Serological biomarkers detect active joint destruction and inflammation in patients with haemophilic arthropathy.

INTRODUCTION: Progressive arthropathy caused by recurrent joint bleeds is a severe complication in haemophilia. AIM: We investigated whether biomarkers of cartilage and bone degradation, and inflammation were altered in haemophilia patients and whether these biomarkers could identify haemophilia patients with arthropathy. METHODS: Serum from 35 haemophilia patients with varying degrees of arthropathy and 43 age- and gender-matched control subjects were analysed. Biomarkers of cartilage degradation (C2M, COMP, CTX-II, ADAMTS5), cartilage formation (PRO-C2), bone formation (PINP), bone resorption (CTX-I) and inflammation (hsCRP, CRPM) were measured by ELISA. Arthropathy was assessed by radiological evaluation (Pettersson score) and physical examination (Gilbert score). RESULTS: In patients with haemophilia, cartilage degradation, measured by C2M, CTX-II and COMP, was increased by 25% (P < 0.05) compared with control subjects. Levels of the cartilage degradation enzyme, ADAMTS5, were 10% lower in haemophilia patients (P < 0.05). Bone formation (PINP) was reduced by 25% (P < 0.05) in haemophilia patients, whereas bone resorption (CTX-I) was increased by 30% (P < 0.001). Acute inflammation (hsCRP) was increased by 50% (P < 0.01), whereas chronic inflammation (CRPM) was decreased by 25% (P < 0.0001). The hsCRP/CRPM ratio was 60% higher (P < 0.001) in haemophilia patients relative to control subjects. A biomarker panel combining C2M, CRPM, and ADAMTS5 could distinguish haemophilia patients from control subjects with 85.3% accuracy (P < 0.0001). We found no strong correlation between biomarkers and radiological and physical examination of the joint. CONCLUSION: Biomarkers detect increased cartilage and bone degradation, and altered inflammatory activity in haemophilia patients with arthropathy. These biomarkers could potentially be used to identify patients with progressing joint disease.

Altered collagen turnover in factor VIII-deficient rats with hemophilic arthropathy identifies potential novel serological biomarkers in hemophilia.

Essentials Joint bleeding in hemophilia may induce significant remodeling of the extracellular matrix. Biomarkers of collagen turnover were investigated in a F8-/- rat model of hemophilic arthropathy. Biomarkers of cartilage degradation increased significantly during development of arthropathy. Basement membrane and interstitial matrix turnover changed significantly following hemarthrosis. SUMMARY: Background Hemophilic arthropathy is a severe complication of hemophilia. It is caused by recurrent bleeding into joint cavities, which leads to synovial inflammation, fibrosis, cartilage degradation and bone remodeling. Extracellular matrix remodeling of affected tissues is a hallmark of these pathological processes. Objectives The aim of this study was to use serological biomarkers of collagen turnover to evaluate extracellular matrix remodeling in a factor VIII-deficient rat model of hemophilic arthropathy. Methods F8-/- rats and wild-type littermate controls were subjected to repeated knee bleeds induced by needle puncture on days 0 and 14. Development of arthropathy was confirmed by histology after termination on day 28. Serum samples were collected at baseline and throughout the study and analyzed for biomarkers of collagen turnover, including collagens of the basement membrane (type IV collagen), the interstitial matrix (collagen types III, V and VI) and cartilage (type II collagen). Results In F8-/- rats, induced knee bleeding and subsequent development of arthropathy caused significant alterations in collagen turnover, measured as changes in serological biomarkers of basement membrane turnover, interstitial matrix turnover and cartilage degradation. Biomarkers of type II collagen degradation correlated significantly with cartilage degradation and degree of arthropathy. Hemophilic rats had a 50% higher turnover of the basement membrane than wild-type littermates at baseline. Conclusions Joint bleeding and hemophilic arthropathy cause changes in turnover of extracellular matrix collagens in hemophilic rats. Biomarkers of collagen turnover may be used to monitor joint bleeding and development of blood-induced joint disease in hemophilia.

Purification and characterization of a new recombinant factor VIII (N8).

SUMMARY: A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg(-1). Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.

Secretion of osteopontin from MG-63 cells under a physiological level of mechanical strain in vitro--a [35S] incorporation approach.

To gain insight into the early response of osteoblastic cells to a physiological level of mechanical strain in vitro, the secretion of osteopontin by MG-63 osteosarcoma cells was assessed by [35S] incorporation and autoradiography. First, osteopontin secreted from MG-63 cells was immunolocalized at 60-64 kDa (Mr) by polyacrylamide gel electrophoresis. A uniform physiological level of strain was generated by a vacuum added to the convex side of a half-ball shaped silicon rubber membrane on which the cells were cultured on the concave side. After labelling proteins with [35S]-methionine/cysteine (147 microCi/ml), the membranes were exposed to a strain of 0.5 per cent (5000 microepsilon), 3 cycles/minute (sine wave) with 10 minutes on and off. At 1, 2 and 4 hours after strain, the supernatants were collected and analysed by 10 per cent sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. The results showed that osteopontin was secreted by the strained cells at significantly higher amounts than the non-strained cells at all three time points (P < 0.05), with the first hour being the most prominent. A physiological level of mechanical strain increased the secretion of osteopontin from MG-63 cells in an early phase. This finding implies an accelerated process of bone remodelling, which suggests that the application of light and intermittent forces would result in the cellular reaction identified in relation to orthodontic tooth movement. The results indirectly indicate that the level of force presently used might be too high.

Time-dependent expression and processing of a hypothetical protein of possible importance for regulation of the Chlamydia pneumoniae developmental cycle.

Chlamydia pneumoniae is an obligate intracellular human pathogen infecting epithelial cells of the upper respiratory tract. It is a Gram-negative bacteria and has a unique biphasic developmental cycle. In this study, we use two-dimensional gel electrophoresis in combination with radioactive labeling to investigate time-dependent expression and processing of C. pneumoniae proteins. We report on (i) the identification of a hypothetical protein which is expressed late in the developmental cycle and subsequently processed; we speculate that this protein may be of importance for the developmental cycle of Chlamydia; (ii) the identification of the major outer membrane protein in three different variants, which may all be present in vivo.

Proteome analysis of the Chlamydia pneumoniae elementary body.

Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniae cultivated in Hep-2 cells were 35S-labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two-dimensional gel electrophoresis. Excised protein spots were in-gel digested with trypsin and peptides were concentrated on reverse-phase chromatographic beads for identification analysis by matrix-assisted laser desorption/ionization-mass spectrometry. In the pH range from 3-11, 263 C. pneumoniae protein spots encoded from 167 genes were identified. These genes constitute 15% of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.

Potential relevance of Chlamydia pneumoniae surface proteins to an effective vaccine.

The surface of Chlamydia pneumoniae is covered with proteins but their exact identification is not known probably because of the presence of conformational epitopes. A family of 21 pmp genes has been found by DNA sequencing. In common, these genes have the capacity to encode the amino acid motif GGAI. Several of the genes have the capacity to encode outer membrane proteins of about 100 kDa. Thus, they are candidate genes to encode the protein(s) present in the 98-kDa protein band of the C. pneumoniae outer membrane complex. The production of recombinant GGAI proteins is described as is the use of polyclonal antibodies raised against the recombinant GGAI proteins to determine their expression in C. pneumoniae elementary bodies. At least three of the proteins, Omp4, 5, and 11, are expressed.