rFVIII concentrate with respect to the duration of prophylactic efficacy in a murine model of haemophilic joint bleeding. Mice were pretreated with BAX 855 or unmodified rFVIII at specified times before right knee puncture to induce haemarthrosis; left knee joints served as controls. Joint bleeding was evaluated using a combination of visual and histological assessments. Administration of a single dose of unmodified rFVIII before joint puncture prevented haemarthrosis in mice up to 24 h, whereas pretreatment with BAX 855 protected the joint from bleeding up to 48 h. This pharmacodynamic study showed prolonged efficacy of BAX 855 compared to ADVATE in a haemophilia A mouse joint bleeding model. This finding supports the possibility of using BAX 855 to increase FVIII trough levels and/or extend the dosing interval in patients with haemophilia A on prophylaxis, which may potentially improve prophylactic efficacy and long-term adherence.

Exploring the biological basis of haemophilic joint disease: experimental studies.

Haemophilia has been recognized as the most severe among the inherited disorders of blood coagulation since the beginning of the first millennium. Joint damage is the hallmark of the disease. Despite its frequency and severity, the pathobiology of blood-induced joint disease remains obscure. Although bleeding into the joint is the ultimate provocation, the stimulus within the blood inciting the process and the mechanisms by which bleeding into a joint results in synovial inflammation (synovitis) and cartilage and bone destruction (arthropathy) is unknown. Clues from careful observation of patient material, supplemented with data from animal models of joint disease provide some clues as to the pathogenesis of the process. Among the questions that remain to be answered are the following: (i) What underlies the phenotypic variability in bleeding patterns of patients with severe disease and the development of arthropathy in some but not all patients with joint bleeding? (ii) What is the molecular basis underlying the variability? (iii) Are there strategies that can be developed to counter the deleterious effects of joint bleeding and prevent blood-induced joint disease? Understanding the key elements, genetic and/or environmental, that are necessary for the development of synovitis and arthropathy may lead to rational design of therapy for the targeted prevention and treatment of blood-induced joint disease.

Prevention of haemarthrosis in a murine model of acute joint bleeding.

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.

Experimental haemophilic arthropathy in a mouse model of a massive haemarthrosis: gross, radiological and histological changes.

Recurrent haemarthrosis results in chronic synovitis and destructive arthropathy. The long-term effect of a single haemorrhage is not known. To investigate the histopathological changes following a single, but major joint haemorrhage, an animal model of massive haemarthrosis without mechanical trauma was developed and is described in this manuscript. The knee joint capsule of mice deficient in coagulation factor VIII or IX and non-haemophilic wild type mice was punctured to induce a one time, but massive haemorrhage. The single joint puncture resulted in acute haemarthrosis in both types of haemophilic mice but not in wild type mice. Subsequent to injury, the changes in the knee joints were analysed using gross, histological and radiographic assessments and compared with the uninjured knee. In addition, a novel imaging modality, micro-computed tomography, was used to document the structural damage to the joint. Our results indicate that the long-term changes classically observed in patients with advanced haemophilic arthropathy are evident following a single massive haemarthrosis. This model will allow a thorough investigation of the pathobiology of blood-induced joint disease and will be useful to test the efficacy of innovative therapeutic strategies to prevent haemophilic synovitis and arthropathy.

Hemostatic effect of recombinant factor VIIa, NN1731 and recombinant factor VIII on needle-induced joint bleeding in hemophilia A mice.

BACKGROUND: Hemophilia A is the most common serious bleeding disorder, and the hallmark of this disease is joint bleeding episodes. These result in hemophilic synovitis, an inflammatory and proliferative condition of the joint, which progresses into a chronic degenerative arthritis, hemophilic arthropathy. METHODS: In this paper, we describe the effect of recombinant factor VIIa (rFVIIa), and an analogue NN1731 as well as rFVIII on needle-induced bleeding in hemophilia A mice. CONCLUSIONS: Here we show a reducing effect of rFVIIa and NN1731 on bleeding induced in hemophilic mice, and we show that preventive treatment with rFVIII normalizes bleeding.

The impact of joint bleeding and synovitis on physical ability and joint function in a murine model of haemophilic synovitis.

Haemophilia is a congenital disorder that commonly results in musculoskeletal bleeding and orthopaedic complications. After an acute joint haemorrhage, an increase in intra-articular pressure and inflammation cause pain, swelling and limited motion. Blood in the joint space provokes a proliferative disorder known as haemophilic synovitis. Overgrowth of the synovial membrane causes mechanical dysfunction. Eventually, there is destruction of the articular surface and underlying bone. The aim of this project was to test the hypothesis that a minimum number of haemarthroses negatively impacts on joint function and that this would be reflected by decreased physical performance of experimental animals. Mice deficient in factor VIII coagulant activity were trained to ambulate on a rotating rod then injured three times at weekly intervals. Their ability to walk was then compared to a group of uninjured mice. Cohorts of mice were killed after 1, 2 or 3 months and the knee joints examined by gross and histological methods. The results supported the following conclusions: (i) haemophilic mice can be trained to ambulate on a rotating rod; (ii) acute hemarthrosis temporarily impairs their ability to ambulate and (iii) following recovery from acute injury, mice developing synovitis demonstrated inferior physical ability compared to mice not developing synovitis. This is the first description of a quantitative assay to monitor joint function in experimental animals and should be useful to evaluate the efficacy of new therapies developed to prevent and treat bleeding and to test strategies to counter the devastating effects of synovitis.

Pathogenesis of haemophilic synovitis: experimental studies on blood-induced joint damage.

Hemarthrosis is a common manifestation of haemophilia, and joint arthropathy remains a frequent complication. Even though the exact mechanisms related to blood-induced joint disease have not yet been fully elucidated, it is likely that iron deposition in the synovium induces an inflammatory response that causes not only immune system activation but also stimulates angiogenesis. This process ultimately results in cartilage and bone destruction. Investigating the processes that occur in the early stages of blood-induced joint disease in humans has been very limited. Therefore, the use of haemophilic animal models is critical to augment the understanding of this phenomenon. This article discusses three cellular regulators (p53, p21 and TRAIL) induced in synovial tissue that are important for iron metabolism. A cartilage remodelling programme induced by the release of cytokines and growth factors that result in articular damage is also discussed. Full elucidation of the pathogenesis of haemophilic joint disease is required to identify new avenues for prevention and therapy.

Histological changes in murine haemophilic synovitis: a quantitative grading system to assess blood-induced synovitis.

Haemophilia is a congenital disorder that results in frequent bleeding into joints, in which a chronic and debilitating arthritis develops. The presence of blood evokes an inflammatory and proliferative synovial reaction. Although the molecular mechanisms and biochemical pathways which underlie this disorder are not known, significant advances have been made by studying a murine model of human haemophilic synovitis. In order to better understand and correlate the pathological, molecular and biochemical changes, it has become necessary to grade the histological changes observed. Despite a search of the literature and review of relevant publications, none of the currently utilized schemes were appropriate, and therefore a novel grading scheme was developed. After review of over 1000 histological sections, six characteristic changes were identified: (i) synovial hyperplasia; (ii) vascularity; (iii) discolouration by haemosiderin; (iv) the presence of blood (erythrocytes); (v) villus formation; and (vi) cartilage erosion. Synovial hyperplasia and vascularity were present in variable amounts and were quantitatively scored (0-3), while the other changes were qualitatively scored as absent or present (0 or 1). Application of the grading scheme was tested and a high interobserver correlation (greater than 80%) was found. The scheme was easy to learn even by novices, with no prior experience. The availability of the histological grading scheme for murine synovitis will allow for precise evaluation of the pathological changes following joint bleeding, and facilitate correlations with molecular and biochemical changes that lead to these changes.

Needles and needleless devices for infusion of anti-haemophilic factor concentrate: impact on protein structure and function.

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.

Synovitis in a murine model of human factor VIII deficiency.

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.

Transfusion-transmitted virus is not present in factor IX concentrates commonly used to treat haemophilia B.

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.

Experimental haemophilic synovitis: rationale and development of a murine model of human factor VIII deficiency.

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.