A fusion protein of interleukin-4 and interleukin-10 protects against blood-induced cartilage damage in vitro and in vivo.

Essentials Targeted treatment for hemophilic arthropathy, still causing significant morbidity, is lacking. This study evaluates the efficacy of a fusion of protein of interleukin(IL)-4 and IL-10. In vitro the fusion protein prevents blood-induced cartilage damage in a dose-dependent manner. In hemophilic mice, the IL4-10 fusion protein ameliorates cartilage damage upon joint bleeding. SUMMARY: Background Joint damage still causes significant morbidity in hemophilia. It results from synovial inflammation and direct cartilage-degenerating properties of blood components. Interleukin (IL)-4 and IL-10 have been shown to protect cartilage from blood-induced damage. Recently an IL4-10 fusion protein has been developed to combine the function of IL-4 and IL-10 and increase their bioavailability. Objectives In this study we evaluate whether this IL4-10 fusion protein protects against blood-induced joint damage. Methods In vitro, human cartilage explants were exposed to whole blood and simultaneously to a broad concentration range of the IL4-10 fusion protein. Effects on cartilage matrix turnover were compared with the individual cytokines. Moreover, the influence of the fusion protein and its individual components on IL-1ß and IL-6 production was investigated. In hemophilia A mice, the effect of intra-articular treatment on synovitis and cartilage damage resulting from joint bleeding was evaluated by histochemistry. Results In vitro, the fusion protein prevented blood-induced cartilage damage in a dose-dependent manner, with equal effectiveness to the combination of the separate cytokines. In whole blood cultures 10 ng mL-1 fusion protein completely blocked the production of IL-1ß and IL-6 by monocytes/macrophages. In hemophilic mice, intra-articular injection of IL-4 and IL-10 did not influence synovitis or cartilage degeneration. In contrast, equimolar amounts of the fusion protein attenuated cartilage damage upon repeated joint bleeding, although synovial inflammation was hardly affected. Conclusions Overall, this study shows that the IL4-10 fusion protein prevents blood-induced cartilage damage in vitro and ameliorates cartilage degeneration upon joint bleeding in hemophilic mice.

Clinical phenotype in genetically confirmed von Willebrand disease type 2N patients reflects a haemophilia A phenotype.

INTRODUCTION: Von Willebrand disease (VWD) type 2N is characterized by a defective binding of factor VIII (FVIII) to von Willebrand factor (VWF) resulting in diminished plasma FVIII levels and a clinical phenotype mimicking mild haemophilia A. Several mutations in the FVIII binding site of VWF have been reported. AIM: This study aims to examine the effect of genotype on clinical phenotype in a cohort of VWD 2N patients. METHODS: Patients with at least one genetically confirmed 2N mutation were selected retrospectively from a cohort of patients with suspected VWD. Clinical and laboratory phenotypes including bleeding scores (BS) were obtained and analysed. RESULTS: Forty-two VWD 2N patients with a mean age of 44 years were included. Eleven patients were homozygous or compound heterozygous (genetically confirmed group) and 31 patients were heterozygously affected (carriers group). Statistically significant differences between genetically confirmed VWD 2N patients and carriers were found in FVIII activity, VWF antigen levels, VWF-FVIII binding capacity, FVIII/VWF antigen ratio (all P<0.001), VWF-ristocetin activity (p=0.001) and VWF collagen binding (P = 0.002). Median BS was 6 in genetically confirmed VWD 2N patients compared with 3 in carriers (P = 0.047). Haemarthrosis, muscle haematomas and postpartum haemorrhage were only reported in genetically confirmed 2N patients. CONCLUSION: Phenotypic analysis showed that all laboratory parameters are lower in genetically confirmed VWD 2N patients compared with heterozygous 2N carriers. The clinical phenotype in genetically confirmed VWD 2N patients is comparable to mild haemophilia A patients and more severe than heterozygous 2N carriers.

Biochemical markers of joint tissue damage increase shortly after a joint bleed; an explorative human and canine in vivo study.

OBJECTIVE: Evaluation whether biomarkers of joint damage are sensitive to change shortly after a joint bleed in hemophilia patients and in a canine model of blood-induced joint damage. METHODS: Blood and urine samples were collected from 10 hemophilia patients after they reported a joint bleed: within 2 days, after 3-5 days, and 12-14 days. Additionally, 90 days after the bleed a blood and urine sample was taken and considered to represent baseline condition. Commercial serum and urine biomarker assays were performed: urinary C-terminal telopeptide of type II collagen (uCTX-II), serum cartilage oligomeric matrix protein (sCOMP), serum cartilage cleavage product C1,2C, and serum chondroitin sulfate 846 (sCS846). The same panel of biomarkers was explored in dogs (n = 7) after induction of a first joint bleed by intra-articular blood injections. Biosamples were collected at baseline, day 2, 1 and 2 weeks later. RESULTS: In hemophilia patients, levels of uCTX-II and sCS846 increased 5 days after joint bleeding when compared with baseline (+52%; P = 0.021 and +14%; P = 0.011, respectively). In dogs, uCTX-II increased statistically significant from day 2 to day 7 (from 75% to 155% of baseline; P = 0.018), and sCOMP from baseline to day 2 (+46%; P = 0.028). CONCLUSIONS: This study demonstrates that biochemical markers of joint tissue damage increase shortly after a single joint bleed, both in humans with established hemophilic arthropathy (HA) and in an animal model of joint damage upon a first joint bleed. Biomarkers might be useful in monitoring the impact of a joint bleed and in evaluation of treatment of such bleeds.

Bone cysts in patients with afibrinogenaemia: a literature review and two new cases.

Afibrinogenaemia is an autosomal recessive disease with an estimated prevalence of approximately one in a million. The most common symptoms of afibrinogenaemia are umbilical cord bleeding, bleeding into skin, mouth, muscles, gastrointestinal and genitourinary tracts and the central nervous system. Other recognized complications include; haemarthroses, spontaneous splenic rupture, epistaxis, menorrhagia, recurrent abortion and venous and arterial thromboembolism. Bone cysts have also been described as a rare complication of afibrinogenaemia. The aim of this study was to conduct a systematic literature review, summarize the reported cases and to report two new cases. Three electronic databases were searched for relevant publications: PubMed, Medline and EMBASE. The following search criteria were used: '(bone cysts OR intraosseous haematoma OR intraosseous haemorrhage) AND (afibrinogenaemia OR fibrinogen deficiency)'. The reference lists of the selected papers were searched for more relevant literature. In total, eight patients had bone cysts as complication of afibrinogenaemia and six of them suffered from pain in their extremities. Bone cysts were primarily located in the vicinity of the cortex or trabeculae in the diaphysis of the long bones, especially in the femora, tibiae and humeri. Some were regressive, probably due to reactive bone remodelling. A number of cysts were filled with serosanguinous fluid. It might be useful to check for bone cysts when patients with congenital afibrinogenaemia complain of 'rheumatic' pains in their extremities. Whole body magnetic resonance imaging is the diagnostic imaging technique of choice. Recurrent episodes of pain, but not radiological deterioration, appear to benefit from prophylactic therapy with fibrinogen concentrate.

Consequences of intra-articular bleeding in haemophilia: science to clinical practice and beyond.

Blood in the joint causes a number of physiological and pathological events that eventually lead to haemophilic arthropathy. Animal models show that blood in the joint induces inflammation that continues long after blood has been cleared. TNF-alpha, IL-1 beta and IL-6 are inflammatory mediators that increase following haemarthrosis in haemophilic mice. Conventional anti-inflammatory drugs have failed to demonstrate a lasting effect in preventing haemophilic arthropathy. A new TNF-alpha antagonist has shown promising results in haemophilic mice. Similarly, the use of cyclo-oxygenase-2 inhibitors may reduce angiogenesis associated with the healing process following bleeding and the associated tissue damage. Animal models are useful for studying the pathophysiology of haemarthropathy, however, when applying results from animals to humans, the differences in matrix turnover rate, thickness of cartilage and joint biomechanics must be kept in mind. In people with haemophilia, there is a variable response to haemarthrosis as demonstrated by magnetic resonance imaging (MRI). Up to 30% of subjects have normal MRI despite having three or more haemarthroses into the same joint. Once bone damage is present, little can be done to restore anatomic integrity. Several molecules, including members of the bone morphogenic protein subfamily, have been injected into bone defects in non-haemophilic subjects with some evidence of benefit. To achieve the primary goal of reducing blood in the joint and the negative sequelae, it is questionable to use ice to treat haemarthrosis. Indeed low temperature is associated with impairment of coagulation enzyme activity and platelet function.

Joint distraction results in clinical and structural improvement of haemophilic ankle arthropathy: a series of three cases.

The incidence of haemophilic arthropathy in multiple joints decreased due to treatment with clotting factor. Nowadays patients are enabled to live a rather normal life, resulting in more (sports) trauma-induced arthropathy in isolated joints like the ankle. As surgical treatment options, fusion of the tibiotalar joint and total ankle replacement are available. Both standard treatments have complications and therefore an alternative treatment is desired. In this study, treatment of haemophilic ankle arthropathy with joint distraction was explored. Three patients with haemophilic ankle arthropathy were treated with joint distraction using an Ilizarov external fixator. Clinical outcomes like function, participation and pain were evaluated in retrospect with three different questionnaires: haemophilia activities list, impact on participation and autonomy and the Van Valburg questionnaire. Structural changes were assessed blinded on X-ray by the Pettersson score and ankle images digital analysis (AIDA) and by an MRI score. All three patients were very satisfied with the clinical outcome of the procedure. They reported a clear improvement for self-perceived functional health, participation in society and autonomy and pain. Partial ankle joint mobility was preserved in the three patients. The Pettersson score remained the same in one patient and slightly improved in the two other patients, while joint space width measured by AIDA and the MRI score demonstrated improvement for all three patients after ankle distraction. This study suggests that joint distraction is a promising treatment for individual cases of haemophilic ankle arthropathy, without additional risk of bleedings during treatment.

IL-4 alone and in combination with IL-10 protects against blood-induced cartilage damage.

OBJECTIVE: It has been reported that interleukin (IL)-10 limits blood-induced cartilage damage. Our aim was to study the effect of IL-4 alone and in combination with IL-10 on blood-induced cartilage damage. DESIGN: Healthy human full thickness cartilage explants were cultured for 4 days in the presence of 50% v/v blood. IL-4, IL-10, or a combination of both cytokines was added during blood exposure. Cartilage matrix turnover was determined after a recovery period; additionally cytokine production, chondrocyte apoptosis, and expression of the IL-4 and IL-10 receptors were analyzed directly after exposure. RESULTS: Blood-induced damage to the cartilage matrix was limited by IL-4 in a dose-dependent way (P<0.05). Also IL-10 limited this damage, although to a lesser extent (P<0.03). The effect of IL-4 plus IL-10 was more pronounced and protective than IL-10 alone (P<0.05). Production of IL-1ß and tumor necrosis factor (TNF)-α was limited by both IL-4 and IL-10 (P<0.05), but more strongly by IL-4. Blood-induced apoptosis of chondrocytes was limited by IL-4 and the combination, and not by IL-10 alone. No direct beneficial effect of IL-4 or IL-10 on cartilage was found, however, the chondrocyte receptor expression of both cytokine receptors was upregulated by exposure to blood. CONCLUSIONS: This study demonstrates that IL-4 alone and in combination with IL-10 prevents blood-induced cartilage damage. Expectedly, anti-inflammatory effects on monocytes in the blood fraction and protective effects on chondrocytes are both involved. IL-4 in combination with IL-10 might be used to prevent blood-induced joint damage as a result of trauma or surgery.