Fausto Galdo

Evaluation of ankylosing spondylitis spinal mobility measurements in the assessment of spinal involvement in psoriatic arthritis

OBJECTIVE To determine the clinical usefulness of spinal mobility measurements used for ankylosing spondylitis (AS) to assess spinal involvement in patients with psoriatic arthritis (PsA). METHODS We assessed 100 patients with PsA and 103 patients with AS. Patients were classified as having axial PsA if they had grade 2 or higher unilateral sacroiliitis in the presence of spinal symptoms. All PsA patients, without taking the degree of joint involvement into consideration, were evaluated using several measurements for AS. Spinal measurements were compared with axial and peripheral forms of PsA, and the ability of the techniques to discriminate between the 2 forms of PsA was analyzed using the Mann-Whitney U test and the area under the receiver operating characteristic (ROC) curve. A logistic regression model was used to determine the best measurements for evaluating axial PsA. Finally, the results of measurements for axial PsA were compared with those for AS. RESULTS Of the 100 PsA patients, 46 met the classification criteria for axial PsA, which presented more severe spinal measurement assessments compared with peripheral PsA. Modified Schober test, lumbar side flexion, chest expansion, and cervical rotation measurements performed best under the ROC curve. Modified Schober test, lumbar side flexion, and cervical rotation were the more suitable measurements for assessing axial PsA. There were only minor differences between axial PsA and AS. CONCLUSION The spinal measurements used to evaluate AS performed well to assess spinal involvement in PsA. These measurements, notably the modified Schober test, lumbar side flexion, and cervical rotation, should be used in daily clinical practice to assess PsA patients with spinal involvement.

Xeno‐implantation of pig chondrocytes into rabbit to treat localized articular cartilage defects: an animal model

Articular cartilage has only a limited ability to regenerate. The transplantation of autologous chondrocytes is currently used to treat focal defects in human articular cartilage, although use of organs, tissues, or cells from different species is being investigated as an alternative treatment. The object of this study was to use xeno‐transplantation of cultured pig chondrocytes for the repair of rabbit chondral defects, and to analyze the significance of tissue rejection in this animal model. Partial chondral defects, including removal of cartilage tissue and a part of the subchondral bone, were created in the lateral femoral condyles of 30 adult New Zealand White rabbits. A periosteal flap was sutured to the native cartilage with the cambium layer facing the defect. As a control, culture medium was injected into the defect void of one group of rabbits while in a treatment group, chondrocytes, isolated from normal femoral pig cartilage, were injected into the defect void. All rabbits were killed by 24 weeks. Macroscopic changes of the cartilage were analyzed using Mankins score. The distal femoral portion was studied histologically using hematoxylin and eosin, alcian blue, toluidine blue, and Masons trichrome. Pig cells and pig genetic material were detected in the neo‐synthesized tissue by immunohistochemical detection of SLA‐II‐DQ and polymerase chain reaction analysis of the gene SLA‐II‐DQB. The synovial membrane was studied histologically by hematoxylin and eosin staining. In the control group, on average, less than 25 percent of the chondral defect was filled. The repair tissue had an irregular surface with few cells similar to chondrocytes or fibroblasts and a minimal formation of extracellular matrix. In the treatment group, the chondral defect was approximately 90 percent filled with good integration between the neo‐synthesized cartilage and the native cartilage. The repair tissue had a smooth surface with cells similar to chondrocytes and a hyaline‐like extracellular matrix. The neo‐synthesized cartilage was morphologically similar to hyaline cartilage. Importantly, there were no signs of graft‐vs.‐host rejections or infiltration by immune cells. In the neo‐synthesized tissue, pig genetic material was detected in 27 ± 5 percent of all cells. These cells containing pig genetic material were distributed throughout the neo‐synthesized cartilage. We conclude that the xeno‐transplantation of chondrocytes could be an alternative method for the repair of articular cartilage defects.

Validity of the bath ankylosing spondylitis disease activity index for the evaluation of disease activity in axial psoriatic arthritis

To assess the validity of the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) for the evaluation and definition of disease activity of axial psoriatic arthritis (PsA).

Pig chondrocyte xenoimplants for human chondral defect repair: an in vitro model

The objective of this study was to evaluate the use of cultured porcine chondrocyte xenotransplantation for the repair of human chondral defects. Two‐millimeter‐diameter defects were drilled into explants of femoral cartilage from healthy adult donors. No cells were implanted in the chondral defects of the control group, while pig chondrocytes from normal femoral cartilage were deposited into the treated chondral defects. Cartilage explants were cultured for 4, 8, and 12 weeks. Tissue sections were processed for standard histologic staining and immunostaining with monoclonal antibodies against types I and II collagen, chondroitin‐4‐sulfate, chondroitin‐6‐sulfate, keratan sulfate, and integrin subunit β1. The porcine origin of chondrocytes was confirmed using a specific pig monoclonal anti‐CD46. Repair was only observed in the cell‐treated defects. Mono‐ or bilayers of cells were detected after 4 culture weeks on the bottom of the defects, while after 8–12 weeks a repair tissue filled near 30–40 percent of the defect. At 8 weeks, the newly synthesized tissue was composed of a fibrous mesh including some cells. However, at 12 weeks it showed a hypercellular hyaline‐like region. This hypercellular region showed excellent bonding with the native cartilage, cells were located in numerous lacunae, and a high content of proteoglycans as indicated by an intense toluidine blue stain was observed. The repaired tissue showed positive immunostaining for both type I and II collagen, as well as chondroitin‐4‐sulfate, chondroitin‐6‐sulfate, keratan sulfate, and integrin subunit β1. Positive staining for porcine anti‐CD46 was localized exclusively in the neo‐synthesized tissue. We conclude that xenotransplantation of pig chondrocytes can repair, in an in vitro model, defects in human articular cartilage.

Anti-apoptotic effect of transforming growth factor-β1 on human articular chondrocytes: role of protein phosphatase 2A

OBJECTIVE To study whether transforming growth factor-beta1 (TGF-beta1) is able to protect human chondrocytes from apoptosis and to analyze the role of phosphatases in the possible anti-apoptotic effect of TGF-beta1. METHODS Cartilage was obtained from patients with osteoarthritis (OA) who were undergoing joint replacement; normal cartilage was obtained from cadavers who had no history of joint disease. Chondrocytes stimulated with tumor necrosis factor-alpha (TNF-alpha) plus Ro 31-8220 (a specific inhibitor of mitogen-activated kinase phosphatase-1 - MKP-1) were employed as an in vitro model of apoptosis. Apoptosis was assessed by flow cytometry and a cell death immunoassay. Protein phosphatase 2A (PP2A) activity was estimated by measuring the absorbance of a molybdate:malachite green:phosphate reaction complex. MKP-1, bcl-2 and bax expressions were quantified by western blot. RESULTS In OA cells, TGF-beta1 significantly reduced the percentage of hypo-diploid chondrocytes, as well as the percentage of internucleosomal DNA breakage. However, in normal chondrocytes, TGF-beta1 did not reduce apoptosis, as assessed by both the percentage of hypo-diploid chondrocytes and internucleosomal DNA breakage. MKP-1 expression did not show significant modulation in OA or normal chondrocytes. However, PP2A activity was differentially modulated in normal and OA chondrocytes. In OA chondrocytes, PP2A activity was not altered by TGF-beta1 stimulation; however in normal chondrocytes PP2A activity was significantly activated by TGF-beta1. The preincubation of normal chondrocytes with TGF-beta1 plus the PP2A inhibitor protein, IPP2A, reduced internucleosomal DNA breakage when compared with TGF-beta1 stimulation alone. The bcl-2/bax protein ratio was significantly higher in TGF-beta1 plus IPP2A preincubated normal chondrocytes than in cells stimulated with TGF-beta1 alone. CONCLUSION By manipulating the degree of PP2A activity, these results show the major role that PP2A plays in the outcome of TGF-beta1 signal transduction. These data suggest that PP2A could be a pivotal regulator of anti-apoptotic TGF-beta1-induced effects.

Paniculitis facticia y síndrome de Löfgren inducidos por silicona: a propósito de un caso clínico

We herein report a patient who came to the hospital because of a polyarticular joint pain, fever and cutaneous lesions. She had silicone implants in her buttocks, a surgery performed 3 years before. We made a biopsy of the skin of the buttocks (facticial panniculitis due to silicone) and of the pretibial surface of the inferior extremities (erythema nodosum). A chest X- ray and a CT scan revealed bilateral hiliar lymphadenopathy, and a transbronquial biopsy showed granulomatous inflammation. She had a good response to rest and anti-inflammatory drugs, so the removal of the silicone implants has not been necessary yet.

224 IMPROVED TISSUE REPAIR IN ARTICULAR CARTILAGE DEFECTS IN VITRO WITH MSC CD271

genetic processes. Functional studies in primary human chondrocytes have long been hampered by the low transfection efficiencies of non-viral gene delivery methods in these cells. Only with the development of specific electroporation methods like nucleofection and radiofrequency (RF) electroporation highly efficient non-viral transfection protocols for human chondrocytes became available, but these methods require relatively high cell numbers and cannot be easily adjusted to the analysis of a larger number of samples. The purpose of our work was therefore the establishment of a high-throughput transfection protocol for primary human articular chondrocytes (PHC) that allows the simultaneous transfection of multiple cell samples while reducing the number of cells necessary per experiment. In parallel analogous protocols should be established for the human chondrocytic cell lines T/C-28a2 and HCS-2/8. Methods: Primary human chondrocytes were isolated from adult knee cartilage by an optimized enzymatic digestion protocol and cultivated in high-density monolayer culture for three to five days. T/C-28a2 and HCS2/8 cells were cultivated under standard conditions. Chondrocytes and chondrocytic cell lines were transfected with an GFP expressing reporter construct applying amaxa’s nucleofector and 96-well shuttle devices with experimental variation of transfection parameters, pulse solutions and cell numbers. Transfection efficiencies were measured by FACS and cell viability was determined by an ATP assay. SiRNA constructs targeting GAPDH expression were transfected into the cells using the established nucleofection protocols and mRNA knockdown values were determined by a branched-DNA assay or by quantitative real-time PCR. Results: For high-throughput transfection of primary human chondrocytes transfection efficiencies of more than 70% of surviving cells could be achieved routinely with the presented nucleofection protocol. Cell viability after transfection was around 80%. Transfection efficiency and survival rate were highly dependent on the proper preparation of the PHCs prior to transfection. The cell number used for transfection could be reduced to 2×105 per sample in comparison to the established single cuvette protocol. The protocol proved also well suited for the transfer of siRNA molecules to primary human chondrocytes with suppression rates of more than 95% for GAPDH mRNA levels. Transfection efficiencies for T/C-28a2 cells surpassed 90% for both single cuvette and high-throughput formats with survival rates of more than 80%. Single cuvette nucleofection of HCS-2/8 cells reached transfection efficiencies of around 40%. Conclusions: We present the successful adaptation of the nucleofection procedure for primary human chondrocytes to a high-throughput format. The protocol allows the efficient transfection of up to 96 cell samples in parallel and is not limited to the transfection of DNA but can be easily adapted to the transfection of siRNA constructs. The protocol requires substantially lower cell numbers than single cuvette protocols and is therefore very well suited for applications requiring multiple experimental replicates. Likewise, the established protocols for T/C-28a2 cells allow the highly efficient transfection in single cuvette and 96-well formats opening novel application areas. In summary, we present efficient high-throughput transfection protocols for primary human chondrocytes and chondrocytederived cell lines with widespread application potential in osteoarthritis research (siRNA screening assays etc.).

A27 DECREASED LEVELS OF MITOCHONDRIAL SUPEROXIDE DISMUTASE IN OSTEOARTHRITIC HUMAN ARTICULAR CHONDROCYTES REVEALED BY MITOCHONDRIAL PROTEOMIC ANALYSIS

by the C/EBPb dificiency in the chondrocytes. p57 was co-localized with C/EBPb in late proliferative and pre-hypertrophic chondrocytes of the growth plate, which was dramatically decreased by the C/EBPb deficiency. The promoter activity of p57 was enhanced by C/EBPb transfection. Deletion, mutagenesis, and tandem-repeat analyses of the luciferase assay identified the core responsive element to be between the −150 and −130 bp region containing a putative C/EBP-binding motif. EMSA revealed the binding of C/EBPb-overexpressed nuclear extracts with the oligonucleotide including this region, whose specificity was verified by the C/EBPb antibody supershift. The knockdown of p57 by the siRNA inhibited the C/EBPb-induced hypertrophic differentiation in cultured chondrocytes. Conclusions: C/EBPb directly transactivates p57 at a specific C/EBP motif to promote transition from proliferation to hypertrophic differentiation of chondrocytes during skeletal growth, indicating the essential role of the C/EBPb/p57 signal during endochondral ossification.

476 GLUCOSAMINE SULFATE AND CHONDROITIN SULFATE: THEIR EFFECT ON CHONDROCYTE PROTEOME

and connective tissue growth factor. Il-1 stimulated release of the cartilage degeneration marker MMP-3, as well as proteins with uncharacterized roles in cartilage pathology, such as neutrophil gelatinase-associated lipocalin. RetA stimulated release of extracellular matrix proteins COMP, link protein and matrilin-3 into the media, accompanied by dramatic reduction in corresponding mRNA transcripts levels. Gelsolin, implicated in cytoskeletal reorganization in arthritic synovial fibroblasts but not previously associated with cartilage pathology, was regulated by Il-1 and RetA. Conclusions: This first analysis of mouse cartilage degradation and protein release using proteomics has identified proteins and fragments, some of which represent novel candidate biomarkers for cartilage degradation. Applying these proteomic techniques to wild-type and geneticallymodified mouse cartilage will provide insights into mechanisms of cartilage degeneration.