Mammalian meiotic silencing exhibits sexually dimorphic features.

During mammalian meiotic prophase I, surveillance mechanisms exist to ensure that germ cells with defective synapsis or recombination are eliminated, thereby preventing the generation of aneuploid gametes and embryos. Meiosis in females is more error-prone than in males, and this is in part because the prophase I surveillance mechanisms are less efficient in females. A mechanistic understanding of this sexual dimorphism is currently lacking. In both sexes, asynapsed chromosomes are transcriptionally inactivated by ATR-dependent phosphorylation of histone H2AFX. This process, termed meiotic silencing, has been proposed to perform an important prophase I surveillance role. While the transcriptional effects of meiotic silencing at individual genes are well described in the male germ line, analogous studies in the female germ line have not been performed. Here we apply single- and multigene RNA fluorescence in situ hybridization (RNA FISH) to oocytes from chromosomally abnormal mouse models to uncover potential sex differences in the silencing response. Notably, we find that meiotic silencing in females is less efficient than in males. Within individual oocytes, genes located on the same asynapsed chromosome are silenced to differing extents, thereby generating mosaicism in gene expression profiles across oocyte populations. Analysis of sex-reversed XY female mice reveals that the sexual dimorphism in silencing is determined by gonadal sex rather than sex chromosome constitution. We propose that sex differences in meiotic silencing impact on the sexually dimorphic prophase I response to asynapsis.

Total knee replacement with retention of both cruciate ligaments: a 22-year follow-up study.

We report on the long-term results of 163 bicruciate-retaining Hermes 2C total knee replacements in 130 patients at a mean follow-up of 22.4 years (20.3 to 23.5). Even when the anterior cruciate ligament had a partially degenerative appearance it was preserved as long as the knee had a normal anterior drawer and Lachman's test pre-operatively. The description and surgical technique of this minimally constrained prosthesis were published in 1983 and the ten-year clinical results in 1999. A total of 12% of the knees (20 of 163) in this study were revised because of wear of the polyethylene tibial insert. Excellent stability was achieved and the incidence of aseptic component loosening was 4.3% (seven of 163). The survival rate using revision for any reason as the endpoint was 82% (95% confidence interval 76.2 to 88.0). Although this series included a relatively small number of replacements, it demonstrated that the anterior cruciate ligament, even when partially degenerated at the time of TKR, remained functional and provided adequate stability at a long-term follow-up.

Modulation of collagenase 3 in human osteoarthritic cartilage by activation of extracellular transforming growth factor beta: role of furin convertase.

OBJECTIVE: Treatment of normal cartilage with transforming growth factor beta (TGFbeta) can increase the synthesis of collagenase 3 by chondrocytes and mimic the in situ distribution of this enzyme in osteoarthritic (OA) cartilage, which occurs predominantly in the deep zone. In this study, we examined the elements of the TGFbeta system that are potentially relevant to this effect. METHODS: TGFbeta1 and TGFbeta2 levels in cultured cartilage explants were determined by enzyme-linked immunosorbent assay (ELISA). OA cartilage explants were treated with small latent TGFbeta1 complex in the presence of various inhibitors, and collagenase 3 levels were determined by ELISA. The inhibitors were against serine proteases, plasmin, cathepsins, furin, and a neutralizing antibody against the mannose-6 phosphate/ insulin-like growth factor 2 receptor (M6P/IGF-2R). Small latent TGFbeta1, TGFbeta receptor types I, II, and III (TGFbetaRI, RII, and RIII), M6P/IGF-2R, and furin were immunolocalized in cartilage. RESULTS: Our data showed that latent TGFbeta1 is the major isoform that is synthesized; levels of 17.2 +/-1.7 pg/mg and 1.1 +/- 0.3 pg/mg tissue wet weight (mean +/- SEM) were found for total TGFbeta1 and TGFbeta2, respectively, in OA cartilage. A general serine protease inhibitor abrogated activation of both endogenous and exogenous small latent TGFbeta1. Plasmin and furin inhibitors and anti-M6P/IGF-2R reduced the levels of exogenous small latent TGFbeta1 complex-induced collagenase 3 by 33%, 95%, and 76%, respectively, but the cathepsin inhibitor had no effect. Immunolocalization of the small latent TGFbeta1 complex as well as of TGFbetaRI and RII revealed a statistically significant increase in the chondrocyte score in only the deep zone of OA cartilage. The M6P/IGF-2R level was significantly higher in OA cartilage in both the superficial and deep zones. Furin was found in normal cartilage exclusively in the superficial zone, whereas in OA cartilage, a level similar to that in normal cartilage was found in the superficial zone, but a significantly higher cell score (mean +/- SEM 23.6 +/- 4.7%) was registered in the deep zone. CONCLUSION: The mechanisms of TGFbeta activation/ activity with regard to collagenase 3 modulation in cartilage appear to be controlled by furin convertase with or without M6P/IGF-2R. These factors and the small latent TGFbeta complex are increased in the deep zone of OA cartilage, corresponding to the preferential site of collagenase 3 production.

The cruciate ligaments in total knee arthroplasty: a kinematic analysis of 2 total knee arthroplasties.

In vivo weight-bearing fluoroscopic kinematic analysis using an interactive model fitting technique with 3-dimensional computer-aided design solid models was done using 16 anterior and posterior (bicruciate)-sparing and 6 posterior cruciate-sparing total knee arthroplasties (TKAs). All patients had a satisfactory clinical result with a minimum of 12 months' follow-up. The femorotibial contact position of TKAs started posterior to the midline in extension. Bicruciate TKAs revealed gradual posterior femoral rollback and limited anterior-posterior translation but remained posterior to the sagittal plane midline in all positions. Posterior cruciate-sparing TKAs began significantly posterior in extension, demonstrated progressive anterior translation with flexion, and had exaggerated medial condyle translation on deep knee bend. The posterior cruciate-retaining TKAs of this study had the most abnormal kinematic performance.

Diacerhein and rhein reduce the ICE-induced IL-1beta and IL-18 activation in human osteoarthritic cartilage.

OBJECTIVE: IL-1beta plays a fundamental role in osteoarthritis (OA) pathophysiology and cartilage destruction. Targeting the activation mechanism of this cytokine appears to be important as a therapeutic approach. As the interleukin-1 converting enzyme (ICE) is the physiologic modulator of the production of active IL-1beta, we investigated the effect of diacerhein and its active metabolite rhein used in the treatment of OA patients, on the enzyme expression and synthesis on human OA cartilage. Further, we looked at the effect of both drugs on the production of the active form of IL-1beta and IL-18. METHODS: The expression and synthesis of ICE were investigated on human OA cartilage explants using in-situ hybridization and immunohistochemical methods, respectively. The effect of the drugs on ICE OA chondrocytes was also determined by Northern blotting and a specific ELISA assay. Furthermore, the effect of both drugs on the level of active IL-1beta and IL-18 was examined by immunohistochemistry. RESULTS: Data showed that diacerhein and rhein have no true effect on reducing total ICE mRNA by both Northern blotting analysis and in-situ hybridization. A marked and statistically significant decrease was, however, found for protein production. ELISA showed a reduction of 31% (P< 0.04) for diacerhein and 50% (P< 0.02) for rhein. The drugs' immunohistological cell score reduction was similar to data from the ELISA, and a statistical significant reduction of ICE production was found at both superficial and deep zones of the cartilage. IL-1beta and IL-18 were both preferentially produced in chondrocytes of the superficial zone. For each of these cytokines, both drugs demonstrated a statistically significant decrease in this zone. A marked decrease was also noted in the deep zone, but statistical significance was reached only for rhein. CONCLUSION: These results provide a novel regulatory mechanism by which diacerhein and rhein could exert a down-regulation on IL-1's effect on OA cartilage.

Interleukin-1beta-converting enzyme/caspase-1 in human osteoarthritic tissues: localization and role in the maturation of interleukin-1beta and interleukin-18.

OBJECTIVE: To study the expression and production of interleukin-1beta-converting enzyme (ICE) in human normal and osteoarthritic (OA) cartilage and synovium, quantitate the level of ICE in OA chondrocytes, and examine the relationship between the topographic distribution of ICE, interleukin-1beta (IL-1beta), and IL-18, as well as apoptosis of chondrocytes. METHODS: The expression and synthesis of ICE were investigated in human normal and OA cartilage and synovial membrane using in situ hybridization and immunohistochemical methods. The intracellular level of ICE in OA chondrocytes was also measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, the topographic relationship between the presence of ICE and mature IL-1beta and IL-18 was examined by immunohistochemistry, and apoptotic chondrocytes by the TUNEL technique. RESULTS: ICE was expressed and synthesized in both human synovial membrane and cartilage, with a significantly greater number of cells staining positive in OA tissue than in normal tissue. ICE production was preferentially located in the superficial and upper intermediate layers of articular cartilage. With a specific ELISA, a level of 230.2+/-22.5 pg/5 x 10(5) cells (mean +/- SEM) of ICE was found in OA chondrocytes. In cartilage, IL-1beta and IL-18 stained positive at a topographic location similar to that of ICE. The production of mature IL-1beta in OA cartilage explants and chondrocytes was completely blocked by treatment with a specific ICE inhibitor, which also markedly diminished the number of IL-18-positive cells. The data show that there was no close relationship between the presence of ICE and the presence of apoptotic chondrocytes in OA cartilage. CONCLUSION: This study shows, for the first time, the presence of active ICE in human articular cartilage, with a markedly increased cellular level in OA tissue. The relationship between active IL-1beta and ICE suggests that ICE may promote OA progression by activating this proinflammatory cytokine. The role of IL-18 in pathologic cartilage is discussed.

Collagenase 3 production by human osteoarthritic chondrocytes in response to growth factors and cytokines is a function of the physiologic state of the cells.

OBJECTIVE: We investigated the response of human osteoarthritic (OA) chondrocytes, in terms of collagenase 3 production, to growth factors and cytokines involved in the anabolism and catabolism of articular cartilage, and explored the major signaling pathways leading to its up-regulation. METHODS: Human OA chondrocytes were treated with the following factors: the proinflammatory cytokine interleukin-1beta (IL-1beta), the growth factors basic fibroblast growth factor (bFGF), platelet-derived growth factor BB (PDGF-BB), parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), transforming growth factor gamma1 (TGFbeta1), and TGFbeta2, the protein kinase (PK) activator antagonists for PKC, PKA, and PKG pathways, and phospholipase A2 and tyrosine kinases, as well as the antiinflammatory cytokines IL-4, IL-10, and IL-13. Collagenase 3 expression and synthesis were determined. Comparison was made with collagenase 1. RESULTS: The human OA chondrocyte population could be divided into 2 categories: the L chondrocytes, showing low collagenase 3 basal synthesis levels and high sensitivity to IL-1beta stimulation; and the H chondrocytes, high collagenase 3 basal synthesis levels and low IL-1beta inducibility. In L chondrocytes, all growth factors stimulated collagenase 3 production. In H chondrocytes, PTH, IGF-1, and TGFbeta had little or no impact; bFGF slightly stimulated it and PDGF-BB showed the same pattern as in the L chondrocytes. The effects of all growth factors, except TGFbeta, on collagenase 1 synthesis followed those of collagenase 3, albeit to a higher degree. Interestingly and unlike collagenase 3, the effects of TGFbeta on collagenase 1 could not be related to the state of the cells, but rather, depended on the isoform. Indeed, TGFbeta2 did not induce collagenase 1 synthesis, whereas TGFbeta1 stimulated it. Among the PK activators tested, phorbol myristate acetate was the strongest inducer, suggesting a major involvement of the PKC pathway. IL-13 inhibited collagenase 3 production, IL-4 had little effect, and IL-10 had none. CONCLUSION: This study shows that collagenase 3 production in human OA chondrocytes depends on the physiologic state of the cell. TGFbeta might be responsible for the change in cells from the L to the H state. Importantly, our in vitro data implicate TGFbeta2 as a possible in vivo agent capable of specifically triggering collagenase 3 production over that of collagenase 1 in OA cartilage.

Total knee arthroplasty with retention of both cruciate ligaments. A nine to eleven-year follow-up study.

BACKGROUND: Although many early designs of total knee arthroplasty allowed the retention of both cruciate ligaments, in most current designs of knee replacement systems, either both cruciate ligaments are removed or the posterior cruciate ligament alone is retained. This report is a review of a series of total knee arthroplasties in which both cruciate ligaments were retained. METHODS: The results of 163 total knee arthroplasties (130 patients) in which both cruciate ligaments were retained were assessed prospectively. One hundred and seven knees (eighty-nine patients) were followed for an average of ten years. There were thirty-four men and ninety-six women, and the average age at the time of the index arthroplasty was sixty-seven years (range, forty-two to eighty-four years). The diagnosis was osteoarthritis in 122 (75 percent) of the knees and rheumatoid arthritis in forty-one (25 percent). Twenty-six knees had a valgus deformity, 109 had a varus deformity, and twenty-eight had a normal alignment of 5 to 10 degrees of valgus. The anterior cruciate ligament was relatively normal in ninety-six knees and was partly degenerated in sixty-seven knees. With use of the rating system of the Knee Society, all 163 knees were prospectively evaluated at yearly intervals; fifty-six of these knees (in forty-one patients) were followed in this manner until the patient died or was lost to follow-up. RESULTS: One hundred and four (97 percent) of the 107 knees available for study at an average of ten years had an excellent or good result. At the time of the latest follow-up, pain was adequately relieved in ninety-seven knees (91 percent) and the average range of flexion was 107+/-12.6 degrees (range, 65 to 135 degrees). Ninety-five knees (89 percent) had normal anteroposterior stability (less than five millimeters of movement in this plane), and twelve knees (11 percent) had five to ten millimeters of movement as demonstrated by the drawer sign. Ninety-six knees (90 percent) had normal mediolateral stability, and eleven (10 percent) had 5 to 10 degrees of laxity. Ninety-four knees (88 percent) had valgus alignment of 5 to 10 degrees. The average knee score was 91+/-8.4 points (range, 54 to 100 points), and the average functional score was 82+/-21 (range, 10 to 100 points). The survival rate at ten years, with revision as the end point, was 95+/-2.0 percent. Seven (4 percent) of the 163 knees in this series were revised. There were no revisions for patellar problems or aseptic loosening of the tibial component. CONCLUSIONS: The good anteroposterior stability in this series after an average follow-up period of ten years indicates that both the anterior and the posterior cruciate ligaments, even when partly degenerated, remain functional when they are preserved in a total knee arthroplasty.

Inhibition of tumor necrosis factor alpha-induced prostaglandin E2 production by the antiinflammatory cytokines interleukin-4, interleukin-10, and interleukin-13 in osteoarthritic synovial fibroblasts: distinct targeting in the signaling pathways.

OBJECTIVE: To investigate the effects of the antiinflammatory cytokines interleukin-4 (IL-4), IL-10, and IL-13 on tumor necrosis factor alpha (TNFalpha)-induced prostaglandin E2 (PGE2) release in the cellular signaling cascade on human osteoarthritis (OA) synovial fibroblasts. METHODS: Human OA synovial fibroblasts were cultured to explore the impact of IL-4, IL-10, and IL-13 on TNFalpha binding to TNF receptors (TNFR), soluble TNFR (sTNFR), cytoplasmic phospholipase A2 (cPLA2), and cyclooxygenase-2 (COX-2) production, and on the binding activity of the transcription factors nuclear factor kappaB (NF-kappaB), CCAAT-enhancer binding protein (C/EBP), activator protein 2 (AP-2), and cyclic AMP response element-binding protein (CREB). RESULTS: IL-4, IL-10, and IL-13 at 5 ng/ml dramatically reduced TNFalpha-induced PGE2 release by approximately 90% (P < 0.0001). IL-4 up-regulated the level of TNFalpha-induced TNFR by 47% (P < 0.06), while IL-10 down-regulated it by 71% (P < 0.02); IL-13 had no effect. Although statistical significance was not reached, all 3 cytokines up-regulated the basal level of sTNFR-55. IL-4 and IL-10, while not altering the basal level of sTNFR-75, significantly increased the TNFalpha-stimulated release of sTNFR-75. IL-4, IL-10, and IL-13 reduced the TNFalpha-induced COX-2 level, and IL-4 and IL-10 reduced the cPLA2 level. IL-4 had no effect on TNFalpha up-regulation of NF-kappaB, and a slight decrease was noted with IL-10 and IL-13 at the highest concentration used (5 ng/ml). IL-4 and IL-13 decreased the TNFa-induced C/EBP accumulation in a dose-dependent manner, while IL-10 up-regulated its basal level. AP-2 and CREB were not induced by TNFalpha. CONCLUSION: The results indicate that these antiinflammatory cytokines reversed the TNFalpha-induced release of PGE2 by OA synovial fibroblasts, by acting at various levels of the TNFa-dependent signaling cascade. These data shed new light on the mechanisms by which these cytokines reduce inflammatory processes.

Differential effects of IL-8, LIF (pro-inflammatory) and IL-11 (anti-inflammatory) on TNF-alpha-induced PGE(2)release and on signalling pathways in human OA synovial fibroblasts.

Tumour necrosis factor alpha (TNF-alpha) inflammatory activity is mediated, at least in part, by prostaglandin E(2)(PGE(2)). In osteoarthritis (OA), other cytokines are believed to play a role by interacting with TNF-alpha. Using OA synovial fibroblasts, we investigated the effects of interleukin 8 (IL-8), leukaemia inhibitory factor (LIF) and IL-11 on the level of TNF-alpha-induced PGE(2), and their impact on the TNF-alpha-induced cellular signalling cascades including the TNF-receptor (TNF-R), soluble TNF-R (TNF-sR), cytoplasmic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX-2), and the transcription factors NF-kappaB, C/EBP, CREB and AP-1.IL-8 increased in a synergistic manner (282% at 5 ng/ml) and LIF in an additive fashion (69% at 50 ng/ml) the TNF-alpha-induced PGE(2)release, while IL-11 reduced it (52% at 5 ng/ml). IL-8 (5 ng/ml) and LIF (50 ng/ml) alone upregulated (30%) the TNF-R binding level, but significantly downregulated the TNF-alpha-induced levels (P<0.007 and P<0.004, respectively) and the TNF-sR55 level. In contrast, IL-11 reduced the basal level by 18% (P<0.005) and the TNF-alpha-induced level of TNF-R by 51% (P<0.01) as well as decreasing both TNF-sR55 and TNF-sR75. The COX-2 synthesis level was increased by IL-8 and LIF under TNF-alpha treatment but downregulated by IL-11. IL-8 and LIF either alone or under TNF-alpha treatment increased the cPLA2 synthesis, while IL-11 decreased the level under both conditions. Interestingly, IL-8 induced in a synergistic manner and LIF in an additive fashion, the level of cPLA2 activity. IL-8 and LIF had no effect on the TNF-alpha-induced NF-kappaB accumulation, while IL-11 significantly decreased it (P<0. 02). All three cytokines inhibited TNF-alpha-induced C/EBP, but no true effect was noted for AP-1 and CREB in the presence of TNF-alpha. These results indicate that IL-8 synergizes and LIF potentiates the TNF-alpha PGE(2)effect which appears to be mediated mostly by increasing cPLA2 activity level. On the other hand, IL-11 alone had no effect on the PGE(2)release, but in conjunction with TNF-alpha, this cytokine showed anti-inflammatory properties. This study provides a rational foundation to develop therapeutic strategies for the treatment of OA by shedding light on the mechanisms of action of three prominent cytokines at work in articular joint tissues.

In vitro effects of diacerhein and rhein on interleukin 1 and tumor necrosis factor-alpha systems in human osteoarthritic synovium and chondrocytes.

OBJECTIVE: To evaluate the in vitro effects of diacerhein, a new drug for the treatment of osteoarthritis (OA), and its active metabolite, rhein, on interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) synthesis and expression in human OA synovial membrane, and on the IL-1beta and TNF-alpha receptors on human OA chondrocytes. METHODS: Levels of IL-1beta and TNF-alpha were determined using specific ELISA in culture medium of human synovial membrane explants incubated in the presence of 1 microg/ml of lipopolysaccharide with or without therapeutic concentrations of diacerhein (1.4, 2.7, 5.4 x 10(-5) M) and rhein (1.7, 3.5, 7.0 x 10(-5) M). IL-1beta mRNA level was quantitated by Northern blotting. Using radioligand binding experiments, we determined the effects of these agents on the density and affinity of chondrocyte IL-1 and TNF receptors. RESULTS: IL-1beta synthesis was significantly inhibited by diacerhein and rhein, with maximum inhibition at 5.4 x 10(-5) M for diacerhein (p < 0.02) and 3.5 x 10(-5) M for rhein (p < 0.05). The effect of both agents on IL-1beta was found to be translational and/or post-translational, judging by the absence of effect on gene expression level. Both agents produced dose and time dependent decreases in the number of IL-1 receptors (IL-1R) on OA chondrocytes. This effect was mediated through a reduction in the level of the type I IL-1R as shown by experiments using a blocking monoclonal antibody against this receptor type. Both agents also markedly reduced the IL-1 induced synthesis and expression of stromelysin 1. Neither diacerhein nor rhein significantly affected the level of synthesis of TNF-alpha or the level of TNF-R. CONCLUSION: Diacerhein and rhein can effectively inhibit the synthesis of IL-1beta on human OA synovium, as well as the action of this cytokine at the cartilage level, by reducing the number of chondrocyte IL-1R. The effects of these agents seemed "selective" to the IL-1 system.

Collagenase-3 (matrix metalloprotease 13) is preferentially localized in the deep layer of human arthritic cartilage in situ: in vitro mimicking effect by transforming growth factor beta.

OBJECTIVE: To examine, by immunohistochemistry, the localization and distribution of human collagenase-3 in normal, osteoarthritis (OA), and rheumatoid arthritis (RA) cartilage, and to investigate the effects of interleukin-1beta (IL-1beta) and transforming growth factor beta (TGFbeta) on the synthesis and distribution of collagenase-3. METHODS: Human cartilage specimens were obtained from tibial plateaus. In the first series of experiments, the OA specimens were excised from fibrillated and nonfibrillated areas of cartilage, and RA specimens were excised from lesional areas, including the cartilage-pannus junction when present. In the second series, full strips of cartilage were processed for culture in the presence or absence of IL-1beta (100 units/ml) or TGFbeta (150 ng/ml). Each specimen was processed for immunohistochemical analysis using a collagenase-3 monoclonal antibody. RESULTS: The number of cells that stained for collagenase-3 in normal cartilage was very low (approximately 3%). In OA cartilage, the percentage increased dramatically, and no difference was found between fibrillated and nonfibrillated areas. A statistically significant increase in the percentage of cells staining for collagenase-3 was found in the deep layer compared with the superficial layer. This finding was noted in both the fibrillated areas (mean +/- SEM 58.4 +/- 1.6% and 40.1 +/- 3.9%, respectively; P < 0.007) and the nonfibrillated areas (55.4 +/- 3.2% and 43.2 +/- 2.7%; P < 0.01). Similarly, RA cartilage showed a statistically significant (P < 0.001) increase in the level of chondrocytes staining positive for collagenase-3 in the deep layers (46.4 +/- 4.1%) compared with the superficial layers (26.2 +/- 3.4%). In these RA specimens, the numbers of positively staining chondrocytes were similar both close to and at a distance from the pannus junction. Both IL-1beta and TGFbeta increased the number of chondrocytes producing collagenase-3. Interestingly, in normal specimens, TGFbeta had a predominant effect in the deep layers, while IL-1beta had a greater effect on the superficial layers. CONCLUSION: This study demonstrates that, in situ, the increase in the level of chondrocytes synthesizing collagenase-3 in arthritic cartilage is predominant in the deep layers. The results further indicate that TGFbeta can up-regulate the level of this enzyme and, in normal cartilage in vitro, can cause a mimicking of the in situ distribution observed in arthritic cartilage.

Osteoarthritic synovial fibroblasts possess an increased level of tumor necrosis factor-receptor 55 (TNF-R55) that mediates biological activation by TNF-alpha.

OBJECTIVE: To investigate the presence, number, and level of expression of tumor necrosis factor receptors (TNF-R) in normal and osteoarthritic (OA) human synovial fibroblasts; to examine which receptor isotype mediates the biological response of these cells to TNF-alpha; and to study homologous regulatory mechanisms of TNF-R by TNF-alpha. METHODS: We used radioligand binding assay with [125I]TNF-alpha and flow cytometric analysis with specific antireceptor antibodies to characterize receptor populations, densities, and ligand induced internalization of TNF-R. Inducible cyclooxygenase (COX-2) synthesis, prostaglandin E2 (PGE2) release, and TNF-R shedding (soluble receptors, TNF-sR) were measured after incubation with TNF-alpha the presence or absence of receptor specific blocking antibodies. RESULTS: Although radioligand binding assays showed no difference in the density or affinity of TNF-R in OA synovial fibroblasts compared with normal cells, flow cytometric analysis revealed that OA cells express a significantly higher level of TNF-R55 (p < 0.04) than normal cells. The TNF-R55 was found to be the major receptor species responsible for ligand binding activity, such as COX-2 induction and PGE2 synthesis, since a specific antireceptor TNF-R55 blocking antibody inhibited about 76% of TNF-alpha binding and TNF-alpha stimulated COX-2 induction and PGE2 production. Further experiments revealed that TNF-R55 was the only receptor type internalized after binding TFN-alpha, whereas TNF-R75 was concomitantly shed. Moreover, reducing the shedding of TNF-sR, particularly the TNF-sR75, with a synthetic inhibitor decreased TNF-alpha induced PGE2 production. CONCLUSION: TNF-R55 is the major receptor isoform transducing PGE2 and COX-2 responses to TNF-alpha in OA synovial fibroblasts; soluble receptors could be involved in facilitating the binding of TNF-alpha to its receptor.

Normal expression of type 1 insulin-like growth factor receptor by human osteoarthritic chondrocytes with increased expression and synthesis of insulin-like growth factor binding proteins.

OBJECTIVE: Our previous research demonstrated that, in contrast to normal chondrocytes, human osteoarthritic (OA) chondrocytes were hyporesponsive to stimulation by insulin-like growth factor 1 (IGF-1). The aim of the present investigation was to examine whether this finding was due to an alteration in the level of IGF receptors (IGFRs) and/or IGF binding proteins (IGFBP). METHODS: A quantitative reverse transcriptase polymerase chain reaction technique (RT-PCR) was used to measure the type 1 IGFR messenger RNA (mRNA) level, and Northern blotting was used to measure type 2 IGFR and IGFBP mRNA levels. Western immunoblotting was used to identify and measure IGFBP levels. RESULTS: There were similar levels of type 1 IGFR mRNA in normal and OA chondrocytes. The level of type 2 IGFR mRNA, in which an increased amount of which can interfere with the biologic effects of IGF-1, was lower in OA chondrocytes compared with normal chondrocytes. Articular chondrocytes produced IGFBP-2, IGFBP-3, and IGFBP-4, and OA chondrocytes secreted and expressed higher amounts than did normal chondrocytes. There was also an increased level of IGFBP-3 in the OA chondrocyte lysates. IGFBPs 1, 5, and 6 were not detectable. CONCLUSION: OA chondrocytes synthesize and express a larger amount of 3 IGFBPs. This observation, along with a lack of detectable change in type 1 IGFR mRNA level, suggests that the hyporesponsiveness of OA chondrocytes to IGF-1 might implicate the involvement of IGFBPs in this pathologic process.

The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. A role in osteoarthritis.

Recently, a new human collagenase, collagenase-3 has been identified. Since collagen changes are of particular importance in cartilage degeneration, we investigated if collagenase-3 plays a role in osteoarthritis (OA). Reverse transcriptase-PCR analysis revealed that in articular tissues collagenase-3 was expressed by the chondrocytes but not by the synoviocytes. Northern blot analysis of the chondrocyte mRNA revealed the presence of two major gene transcripts of 3.0 and 2.5 kb, and a third one of 2.2 kb was occasionally present. Compared to normal, OA showed a significantly higher (3.0 kb, P < or = 0.05; 2.5 kb, P < or = 0.03) level of collagenase-3 mRNA expression. Collagenase-3 had a higher catalytic velocity tate (about fivefold) than collagenase-1 on type II collagen. With the use of two specific antibodies, we showed that human chondrocytes had the ability to produce collagenase-3 as a proenzyme and as a glycosylated doublet. The chondrocyte collagenase-3 protein is produced in a significantly higher (P < or = 0.04) level in OA (approximately 9.5-fold) than in normal. The synthesis and expression of this new collagenase could also be modulated by two proinflammatory cytokines, IL-1 beta and TNF-alpha, in a time- and dose-dependent manner. This study provides novel and interesting data on collagenase-3 expression and synthesis in human cartilage cells and suggest its involvement in human OA cartilage patho-physiology.

[Influence of the tibial slope on tibial translation and mobility of non-constrained total knee prosthesis]. / Influence de la pente tibiale sur la translation tibiale et la mobilité d'une prothèse totale du genou non contrainte.

PURPOSE OF THE STUDY: We determined retrospectively the influence of posterior tibial slope and anterior cruciate ligament (ACL) sparing on anterior tibial translation in 68 Cloutier total knee prosthesis. We also precised the influence of posterior tibial slope on knee functional score and appearance of tibial prosthetic interfaces. MATERIAL: 38 Cloutier total knee prosthesis (62 patients mean aged 62 +/- 10 years (36-76) at surgery) reviewed at systematic follow-up control, after a mean period of 5.5 +/- 3 years (2-15), were included in the study. The ACL was preserved in 38 knees and sacrified in 30 knees, the posterior cruciate ligament was preserved in all cases. The prosthetic design was the same whatever the number of cruciate ligament preserved. Osteoarthritis was the reason for surgery in 54 knees, and rheumatoid arthritis in 14 knees. Mean HSS knee score was 54 +/- 10 (29-80) before surgery and 89 +/- 10 (35-100) at follow-up. The mean range of motion was 103 +/- 24 degrees (30-130) before surgery and 110 +/- 14 degrees at follow-up (40-130). METHODS: Anterior tibial translation was determined on two profil x-rays (non weight bearing and weight bearing) at 20 degrees of flexion by comparing the position of tibial tray with regard to posterior edge of femoral prosthesis. Tibial slope was measured on lateral view with regard to peroneus axis. Appearance of tibial prosthetic interface was studied in 48 knees on AP and lateral x-rays orientated with an image intensifier in order to obtain the x-ray would be parallel to the tibial interface. RESULTS: Posterior tibial slope (mean value 6.2 degrees +/- 4.2 degrees) was the main factor influencing the anterior tibial translation (mean value 3.9 +/- 4.6 mm) (p = 0.0007). A 10 degree increase of posterior tibial slope makes the anterior tibial translation rise by 5.6 mm in weight bearing situation. When ACL was preserved, the anterior tibial translation was lower but the decrease was not significant. Likewise, preservation of ACL or the degree of posterior tibial slope had no influence on: 1) HSS knee functional score, 2) range of motion. Radiolucent lines were observed in 18 out of 48 knees, but their occurrence was not influenced by the degree of posterior tibial slope or preservation of ACL. DISCUSSION: Posterior tibial slope has a higher influence than ACL preservation on anterior tibial translation. The increase of posterior tibial slope in order to improve range of motion and to protect the bone-prosthetic tibial interface appeared unjustified with this non-constrained prosthesis. Moreover, implantation of tibial tray (whatever the preservation of ACL) with an important posterior inclination exposes to high anterior tibial translation in weight bearing situation. This last condition could reduce the survivorship of tibial polyethylene.

Human synovial fibroblasts coexpress IL-1 receptor type I and type II mRNA. The increased level of the IL-1 receptor in osteoarthritic cells is related to an increased level of the type I receptor.

BACKGROUND: Cytokines, in particular IL-1, are believed to be responsible for mediating cartilage degradation in osteoarthritis (OA). To investigate the role of the IL-1 system in this disease, we studied in normal and OA human synovial fibroblasts the nature, the number, and the level of expression of the IL-1 receptor (IL-1R) and through which receptor the biologic stimulation of these cells by IL-1 is mediated. EXPERIMENTAL DESIGN: We determined the IL-1R level by radioligand assay, the type of IL-1R with the use of specific antibodies and by the reverse transcriptase-PCR (RT-PCR), and the mRNA level of the type I IL-1R by slot blot analysis. Biologic activity was measured on the synovial fibroblasts via IL-1 binding and prostaglandin E2 production. RESULTS: Binding data revealed the presence of a single class of high affinity IL-1R in both normal (kD, 21 +/- 4.5 pM) and OA (kD, 23 +/- 5.0 pM) human synovial fibroblasts. The number of receptors was significantly higher (p < 0.004) in OA synovial fibroblasts (2534 +/- 187 sites/cell) than in normal cells (1310 +/- 96 sites/cell). This increase was transient; OA synovial fibroblasts in second and third passages had a normal level of IL-1R. Analysis of the mRNA species by RT-PCR revealed that both type I and type II IL-1R are coexpressed in normal and OA synovial fibroblasts; the type I mRNA was the most predominant in all samples. No difference in the relative amount of type I IL-1R mRNA level was found between normal and OA cells. A blocking Ab against the type I IL-1R completely inhibited, in both normal and OA cells, the receptor binding and IL-1 beta stimulated PGE2 production, whereas the treatment with anti-type II IL-1R was ineffective. CONCLUSIONS: These results indicate that the type I IL-1R is up-regulated in OA synovial fibroblasts and is responsible for mediating the biologic activation of these cells by IL-1. This phenomenon is probably secondary to an abnormality in the post-transcriptional regulation of the type I IL-1R. Although type II IL-1R is also expressed, its translation seems to be inoperative, or this receptor is already shed.

Increased insulin-like growth factor 1 production by human osteoarthritic chondrocytes is not dependent on growth hormone action.

OBJECTIVE: To investigate insulin-like growth factor 1 (IGF-1) production in normal and osteoarthritis (OA) chondrocytes and to further examine the role of growth hormone (GH) in adult human cartilage and, in particular, in diseased tissue. METHODS: IGF-1 production was measured with a radioimmunoassay. Binding assay, Northern blot, and reverse transcriptase polymerase chain reaction (RT-PCR) techniques were used for GH receptor (GHR) detection. The biological response to GH was estimated via IGF-1 production. RESULTS: We observed that basal levels of IGF-1 production were significantly higher in OA chondrocytes than in normal cells (P < 0.005). Adult human chondrocytes, however, were unresponsive to GH stimulation with regard to IGF-1 production, as shown in dose-response (0-1,000 ng/ml) and time-course (days 1-8) studies. In addition, no specific 125I-GH binding was detected in either cell type. Northern blot analysis revealed a 5.5-kb GHR messenger RNA (mRNA) species, but semiquantitative RT-PCR revealed no difference in GHR mRNA expression by normal and OA chondrocytes. CONCLUSION: This study indicates that the elevated synthesis of IGF-1 by adult human OA chondrocytes occurs through a GH/GHR-independent mechanism, suggesting that other factors are capable of controlling local IGF-1 production in these cells.

Synthesis of metalloproteases and interleukin 6 (IL-6) in human osteoarthritic synovial membrane is an IL-1 mediated process.

We investigated the nature of cytokines synthesized by human osteoarthritic (OA) synovium, particularly interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF alpha). We examined the capacity of recombinant human interleukin 1 receptor antagonist (rhIL-1ra) to block the synthesis of metalloproteases (collagenase and stromelysin), IL-1 beta, and IL-6 in osteoarthritis (OA) synovium. Human OA synovium were incubated in the presence or absence of lipopolysaccharide (LPS) or increasing concentrations of rhIL-1ra. The determinations of IL-1 alpha, IL-1 beta, TNF alpha, IL-6, and IL-1ra in culture medium were carried out using specific ELISA. Although both IL-1 isoforms and TNF alpha could be produced by OA synovium, IL-1 beta was the predominant cytokine synthesized either in the presence or absence of LPS. Treatment of the OA synovium with an increasing concentration of rhIL-1ra (0-10 micrograms/ml) showed a dose dependent reduction of both metalloproteases and IL-6. Maximal inhibition was 70% for collagenase, 80% for stromelysin, and 76% for IL-6. LPS treated synovium also showed a consistent suppression of metalloproteases and IL-6, although a higher IL-1ra concentration was required. Conversely, IL-1 beta production was not inhibited by IL-1ra, irrespective of the concentration used and whether the membranes were LPS stimulated. These data showed that IL-1 appears to be the major autocrine cytokine involved in the stimulation of metalloproteases and IL-6 synthesis in OA synovium.

Excess of metalloproteases over tissue inhibitor of metalloprotease may contribute to cartilage degradation in osteoarthritis and rheumatoid arthritis.

BACKGROUND: In an attempt to identify the factor(s) involved in the modulation of the degradative pathway of articular cartilage, we previously reported a possible imbalance between the levels of biologically active forms of metalloproteases and tissue inhibitor of metalloprotease (TIMP) in osteoarthritis (OA) cartilage. EXPERIMENTAL DESIGN: We extended our analysis on the protein level and the synthesis of stromelysin-1, collagenase, TIMP-1, and TIMP-2 in normal, OA, and RA cartilages, and provided information on the synthesis pattern of these proteins in respect to the action of interleukin-1 (IL-1). These protein concentrations were determined by specific sandwich EIA assays. RESULTS: This study allowed us to establish that the concentration of stromelysin-1 and collagenase is elevated in both OA and rheumatoid arthritis (RA) cartilages when compared with normal, with significantly higher levels of collagenase found in OA (p < 0.0003) and RA (p < 0.0001), and of stromelysin-1 in RA (p < 0.02). In all cases, the level of stromelysin-1 significantly exceeded (a few 100-fold) the collagenase level. The cartilage TIMP-1 level was notably enhanced only in RA, whereas TIMP-2 was increased in both OA and RA cartilage. RA patients with active disease had a higher level of metalloproteases and TIMP than those patients with inactive disease. Moreover, patients taking steroids alone or in combination with methotrexate had a markedly lower metalloprotease level without any changes in the TIMP-1 level. In culture cartilage explants, the synthesis of stromelysin-1 was enhanced in RA cartilage, whereas the level of collagenase was increased both in OA and RA explants. When compared with normal patients, the TIMP-1 synthesis was essentially unchanged in arthritic explants, whereas the level of TIMP-2 was decreased in RA explants when compared to OA. IL-1 induced a statistically significant increased synthesis of metalloproteases with the highest level found in arthritic explants. IL-1 also significantly decreased the TIMP-1 synthesis in OA and RA explants, and the TIMP-2 synthesis in OA. CONCLUSIONS: This study demonstrates that stromelysin-1 is the predominant metalloprotease synthesized in human articular cartilage and that both TIMP-1 and TIMP-2 are present in this tissue. The differential regulation of metalloprotease and TIMP syntheses by IL-1 suggests that this cytokine, during inflammatory conditions, may promote cartilage degradation by creating an imbalance between the level of these enzymes and their inhibitors.