Stijn Lambrecht

Proof of concept: enthesitis and new bone formation in spondyloarthritis are driven by mechanical strain and stromal cells

Objectives Spondyloarthritides (SpA) are characterised by both peripheral and axial arthritis. The hallmarks of peripheral SpA are the development of enthesitis, most typically of the Achilles tendon and plantar fascia, and new bone formation. This study was undertaken to unravel the mechanisms leading towards enthesitis and new bone formation in preclinical models of SpA. Results First, we demonstrated that TNFΔARE mice show typical inflammatory features highly reminiscent of SpA. The first signs of inflammation were found at the entheses. Importantly, enthesitis occurred equally in the presence or absence of mature T and B cells, underscoring the importance of stromal cells. Hind limb unloading in TNFΔARE mice significantly suppressed inflammation of the Achilles tendon compared with weight bearing controls. Erk1/2 signalling plays a crucial role in mechanotransduction-associated inflammation. Furthermore, new bone formation is strongly promoted at entheseal sites by biomechanical stress and correlates with the degree of inflammation. Conclusions These findings provide a formal proof of the concept that mechanical strain drives both entheseal inflammation and new bone formation in SpA.

A Pilot Study of the Use of an Osteochondral Scaffold Plug for Cartilage Repair in the Knee and How to Deal With Early Clinical Failures

PURPOSE To present our short-term experience with an osteochondral scaffold plug (TruFit plug; Smith & Nephew, Andover, MA) for cartilage repair in the knee and, more importantly, to discuss our approach to treat early clinical failures. METHODS Twenty patients were consecutively treated for their cartilage lesions with the plug technique. These patients were prospectively clinically evaluated at 6 and 12 months of follow-up. Magnetic resonance imaging (MRI) was used for morphologic analysis of the cartilage repair. Biopsy samples were taken from 3 cases during revision surgery, allowing histologic assessment of the repair tissue. RESULTS The short-term clinical and MRI outcome of this pilot study are modest. No signs of deterioration of the repair tissue were observed. Of the 15 patients followed up during 1 year, 3 (20.0%) showed persistent clinical symptoms or even more clinical symptoms after insertion of the plug. These patients were considered as failures and therefore eligible for revision surgery. During revision surgery, the repair tissue was carefully removed. The remaining osteochondral defect was filled with autologous bone grafts. Immediate and persistent relief of symptoms was observed in all 3 patients. Histologic assessment of biopsy specimens taken during revision surgery showed fibrous vascularized repair tissue with the presence of foreign-body giant cells. CONCLUSIONS The overall short-term clinical and MRI outcome of the osteochondral scaffold plug for cartilage repair in the knee is modest. In this pilot study a modest clinical improvement became apparent at 12 months of follow-up. MRI data showed no deterioration of the repair tissue. Of the 15 patients, 3 (20%) had persistent clinical symptoms after surgery. These patients were successfully treated with removal of the osteochondral plug remnants and the application of autologous bone grafts. LEVEL OF EVIDENCE Level IV, therapeutic case series.

Midterm Results of the Treatment of Cartilage Defects in the Knee Using Alginate Beads Containing Human Mature Allogenic Chondrocytes

Background: The treatment of chondral lesions is still an important challenge for the orthopaedic surgeon. Attempts have been made to restore cartilage lesions by filling the defects with a temporary biocompatible matrix. Purpose: The authors present their midterm experience with the implantation of alginate beads containing human mature allogenic chondrocytes for the treatment of cartilage lesions in the knee. Study Design: Case series; Level of evidence, 4. Methods: A biodegradable, alginate-based biocompatible scaffold containing human mature allogenic chondrocytes was used for the treatment of cartilage lesions in the knee. Twenty-one patients were clinically prospectively evaluated with use of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and a visual analog scale (VAS). The mean follow-up time was 6.3 years (range, 5-8 years). Magnetic resonance imaging (MRI) data were analyzed based on the MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) system, allowing morphologic assessment of the repair tissue. Magnetic resonance images were taken at 1 year of follow-up and at a mean follow-up of 6.1 years (range, 5-7 years). Results: During the follow-up period, the WOMAC and VAS scores improved significantly. No signs of clinical deterioration or adverse reactions to the alginate beads/allogenic chondrocyte implantation were observed. Four failures occurred during the follow-up period in this study (19.05%). The MOCART scores were moderate and remained stable in time. Conclusion: This investigation provided useful information on the efficacy of the implantation of alginate beads containing human mature allogenic chondrocytes for the treatment of cartilage lesions in the knee. The midterm clinical outcome of the presented technique was satisfactory. However, these results were not confirmed by the MRI findings.

Proteome characterization of human articular chondrocytes leads to novel insights in the function of small heat-shock proteins in chondrocyte homeostasis

OBJECTIVE In recent years, studies have been initiated to disclose the proteome of human chondrocytes and cartilage. Despite these studies, comprehensive information of the chondrocyte proteome remains limited. This study aimed to further explore the proteome expressed by human knee chondrocytes, and to study the functional aspects of heat-shock protein 27 (HSP27), a protein related to the previously described alphaBcrystallin, in chondrocyte biology. METHODS Chondrocytes isolated from human knee articular cartilage were cultured in a three-dimensional alginate culture system. To simplify the protein mixtures, proteins extracted from chondrocyte cell lysates were fractionated based on hydrophobicity and molecular weight. Proteins were digested and the resulting peptides were separated and identified by an on-line two-dimensional (2-D) nanoliquid chromatography (nanoLC)-system coupled to a quadrupole time-of-flight (Qq-TOF) mass spectrometer. Differential expression analysis of HSP27 was performed by Western Blotting and quantitative polymerase chain reaction (QPCR). The effects of HSP27 on chondrocyte biology were explored by suppression of HSP27 expression induced by RNA interference (RNAi). RESULTS In this study, we identified proteins with unknown functions together with membrane proteins, transcription factors and other low abundant proteins, which have not yet been described in chondrocytes. Based on previous knowledge on the related protein alphaBcrystallin, we selected HSP27 from the chondrocyte proteome database. Differential expression analysis revealed a decreased expression of HSP27 in Osteoarthritic (OA) chondrocytes. RNAi experiments revealed that HSP27 is involved in interleukin-1beta (IL-1beta) induced IL-6 secretion. CONCLUSION These findings highlight that small HSPs, especially HSP27, play a prominent role in the maintenance of human articular chondrocyte homeostasis.

Growth differentiation factor 15, a marker of lung involvement in systemic sclerosis, is involved in fibrosis development but is not indispensable for fibrosis development.

Transforming growth factor β superfamily members are involved in the pathogenesis of systemic sclerosis (SSc). Growth differentiation factor 15 (GDF‐15) is a distant member of this family. We undertook this study to evaluate the role of GDF‐15 in SSc pathogenesis.

Differential expression of αB-crystallin and evidence of its role as a mediator of matrix gene expression in osteoarthritis

OBJECTIVE Alpha B-crystallin belongs to the family of small heat-shock proteins (HSPs). The role of this protein family in chondrocytes is not well understood. The present study was undertaken to investigate expression levels of alphaB-crystallin in chondrocytes isolated from healthy subjects and patients with osteoarthritis (OA), and to explore the functional role of this potentially interesting protein in chondrocyte metabolism. METHODS Western blot and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were performed to determine expression levels of alphaB-crystallin in healthy and OA chondrocytes cultured in alginate beads. RNA interference-mediated gene knockdown was used to explore the role of this small HSP in chondrocyte biology, by transfecting low concentrations of small interfering RNA (siRNA) in cultured chondrocytes. RESULTS We initially identified alphaB-crystallin as a small HSP that was differentially expressed between healthy and OA-affected chondrocytes. The decreased abundance of this protein in OA chondrocytes was confirmed by Western blotting. Moreover, real-time RT-PCR confirmed the differential expression between chondrocytes isolated from visibly intact and visibly damaged zones of OA cartilage. The proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha both down-regulated alphaB-crystallin expression. Transfection of low concentrations of siRNA in cultured chondrocytes resulted in efficient knockdown of alphaB-crystallin gene expression. This was accompanied by altered expression of the chondrocyte-specific bone morphogenetic protein 2, aggrecan, and type II collagen genes. CONCLUSION The present findings identify the small HSP alphaB-crystallin as a novel mediator of chondrocyte matrix gene expression that may contribute to altered chondrocyte metabolism during the development of OA.

A20 inhibition of STAT1 expression in myeloid cells: a novel endogenous regulatory mechanism preventing development of enthesitis.

Objectives A20 is an important endogenous regulator of inflammation. Single nucleotide polymorphisms in A20 have been associated with various immune-mediated inflammatory diseases, and cell-specific deletion of A20 results in diverse inflammatory phenotypes. Our goal was to delineate the underlying mechanisms of joint inflammation in myeloid-specific A20-deficient mice (A20myelKO mice). Methods Inflammation in A20myelKO mice was assessed in a time-dependent manner. Western blot analysis and quantitative PCR analysis were performed on bone marrow-derived macrophages from A20myelKO and littermate control mice to study the effect of A20 on STAT1/STAT3 expression and STAT1/STAT3-dependent gene transcription in myeloid cells. The in vivo role of Janus kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signalling in the development of enthesitis in A20myelKO mice was assessed following administration of a JAK inhibitor versus placebo control. Results Enthesitis was found to be an early inflammatory lesion in A20myelKO mice. A20 negatively modulated STAT1-dependent, but generally not STAT3-dependent gene transcription in myeloid cells by suppressing STAT1 but not STAT3 expression, both in unstimulated conditions and after interferon-γ or interleukin-6 stimulation. The increase in STAT1 gene transcription in the absence of A20 was shown to be JAK-STAT-dependent. Moreover, JAK inhibition in vivo resulted in significant reduction of enthesitis, both clinically and histopathologically. Conclusions Our data reveal an important and novel interplay between myeloid cells and tissue resident cells at entheseal sites that is regulated by A20. In the absence of A20, STAT1 but not STAT3 expression is enhanced leading to STAT1-dependent inflammation. Therefore, A20 acts as a novel endogenous regulator of STAT1 that prevents onset of enthesitis.

First insights into human acetabular labrum cell metabolism

OBJECTIVE Hip labrum pathology has only begun to emerge as a significant source of groin pain in the last decade since the development of hip arthroscopy. Few data are available on the anatomy, histology and function of this structure. Moreover, no metabolic data exist at cellular level. The aim of this study was to characterize extracellular matrix (ECM) genes and pro-inflammatory mediators expressed by these cells. METHODS Isolated human acetabular labrum cells were cultured in alginate beads for 10 days and additionally stimulated with interleukin (IL)-1 for 24 h. Gene expression levels and secretion of different ECM genes, enzymes and cytokines were examined by quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) to assess the metabolic characteristics of labrum cells. Articular chondrocytes and meniscus cells served as controls. RESULTS Labrum cells expressed high levels of COL1A1 and low levels of COL2A1, aggrecan and SOX-9 compared to chondrocytes. However, COL2A1 was more expressed by labrum cells than by meniscus cells. The expression of matrix metalloproteinase (MMP)-1/-2/-9, ADAMTS-4 and IL-6 was significantly higher in labrum cells than in chondrocytes. IL-1 suppressed the ECM gene expression levels of labrum cells, but increased the expression levels and release of MMP-1/-3/-9/-13 and ADAMTS-4 and IL-6 by these cells. Remarkably, MMP-9 was only significantly upregulated in acetabular labrum cells. CONCLUSIONS The findings in this study demonstrated that the acetabular labrum is populated with unique highly active fibrochondrocyte-like cells. These cells are capable of expressing and releasing pro-inflammatory enzymes and cytokines and react to a pro-inflammatory stimulus. In this way, they contribute obviously to disturbed tissue function in hip labrum pathology.

Quantitative proteomics to characterize specific histone H2A proteolysis in chronic lymphocytic leukemia and the myeloid THP-1 cell line.

Proteome studies on hematological malignancies contribute to the understanding of the disease mechanism and to the identification of new biomarker candidates. With the isobaric tag for relative and absolute quantitation (iTRAQ) method we analyzed the protein expression between B-cells of healthy people and chronic lymphocytic leukemia (CLL) B-cells. CLL is the most common lymphoid cancer of the blood and is characterized by a variable clinical course. By comparing samples of patients with an aggressive vs. indolent disease, we identified a limited list of differentially regulated proteins. The enhanced sensitivity attributed to the iTRAQ labels led to the discovery of a previously reported but still not clarified proteolytic product of histone H2A (cH2A) which we further investigated in light of the suggested functional properties of this modification. In the exploratory proteome study the Histone H2A peptide was up-regulated in CLL samples but a more specific and sensitive screening of a larger patient cohort indicated that cH2A is of myeloid origin. Our subsequent quantitative analysis led to a more profound characterization of the clipping in acute monocytic leukemia THP-1 cells subjected to induced differentiation.

Proteomics in rheumatology: The beginning of a fairy tale?

One of the major challenges in proteome research is to translate its applications to the setting of human diseases. Proteomics in rheumatology is an area with marked potential including applications ranging from diagnostics, over therapeutic monitoring to discovery of new potential therapeutic targets. Biomarkers will be essential to discriminate between clinical similar rheumatic diseases, to monitor disease‐states or to install the best appropriate therapy. Especially in the field of rheumatology, analysis of specific genes and/or their expression products by pharmacogenetics/‐genomics or pharmacoproteomics could be necessary to enable an effective, patient‐tailored therapy. In rheumatology, direct examination of proteins may be of utmost importance, as it is already known that PTMs, such as citrullination of proteins or peptides, may be involved in certain rheumatic diseases. The discovery and validation of antibodies directed against citrullinated proteins/peptides in rheumatic diseases using proteome analysis approaches has been described. Gel‐free methods, SELDI‐approaches and classic 2‐DE approaches have been deployed on body fluids as well as on target tissues in different rheumatic diseases. Proteomics in rheumatology is on the rise and pilot studies have indicated that the application of proteomics‐based technologies in rheumatic diseases appears to be an exciting example of translational research.