Abstract: Feeding rats with a high-fructose diet induced insulin resistance, leading to hypertension or metabolic disorders. Although hypertension is known to accelerate osteoporosis, it is not obvious whether insulin resistance would accelerate osteoporosis. In this study, we evaluated whether osteoporosis might accelerate in fructose-fed rats (FFR), and examined the effect of fluvastatin through a blockade of the mevalonate pathway and an antioxidant action. Stimulation of recombinant receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) expressed by osteoblasts/ stromal cells and macrophage-colony stimulating factor (M-CSF) significantly increased TRAP-positive multinuclear osteoclasts and pit formation, accompanied by an increase in reactive oxygen species as assessed by dichlorodihydrofluorescein (DCF) staining. Interestingly, it was completely abolished by treatment with fluvastatin, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), but not pravastatin. These actions of fluvastatin were partially abolished by co-treatment with geranylgeranylpyrophosphate (GGPP), but not farnesylpyrophosphate (FPP). In the estrogen-deficient model by ovariectomy, FFR exhibited a decrease in bone mineral density, activation of osteoclasts, and an increase in urinary deoxypyridinoline. Importantly, the treatment of fluvastatin, but not pravastatin, attenuated FFR-induced osteoporosis. The present study demonstrates that fructose fed to rats induced insulin resistance and accelerated osteoporosis, while fluvastatin, but not pravastatin, significantly attenuated osteoclast differentiation and activation through a blockade of the classical mevalonate pathway and an antioxidant action, leading to prevention of osteoporosis.

Abstract: We have recently demonstrated that bone marrow CD34+ cells from rheumatoid arthritis (RA) patients displayed abnormal capacities to respond to TNF-alpha and to differentiate into fibroblast-like cells producing MMP-1 (type B synoviocyte -like cells). The current study examined the effects of representative potent disease-modifying antirheumatic drugs, including bucillamine (BUC) and methotrexate (MTX) on the in vitro generation of fibroblast-like cells from RA bone marrow CD34+ cells. CD34+ cells purified from bone marrow specimens of 8 patients with active RA were cultured in the presence or absence of pharmacologically attainable concentrations of intramolecular disulfide form of bucillamine (BUC-ID, 3 microM), a major metabolite of BUC or MTX (20 nM). After incubation for 28 days, the generation of fibroblast-like cells was assessed under phase-contrast light microscopy and the concentrations of MMP-1 and VEGF in the culture supernatants were measured by ELISA. BUC-ID, but not MTX, significantly suppressed the generation of fibroblast-like cells from RA bone marrow CD34+ cells stimulated with SCF, GM-CSF and TNF-alpha (p=0.024 as determined by Wilcoxon signed rank test). Accordingly, BUC-ID, but not MTX, significantly suppressed the production of MMP-1 (p=0.017) and VEGF (p=0.017) by RA bone marrow CD34+ cells, without inhibition of beta2-microglobulin production. These results demonstrate that BUC-ID, but not MTX, is a potent inhibitor of differentiation of fibroblast-like cells from RA bone marrow CD34+ cells. Since MTX, but not BUC, has been previously shown to influence on type A synoviocytes, the data provide rationale of combination of BUC and MTX in the treatment of RA.

Abstract: With recent technical advancements, the number of operative manipulations in the knee joint by minimally invasive surgery-total knee arthroplasty (MIS-TKA) is now considered to be the same as that using standard TKA (S-TKA). The question still remains, however, if MIS-TKA improves recovery compared to S-TKA. We compared MIS-TKA and S-TKA patients' physical activity as measured by an accelerometer. Physical activity expressed as cumulative acceleration was significantly higher in the MIS-TKA than in the S-TKA group on postoperative days (POD1, 2, 3, 4, 5, 10, 11) (P < .05). The recovery time, defined as the number of days required to achieve cumulative acceleration of 80% of the preoperative level, was significantly shorter (P < .05) in the MIS-TKA (3.0 +/- 3.3 days) group than in the S-TKA (7.0 +/- 3.5 days) group. Minimally invasive surgery-total knee arthroplasty appears to allow an earlier recovery after the operation than S-TKA. Less invasion to muscle during the surgery appears to contribute to shorter convalescence.

Abstract:

Abstract: OBJECTIVE: To evaluate the osteoblastic differentiation of human mesenchymal stem cells (hMSCs) in patients with RA. METHODS: Heparinized bone marrow aspirate was obtained from patients with OA and RA. Mononuclear cells were cultured for 2 weeks and a colony-forming assay was performed. The phenotype of cells was analysed by flow cytometry. Passage 2 cells were cultured with beta-glycerophosphate (bGP) in the control group and bGP, ascorbic acid and dexamethasone in the differentiation group. After 2 weeks, ALP staining and activity were performed. After 3 weeks, Alizarin Red S assay was performed. Total RNA was extracted from cells cultured for 2 and 3 weeks. Gene expression of bone formation factor was examined by real-time PCR. RESULTS: The phenotype of cells was identical in both OA and RA and the content was thought to be hMSCs. The results of ALP activity and Alizarin Red S assay showed higher levels in the differentiation group for both OA and RA samples compared with the control group. The results of a colony-forming assay were identical in both OA and RA samples. Gene expression in the differentiation group was higher than in the control group in both OA and RA samples. There was no significant difference between OA and RA samples in all experiments. CONCLUSION: The function of osteoblastic differentiation of hMSCs is similar between OA and RA.

Abstract: Abnormal reactions accompanied by bone formation in the osteoarticular region induced by long-term administration of etretinate have been reported. We treated a patient who received continuous treatment of psoriatic erythroderma with etretinate for 7 years, and who had an osseous bridge that extended across the acetabulum over the femur on both sides. The patient experienced a major gait disturbance and eventually was unable to walk. Functional gait was restored by resecting the ossified regions and radiotherapy. Histologic sections of the ossified lesions showed enchondral ossification in the ligament attachment site in the joint margin, with advancing ossification along the articular capsule; the pattern was similar to that in diffuse idiopathic skeletal hyperostosis. This is the first report of an osseous bridge associated with long-term administration of etretinate extending across the acetabulum over the femur on both sides.

Abstract: A recent analysis of clinical studies suggests that angiotensin-converting enzyme (ACE) inhibitors might reduce bone fractures. In this study, we examined whether an ACE inhibitor might attenuate osteoporosis in a hypertensive rat model. In spontaneous hypertensive rats (SHRs), estrogen deficiency induced by ovariectomy (OVX) resulted in a significant increase in osteoclast activation as assessed by the tartrate-resistant acid phosphatase (TRAP) activity in the tibia, accompanied by a significant decrease in bone density evaluated by dual-energy X-ray absorptiometry and an increase in urinary deoxypyridinoline. Treatment with an ACE inhibitor, imidapril, attenuated OVX-induced decrease in bone density and increase in TRAP activity and urinary deoxypyridinoline. As ACE inhibitors possess the effects of blockade of the renin-angiotensin system (RAS) and activation of the bradykinin-nitric oxide pathway, we examined the contribution of both pathways in an OVX-induced osteoporosis model. Administration of nitro-L-arginine methylester (L-NAME) did not alter TRAP activity, urinary deoxypyridinoline or bone density, whereas the administration of a subpressor dose of angiotensin II accelerated the increase in TRAP activity in the tibia, accompanied by a significant decrease in bone density and an increase in urinary deoxypyridinoline. Thus, ACE inhibitors prevented osteoporosis, probably because of the inhibition of RAS, but not of nitric oxide. Overall, ACE inhibitors attenuated osteoporosis in a hypertensive rat model through the blockade of RAS.Hypertension Research advance online publication, 10 July 2009; doi:10.1038/hr.2009.99.

Abstract: Recent progress in DNA technologies has provided the strategies to regulate the transcription of disease-related genes in vivo using antisense oligodeoxynucleotide (ODN). Transfection of cis-element double-stranded oligodeoxynucleotides (decoy ODNs) has been reported as a new therapeutic tool of anti-gene strategies for gene therapy. In the field of arthritis, decoy ODNs strategies have been significant therapeutic potential. The concept of regulation the disease related gene expression at the level of transcriptional factor may be more therapeutic effects compared with monotherapy in arthritis. Injection of NFkappaB decoy ODN into the affected joint resulted in marked suppression of joint destruction in CIA models. In vitro studies demonstrated that the inhibitory effect on inflammatory cytokines and matrix metalloproteinase production from stimulated synovial cells derived from rheumatoid arthritis patients. NFkappaB decoy ODN inhibited induction of osteoclasts and bone resorption ability. Parthenolide is one of the main sesquiterpense lactones responsible for the bioactivities of feverfew and recently reported to inhibit NFkappaB activation. Parthenolide has ameliorated the severity of joint destruction in experimental animal model. Based upon these findings, NFkappaB may be one of important therapeutic target for arthritis.

Abstract: The purpose of this study was to clarify variations in patterns of flattening in rheumatoid hindfoot. Out of 232 outpatients with rheumatoid arthritis treated at our hospital from 2001 to 2003, we studied lateral radiographs of feet of 216 patients (423 weight-bearing views). We measured the medial arch angle (MAA) and talar angle (TA) and compared the alignment of the talonavicular joint-sagittal plane of each foot. We also evaluated the relationship between the severity of flattening and inclination of the talus and performed cluster analysis. Three groups were clustered by MAA and TA. In group I, joints were normal or close to normal. In group II, both talonavicular and subtalar joints were affected. In group III, talonavicular joints were minimally affected, and the subtalar joints were primarily affected. Groups II and III were thought to be a different pattern of flattening. The present results suggest that there are at least two patterns of flattening in rheumatoid hindfoot.

Abstract: In inflammatory arthritis such as RA, osteoclastic activity is severely enhanced. GM-CSF was reportedly elevated in synovial fluid, but is a strong inhibitor of osteoclastogenesis; here lies a contradiction. Our objective was to examine what type of osteoclasts generate and resorb bone with resistance to GM-CSF in an inflammatory joint. Monocyte-derived cells generated in GM-CSF were morphologically and immunophenotypically different from both the conventional DC and macrophage. They could differentiate into osteoclasts in the presence of RANKL + M-CSF, acquiring a stronger osteoclastic activity under TNF treatment. Furthermore, their differentiation was not inhibited by GM-CSF, while monocyte-derived osteoclast differentiation was completely inhibited. The resorption was suppressed by GM-CSF, and the existence of another osteoclastic pathway has been suggested. Our findings indicate another type of osteoclast exists in inflammatory arthritis.

Abstract: Recent clinical studies suggest that several antihypertensive drugs, especially angiotensin-converting enzyme inhibitors, reduced bone fractures. To clarify the relationship between hypertension and osteoporosis, we focused on the role of angiotensin II (Ang II) on bone metabolism. In bone marrow-derived mononuclear cells, Ang II (1x10(-6) M) significantly increased tartrate-resistant acid phosphatase (TRAP) -positive multinuclear osteoclasts. Of importance, Ang II significantly induced the expression of receptor activator of NF-kappaB ligand (RANKL) in osteoblasts, leading to the activation of osteoclasts, whereas these effects were completely blocked by an Ang II type 1 receptor blockade (olmesartan) and mitogen-activated protein kinase kinase inhibitors. In a rat ovariectomy model of estrogen deficiency, administration of Ang II (200 ng/kg/min) accelerated the increase in TRAP activity, accompanied by a significant decrease in bone density and an increase in urinary deoxypyridinoline. In hypertensive rats, treatment with olmesartan attenuated the ovariectomy-induced decrease in bone density and increase in TRAP activity and urinary deoxypyridinoline. Furthermore, in wild-type mice ovariectomy with five-sixths nephrectomy decreased bone volume by microcomputed tomography, whereas these change was not detect in Ang II type 1a receptor-deficient mice. Overall, Ang II accelerates osteoporosis by activating osteoclasts via RANKL induction. Blockade of Ang II might become a novel therapeutic approach to prevent osteoporosis in hypertensive patients.

Abstract: The objective of this study was to evaluate in vivo kinematics of a high-flexion, posterior-stabilized fixed-bearing, total knee arthroplasty in weight-bearing deep knee-bending motion. A total of 20 knees implanted with the Scorpio Non-Restrictive Geometry knee system in 17 patients were assessed in this study. The Scorpio Non-Restrictive Geometry is a recent implant design with modifications made to accommodate a higher flexion range of motion and greater axial rotation, particularly during more functionally demanding activities. Patients were examined during a deep knee-bending motion using fluoroscopy, and femorotibial motion was determined using a 2-dimensional to 3-dimensional registration technique. The average flexion angle was 126.5 degrees (110 degrees -149 degrees ). The femoral component demonstrated a mean of 13.5 degrees (5.2 degrees -21 degrees ) external rotation. The external rotation increased up to maximum flexion. The pivot pattern was a medial pivot pattern similar to that reported in normal knee kinematics.

Abstract: To examine the clinical features of vertebral and non-vertebral fractures in patients with rheumatoid arthritis (RA), including insufficiency fractures, and to assess the risk factors for fracture, we prospectively studied 209 outpatients with rheumatoid arthritis for 1 year. The age, gender, Steinbrocker's functional class, glucocorticoid use, history of lower limb surgery, serum C-reactive protein (CRP), and use of bisphosphonates were evaluated. Examination for fractures was performed by radiography, computed tomography (CT), magnetic resonance imaging (MRI), and bone scanning. Thirty-three fractures occurred in 24 patients over the 1-year study period, and the incidence was 15.8 fractures per 100 patient-years. Fractures occurred at various sites. The majority (70%) was insufficiency fracture, and more than 50% caused ambulatory dysfunction. Radiographic findings were absent in 39% of the fractures at the onset of pain. The functional class and glucocorticoid dose were significantly associated with fracture development. This prospective study showed that the incidence of fractures, especially insufficiency fractures, was very high in patients with rheumatoid arthritis and that most of their fractures caused gait disturbance. Early intervention to prevent secondary osteoporosis is recommended to maintain the quality of life in patients with rheumatoid arthritis, especially those with functional impairment or undergoing glucocorticoid therapy.

Abstract: The objective of this study was to evaluate the kinematics of a high-flexion, posterior-stabilized, mobile-bearing total knee arthroplasty (TKA) in weight-bearing, deep knee bending motion. Thirteen patients implanted with the Legacy Posterior Stabilized Flex (Zimmer, Warsaw, IN) mobile-bearing TKA were examined during a deep knee bending motion using fluoroscopy. Femorotibial motion was determined using a 2-dimensional to 3-dimensional registration technique, which used computer-assisted design models to reproduce the position of metallic implants from single-view fluoroscopic images. The average flexion range of motion between the metallic implants was 116 degrees . The average rotation of the femoral component was 9.3 degrees external rotation. The mean kinematic pathway was early rollback, lateral pivot with external rotation, and bicondylar rollback. We found that the kinematic pattern of the Legacy Posterior Stabilized Flex mobile-bearing TKA was different than normal knee kinematics.

Abstract: Knowledge of the pattern of joint destruction is important for planning the therapeutic approach to rheumatoid arthritis (RA) of the elbow. Accordingly, we carried out a large-scale radiographic study with the objective of elucidating the joint destruction pattern in rheumatoid elbows. From 2001 through 2003, we examined and took plain X-rays of both elbows of 193 RA patients (i.e., 386 elbows), consisting of 18 men and 175 women, with a mean age of 57.0 years. Radiographic images of the elbow joints were used to classify the degree of bone loss in various zones on the elbow joint surface into four grades of severity, and joint destruction was compared between the left and right elbows. In addition, correlation in the extent of bone loss between each of the zones of the same elbow and differences in the extent of bone loss were analyzed statistically. The results showed direct correlations for destruction of the elbow joint surface among the zones for the left and right elbow joints and in the same elbow joint. However, more severe destruction was observed on the radial side of the humeral trochlea, and it was surmised that destruction of the elbow joint must begin at that site and gradually spread mediolaterally. In addition, in the same elbow joint, the correlation in the degree of bone loss between the trochlea of humerus and the trochlear notch was especially strong, indicating that the bone destruction at both sites represented mirror lesions. We conclude that when performing radiographic diagnosis of the joint damage in the rheumatoid elbow, knowledge of this pattern of joint destruction will be useful for assessing whether there is joint destruction in the initial stage and for deciding the therapeutic approach.

Abstract: The purpose of this study was to analyze the skeletal alignment of the hindfoot valgus deformity in patients with rheumatoid arthritis using bone models reconstructed from three-dimensional computerized tomography data. Computed tomography was performed on 21 feet of patients with rheumatoid arthritis, and magnetic resonance imaging was taken of 10 normal feet of eight volunteers. An image processing system was used to create bone models and analyze the three-dimensional displacement of the calcaneus, talus, navicular, and cuboid bones. With a standard coordinate system in the distal tibia and a local coordinate system in each bone of the hindfoot, three rotational parameters and three translational parameters were used to evaluate the relative displacement. The talus showed plantar flexion. Both the calcaneus and navicular bones had valgus and lateral shift displacements. However, the cuboid had no displacement relative to the calcaneus, and the navicular showed no displacement relative to the cuboid. The calcaneus, navicular, and cuboid bones have the same pattern of deformity in patients with rheumatoid arthritis. This three-dimensional image-based technique successfully quantified the hindfoot valgus deformity resulting from rheumatoid arthritis and is beneficial for better understanding the deformity pathomechanism.

Abstract: The objective of the present study was to elucidate the mode of rheumatoid arthritis shoulder destruction. The study included 402 shoulders from 201 patients with rheumatoid arthritis. Plain radiographic findings were used to assess and statistically analyze the severity of the glenohumeral joint destruction (GHD) and greater tuberosity destruction (GTD). For both GHD and GTD scores, a statistically significant correlation was found between the left and right sides and also between the GHD and GTD scores within the same shoulder. However, 97 shoulders of 67 patients showed a heterogeneous pattern. An interesting finding was that no patients showed a combination of the GHD type plus the GTD type. Shoulders with rheumatoid arthritis showed statistically significant symmetry and uniform destruction. Even if they showed heterogeneous destruction, there were no cases of a different pattern of heterogeneity on the opposite side. The mode of destruction was not always definite, however.

Abstract: FR167653 is a potent inhibitor of p38 MAP Kinase and inhibits TNF-alpha and IL-1beta production in inflammatory cells. In this study we investigated the effect of FR167653 on CIA. CIA rats were subcutaneously injected with FR167653 (32 mg/kg/day) starting on the day of the booster injection and after the onset of arthritis in the prophylactic and therapeutic treatment groups, respectively. The hind paw swelling, radiolographic and histologic scores, and osteoclast number were evaluated. Serum and tissue cytokine levels were assessed by ELISA. Flow cytometric analysis of T-lymphocytes from bone marrow was also performed. The effect of FR167653 on in vitro osteoclast formation induced by sRANKL and TNF-alpha was examined. Hind paw swelling occurred in CIA rats but not in the prophylactic treatment group. Therapeutic treatment also significantly reduced the paw swelling. The mean radiographic, histologic score, and osteoclast number of the treatment group were significantly lower than those of CIA rats without treatment. FR167653 treatment reduced serum TNF-alpha and IL-1beta levels, ankle IL-1beta concentration, and CD4-CD8a+ T-cell population in bone marrow. Furthermore, FR167653 inhibited the osteoclast-like cell differentiation induced by both sRANKL and TNF-alpha in vitro. FR167653 prevented the onset of arthritis in a prophylactic treatment model and suppresses the progression of joint destruction in a therapeutic treatment model, suggesting that p38 MAP Kinase is a potential therapeutic target for rheumatoid arthritis.

Abstract: BACKGROUND: For the development of molecular therapy based on oligodeoxynucleotides (ODN), ODN have to be stable against nucleases and be specific to the target transcription factor. To decrease non-specific binding and degradation from the 3'-terminus of ODN, we designed partially annealed ODN by binding the extremities of two single strands, resulting in a ribbon-shaped ODN, so called ribbon-type decoy ODN (R-ODN). METHODS: We evaluated the efficiency in the process of enzymatic ligation of R-ODN, the binding activity to nuclear factor-kappaB (NF-kappaB), and the stability against Exonuclease III and nucleases present in serum. The functional activity of R-ODN to inhibit NF-kappaB in vitro was evaluated in human aortic smooth muscle cells (VSMC): TNF-alpha-induced proliferation rate and MMP-9 expression were assessed after R-ODN transfection. RESULTS AND CONCLUSIONS: Although R-ODN have a phosphodiester backbone, their physical conformation was designed to provide nuclease resistance without interfering with their binding activity. As expected, R-ODN showed more resistance to exonucleases and stability in 100% serum than non-modified decoy ODN (N-ODN). Importantly, the R-ODN construction did not interfere with its binding activity to NF-kappaB, similar to N-ODN. Transfection of R-ODN significantly inhibited the expression of MMP-9 induced by TNF-alpha in VSMC as assessed by real-time polymerase chain reaction (PCR), and R-ODN also inhibited the proliferation of VSMC induced by TNF-alpha (10 ng/ml), similar to phosphorothioate decoy ODN. Overall, the development of ribbon NF-kappaB decoy ODN could provide a useful tool for basic and clinical research.

Abstract: The transcription factor, nuclear factor-kappa B (NFkappaB), is believed to play a pivotal role in osteoclast formation. In this study, we focused on NFkappaB decoy oligodeoxynucleotides (ODN) as a new therapeutic strategy to attenuate osteoporosis. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts formed in mononuclear cells including osteoclast precursors from neonatal rabbit bone marrow were increased in the presence of 1,25-dihydroxyvitamin D3, whereas transfection of NFkappaB decoy ODN decreased the number of TRAP-positive cells and attenuated RANKL and M-CSF-induced osteoclast formation. NFkappaB decoy ODN also inhibited the activity of osteoclasts, as assessed by pit formation. In rat ovariectomized model of estrogen deficiency, continuous administration of NFkappaB decoy ODN attenuated the increase of TRAP activity, accompanied by a significant increase in calcium concentration in tibia and femur and decrease in urinary deoxypyridinoline. In additional osteoporosis model using vitamin C-deficient rat, inhibition of NFkappaB by decoy ODN dramatically improved the bone length, weight, density as assessed by dual-energy X-ray absorptiometry. Overall, inhibition of NFkappaB by decoy strategy prevented osteoporosis through the inhibition of bone resorption. Targeting of NFkappaB might be potential therapy in various bone metabolic diseases.

Abstract: Bone marrow CD34+ cells from rheumatoid arthritis (RA) patients have abnormal capacities to respond to tumor necrosis factor (TNF)-alpha and to differentiate into fibroblast-like cells producing matrix metalloproteinase (MMP)-1. We explored the expression of mRNA for nuclear factor (NF)kappaB in RA bone marrow CD34+ cells to delineate the mechanism for their abnormal responses to TNF-alpha. CD34+ cells were purified from bone marrow samples obtained from 49 RA patients and 31 osteoarthritis (OA) patients during joint operations via aspiration from the iliac crest. The mRNAs for NFkappaB1 (p50), NFkappaB2 (p52) and RelA (p65) were examined by quantitative RT-PCR. The expression of NFkappaB1 mRNA in bone marrow CD34+ cells was significantly higher in RA than in OA, whereas there was no significant difference in the expression of mRNA for NFkappaB2 and RelA. The expression of NFkappaB1 mRNA was not correlated with serum C-reactive protein or with the treatment with methotrexate or oral steroid. Silencing of NFkappaB1 by small interfering RNA abrogated the capacity of RA bone marrow CD34+ cells to differentiate into fibroblast-like cells and to produce MMP-1 and vascular endothelial growth factor upon stimulation with stem cell factor, granulocyte-macrophage colony stimulating factor and TNF-alpha without influencing their viability and capacity to produce beta2-microglobulin. These results indicate that the enhanced expression of NFkappaB1 mRNA in bone marrow CD34+ cells plays a pivotal role in their abnormal responses to TNF-alpha and, thus, in the pathogenesis of RA.

Abstract: THR0921 is a novel peroxisome proliferator-activated receptor gamma (PPARgamma) agonist with potent anti-diabetic properties. Because of the proposed role of PPARgamma in inflammation, we investigated the potential of orally active THR0921 to inhibit the pathogenesis of collagen-induced arthritis (CIA). CIA was induced in DBA/1J mice by the injection of bovine type II collagen in complete Freund's adjuvant on days 0 and 21. Mice were treated with THR0921 (50 mg/kg/day) starting on the day of the booster injection and throughout the remaining study period. Both clinical disease activity scores as well as histological scores of joint destruction were significantly reduced in mice treated with THR0921 compared to untreated mice. Proliferation of isolated spleen cells, as well as circulating levels of IgG antibody to type II collagen, was decreased by THR0921. Moreover, spleen cell production of IFN-gamma, tumor necrosis factor (TNF)-alpha and IL-1beta in response to exposure to lipopolysaccharide or type II collagen was reduced by in vivo treatment with THR0921. Steady state mRNA levels of TNF-alpha, IL-1beta, monocyte chemotactic protein-1 and receptor activator of nuclear factor kappaB ligand (RANKL) in isolated joints were all decreased in mice treated with THR0921. Finally, THR0921 inhibited osteoclast differentiation of bone marrow-derived cells stimulated with macrophage colony-stimulating factor and RANKL. In conclusion, THR0921 attenuates collagen-induced arthritis in part by reducing the immune response. As such, PPARgamma may be an important therapeutic target for rheumatoid arthritis.

Abstract: Although skin diseases are one of the target diseases for gene therapy, there has been no practical gene transfer method. First, we examined gene transfer efficiency of the spring-powered jet injector, Shima Jet, which was originally developed as a non-needle jet injector of insulin. Local gene expression was about 100 times higher when the luciferase plasmid was transferred by the Shima Jet than by a needle. Gene transfer of beta-galactosidase revealed gene expression in the epidermis. Based on these results, we then examined the potential of gene therapy using the Shima Jet for wound healing. An increase of cellular proliferation of the epidermis and the number of microvessels in the granulation tissue was observed after hepatocyte growth factor (HGF) gene transfer. An increase in blood flow around the wound was observed after prostacyclin synthase (PGIS) gene transfer. Moreover, promotion on wound healing was observed in HGF gene transferred group, and further promotion was observed in combined gene transferred group as assessed by measuring wound area. These results indicate that co-transfer of HGF and PGIS genes by the Shima Jet could be an effective strategy to wound healing.

Abstract: In this study we examined the effect of ribbon-type (circular-type) NF-kappaB decoy oligodeoxynucleotides (RNODN) on osteoclast induction and activity. We extracted bone marrow cells from the femurs of rats and incubated non-adherent cells with receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). First, transfer efficiency into osteoclasts and their precursors, resistance to exonuclease, and binding activity of decoy to NF-kappaB were examined. Next, to examine the effect of RNODN on osteoclast induction and activity, osteoclast differentiation and pit formation assays were performed. RNODN were injected into the ankle joints of rats with collagen-induced arthritis. Joint destruction and osteoclast activity were examined by histological study. The resistance of RNODN to exonuclease and their binding activity on NF-kappaB were both greater than those of phosphorothionated NF-kappaB decoy oligodeoxynucleotides. The absolute number of multinucleate cells scoring positive for tartrate-resistant acid phosphatase was significantly decreased in the RNODN-treated group. The average calcified matrix resorbed area was significantly decreased in the RNODN-treated group. Histological study showed marked suppression of joint destruction and osteoclast activity by intra-articular injection of RNODN. These results suggest the inhibitory effect of RNODN on the induction and activity of osteoclasts. Direct intra-articular injection of RNODN into the joints may be an effective strategy for the treatment of arthritis.

Abstract: This study examined the ability of E2F decoy oligodeoxynucleotides (ODN) to inhibit proliferation of synovial fibroblasts derived from patients with rheumatoid arthritis (RA). The effect of E2F decoy ODN on cartilage invasion by RA synovium in a murine model of human RA was also investigated. E2F decoy ODN were introduced into synovial tissue and synovial fibroblasts derived from patients with RA using hemagglutinating virus of Japan (HVJ)-liposomes. The effect of E2F decoy ODN on synovial fibroblast proliferation was evaluated by MTT assay and by RT-PCR for the cell cycle regulatory genes proliferating-cell nuclear antigen (PCNA) and cyclin-dependent kinase 2 (cdk2). Changes in production of inflammatory mediators by RA synovial tissue following transfection with E2F decoy ODN were assessed by ELISA. Human cartilage and RA synovial tissue transfected with E2F decoy ODN were co-transplanted in severe combined immunodeficient (SCID) mice. After 4 weeks, the mice were sacrificed and the implants histologically examined for inhibition of cartilage damage by E2F decoy ODN. E2F decoy ODN resulted in significant inhibition of synovial fibroblast proliferation, corresponding with reduced expression of PCNA and cdk2 mRNA in synovial fibroblasts. The production of interleukin-1beta (IL-1beta), IL-6 and matrix metalloproteinase (MMP)-1 by synovial tissue was also significantly inhibited by the introduction of E2F decoy ODN. Further, in an in vivo model, cartilage that was co-implanted with RA synovial tissue transfected with E2F decoy ODN exhibited no invasive and progressive cartilage degradation. These data demonstrate that transfection of E2F decoy ODN prevents cartilage destruction by inhibition of synovial cell proliferation, and suggest that transfection of E2F decoy ODN may provide a useful therapeutic approach for the treatment of joint destruction in arthritis.

Abstract: The purpose of this study was to build a visualization technique of the femorotibial contact in fixed-bearing total knee arthroplasty (TKA) using X-ray fluoroscopy, and to apply this technique to a TKA patient during dynamic motion. In vivo kinametcis of the metallic knee implant was determined using a 2D/3D registration technique, which uses computer assisted design (CAD) model of the implant to estimate the 3D pose of radiopaque metallic femoral and tibial components from a single-plane fluoroscopic image. In fixed-bearing TKA, a 3D pose of radiolucent tibial polyethylene insert can be determined from the estimated pose of the tibial component. To visualize femorotibial contact, the proximity between surfaces of femoral component and tibial insert was calculated, and mapped onto the insert surface model. For the clinical application, dynamic states of contact on the tibial insert were observed including axial rotation and unilateral loading during knee flexion, and post-cam contact of posterior stabilized TKA. The present technique provided us new information and enabled us to better understand the relationship between in vivo knee kinematics and articular shape of the implant.

Abstract: To achieve quantitative assessment of 3D dynamic motion of artificial knee implants under clinical conditions, we developed a 3D kinematic analysis system using X-ray fluoroscopic imaging. The 3D pose-estimation technique for knee implants was built on a 2D/3D registration algorithm, which determines the spatial pose for each femoral and tibial component from the knee implant contours and computer-assisted design (CAD) models of the implant. In order to validate the accuracy of the 3D pose estimation and the system, computer simulation and in vitro tests were performed using images of knee implants taken in 10 different poses with respect to X-ray focus. Computer simulation tests showed that the root mean square errors (RMSE) for all variables were less than 1.0 mm 1.0 degrees. In vitro tests showed that the RMSE for translation perpendicular to the X-ray image plane was about 1.5 mm, while the accuracy of the remaining two translational and three rotational variables was found to be sufficient for analyzing knee kinematics. Computation time in 3D pose estimation was then obtained in less than 30 seconds for each frame. In clinical application, dynamic movement in deep knee bending was quantitatively analyzed, and the feasibility and effectiveness of the system was demonstrated.

Abstract: The purpose of this study was to compare mid-term results of mobile-bearing and fixed-bearing in bilateral total knee arthroplasty (TKA). Twenty-two patients underwent bilateral TKA with a mobile-bearing prosthesis (Rotaglide, Corin, UK) on one side and a fixed-bearing prosthesis (NexGen-CR, Zimmer, USA) on the other. There were 21 female patients, and in 18 patients, the diagnosis was rheumatoid arthritis. The average age was 59.6 (35-78) years. In all procedures, the posterior cruciate ligament was retained and patella re-surfaced. The average follow-up in the mobile-bearing group was 98 (79-107) months and 96 (79-107) months in the fixed-bearing group. At the final follow-up, the knee score was 91.8 points and 91.1 points, respectively, and the function score 65.5 points. The range of motion was similar in the two groups (1.1-106.9 degrees; 0.4-106.9 degrees). Five patients favoured the fixed-bearing prosthesis, but 16 found no difference. In patients with bilateral TKA, there was no difference in the short-term result between mobile-bearing and fixed-bearing prostheses.

Abstract:

Abstract: Although interleukin-1 (IL-1) has been implicated in the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are largely unknown. Recently, a cytokine-driven, stromal cell-free mouse osteoclastogenesis model was established. A combination of macrophage colony stimulating factor (M-CSF) and receptor activator of NFkappaB ligand (RANKL) was proven to be sufficient in inducing differentiation of bone marrow hematopoietic precursor cells to bone-resorbing osteoclasts in the absence of stromal cells or osteoblasts. This study utilizes this model to examine the impact of human IL-1beta on in vitro osteoclastogenesis of bone marrow progenitor cells. We found that osteoclast precursor cells failed to undergo osteoclastogenesis when treated with IL-1 alone. In contrast, IL-1 dramatically up-regulated osteoclastogenesis by 2.5- to 4-folds in the presence of RANKL and M-CSF. The effect can be significantly blocked by IL-1 receptor antagonist (p < 0.01). Tumor necrosis factor-alpha (TNF-alpha) was undetectable in the culture medium of differentiating osteoclasts induced by IL-1. Adding exogenous TNF-alpha neutralizing antibody had no influence on the IL-1-induced effect as well. These results show that in the absence of stromal cells, IL-1 exacerbates osteoclastogenesis by cooperating with RANKL and M-CSF, while TNF-alpha is not involved in this IL-1-stimulated osteoclast differentiation pathway.

Abstract: OBJECTIVE: To investigate the morphology and function of multinucleated bone-resorbing giant cells derived from CD14-positive cells in the synovial fluids (SF) of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). METHODS: CD14-positive cells were obtained by magnetic-activated cell sorting of primary cultures of mononuclear cells from the SF. Multinucleated bone-resorbing giant cells were induced from the CD14-positive cells in the presence or absence of cytokines. We examined various characteristics, including osteoclast markers, fusion index and bone-resorption activities of the multinucleated giant cells. RESULTS: Multinucleated giant cells were induced from the CD14-positive cells in the SF of the RA and OA patients by the addition of interleukin (IL)-3, IL-5 and IL-7, or granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. These multinucleated giant cells were positive for tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, actin, vitronectin receptor and the calcitonin receptor. However, the average values for the number of nuclei, fusion index and bone-resorption functions of the SF cells from the RA patients were significantly higher than those derived from the OA patients. CONCLUSION: These results suggest that the induction and activities of multinucleated bone-resorbing giant cells may play a pivotal role in bone destruction, and that these processes may be enhanced significantly in RA patients.

Abstract: Osteoclastogenesis is a key event of the cellular reaction in prosthetic loosening. Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) were used to study the localization and expression of receptor activator of nuclear factor kappa B ligand (RANKL), a potent factor for osteoclastogenesis in the membranous tissue formed around loosened prosthetic joint implants. RANKL was identified in a wide variety of cells appearing in this membranous tissue. At least three types of RANKL-positive cells were identified, including prolyl 4-hydroxylase (PH)-positive fibroblast lineage cells, CD68 cells, and tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multi-nucleated macrophage lineage cells. Tumor necrosis factor (TNF)-alpha-converting enzyme (TACE) was colocalized with RANKL in these cells, suggesting the in-situ release of this factor. RT-PCR confirmed the actual expression of the RANKL and TACE genes in the tissues around the loosened implant. These observational findings indicate the possible synthesis of RANKL by fibroblast and macrophage lineage cells, and suggest the in-situ involvement of RANKL in both osteoclastogenesis and osteoclastic bone resorptive events occurring in prosthetic joint loosening.

Abstract: Two-dimensional (2-D)/three-dimensional (3-D) registration techniques using single-plane fluoroscopy are highly important for analyzing 3-D kinematics in applications such as total knee arthroplasty (TKA) implants. The accuracy of single-plane fluoroscopy-based techniques in the determination of translation perpendicular to the image plane (depth position), however, is relatively poor because a change in the depth position causes only small changes in the 2-D silhouette. Accuracies achieved in depth position using conventional 2-D/3-D registration techniques are insufficient for clinical applications. Therefore, we propose a technique for improving the accuracy of depth position determination in order to develop a system for analyzing knee kinematics over the full six degrees of freedom (6 DOF) using single-plane fluoroscopy. In preliminary experiments, the behaviors of errors for each free variable were quantified as evaluation curves by examining changes in cost function with variations in the free variable. The evaluation curve for depth position was more jagged, and the curve peak less pointy, compared to the evaluation curves of the other five variables, and the curve was found to behave differently. Depth position is therefore optimized independently of the other variables, using an approximate evaluation curve of depth position prepared after initial registration. Accuracy of the proposed technique was evaluated by computer simulation and in vitro tests, with validation of absolute position and orientation performed for each knee component. In computer simulation tests, root-mean-square error (RMSE) in depth position was improved from 2.6 mm (conventional) to 0.9 mm (proposed), whereas for in vitro tests, RMSE improved from 3.2 mm to 1.4 mm. Accuracy of the estimation of the remaining two translational and three rotational variables was found to be almost the same as that obtained by conventional techniques. Results of in vivo tests are also described in which the possibility of full 6 DOF kinematic analysis of TKA implants is shown.

Abstract: We reviewed 21 patients with rheumatoid arthritis who had a total ankle replacement between 1984 and 2000. The average follow-up was 72 (15-169) months. Clinical results were evaluated using the American Orthopaedic Foot and Ankle Society (AOFAS) score. At the latest review, three ankles had been revised. Two ankles were excellent, seven good, three fair, and 12 poor. Eleven patients with 13 ankles had residual pain, with radiographs showing a high incidence of radiolucent lines. Migration of the tibial component was seen in 13 ankles and collapse of talus in nine. Although clinical results were poor, patient satisfaction was not.

Abstract: OBJECTIVE: To examine the capacity of bone marrow CD34+ cells to generate endothelial cells, in order to assess the role of bone marrow in neovascularization in the synovium of rheumatoid arthritis (RA). METHODS: CD34+ cells purified from the bone marrow of 13 patients with active RA and 9 control subjects (7 osteoarthritis [OA] patients and 2 healthy individuals) were cultured in the presence of stem cell factor (10 ng/ml) and granulocyte-macrophage colony-stimulating factor (1 ng/ml). After 18 days of incubation, the generation of endothelial cells was assessed by flow cytometry. The generation of endothelial cells was compared with the degree of vascularization in the synovial tissues and with the microvessel densities in the synovium, as determined by microscopy. The expression of vascular endothelial growth factor receptor 2/kinase insert domain receptor (KDR) messenger RNA (mRNA) in CD34+ cells was examined by quantitative reverse transcription-polymerase chain reaction. RESULTS: The generation of CD14+ cells from bone marrow-derived CD34+ cells from RA patients was comparable to that from control subjects. However, the generation of von Willebrand factor (vWF)-positive cells and CD31+/vWF+ cells from RA bone marrow-derived CD34+ cells was significantly higher than that from control subjects (P = 0.004 and P = 0.030, respectively). The generation of vWF+ cells from bone marrow CD34+ cells correlated significantly with the microvessel densities in the synovial tissues (r = 0.569, P = 0.021). Finally, RA bone marrow CD34+ cells expressed KDR mRNA at higher levels than OA bone marrow CD34+ cells. CONCLUSION: These results indicate that RA bone marrow CD34+ cells have enhanced capacities to differentiate into endothelial cells in relation to synovial vascularization. The data therefore suggest that bone marrow CD34+ cells might contribute to synovial neovascularization by supplying endothelial precursor cells and, thus, play an important role in the pathogenesis of RA.

Abstract: The current study aimed to analyze kinematics during deep knee bending motion by subjects with fully congruent mobile-bearing total knee arthroplasties allowing axial rotation and anteroposterior (AP) gliding. Twelve subjects were implanted with Dual Bearing Knee prostheses (DBK, slot type: Finsbury Orthopaedics, Surrey, UK). These implants include a mobile-bearing insert that is fully congruent with the femoral component throughout flexion and allows axial rotation and limited AP translation. Sequential fluoroscopic images were taken in the sagittal plane during loaded knee bending motion. In vivo kinematics were analyzed using a two- to three-dimensional registration technique, which uses computer-assisted design models to reproduce the spatial position of femoral and tibial components from single-view fluoroscopic images. The average femoral component demonstrated 13.4 degrees external axial rotation for 0-120 degrees flexion. On average, the medial condyle moved anteriorly 6.2 mm for 0-100 degrees flexion, then posteriorly 4.0 mm for 100-120 degrees flexion. On average, the lateral condyle moved anteriorly 1.0 mm for 0-40 degrees flexion, then posteriorly 8.7 mm for 40-120 degrees flexion. The typical subject exhibited a lateral pivot pattern from extension to 60 degrees flexion and a central pivot pattern from 60 degrees to 100 degrees flexion, patterns that are not usually observed in normal knees. Subsequently from 100 degrees to 120 degrees flexion, a rollback pattern was reproduced in which bilateral condyles moved backward.

Abstract: The inhibition of specific transcription regulatory proteins is a novel approach to regulate gene expression. The transcriptional activities of DNA binding proteins can be inhibited by the use of double-stranded oligonucleotides (ODNs) that compete for binding to their specific target sequences in promoters and enhancers. Transfection of this cis-element double-stranded ODN, referred to as decoy ODN, has been reported to be a powerful tool that provides a new class of anti-gene strategies to gene therapy and permits examination of specific gene regulation. We have demonstrated the usefulness of this decoy ODN strategy in animal models of restenosis, myocardial infarction, glomerulonephritis and rheumatoid arthritis. However, one of the major limitations of decoy ODN technology is the rapid degradation of phosphodiester ODNs by intracellular nucleases. To date, several different types of double-stranded decoy ODNs have been developed to overcome this issue. Circular dumb-bell (CD) double-stranded decoy ODNs that were developed to resolve this issue have attracted a high level of interest. In this review, the applications of decoy ODN strategy and the advantages of modified CD double-stranded decoy ODNs will be discussed.

Abstract: OBJECTIVES: To examine the localization of bone morphogenetic protein (BMP)-2 mRNA and protein in human osteoarthritic (OA) articular cartilage and osteophyte. DESIGN: Five normal, four growing and 14 OA human cartilage samples, graded histomorphologically by Mankin Score, were studied by in situ hybridization and immunohistochemistry for the expression of BMP-2. RESULTS: BMP-2 mRNA was present in chondrocytes in neonatal growing articular cartilage, but was scarcely present in normal adult articular cartilage. In OA articular cartilage, BMP-2 mRNA and protein were detected in both clustering and individual chondrocytes in moderately or severely damaged OA cartilage. In moderately damaged OA cartilage, BMP-2 mRNA was localized in both upper and middle zone chondrocytes, but was not detected in deep layer chondrocytes. In severely damaged OA cartilage, cellular localization of BMP-2 mRNA was extended to the deep zone. In the area of osteophyte formation, BMP-2 mRNA was intensely localized in fibroblastic mesenchymal cells, fibrochondrocytes, chondrocytes and osteoblasts in newly formed osteophytic tissue. The pattern of BMP-2/4 immunolocalization was associated with that of mRNA localization. CONCLUSIONS: BMP-2 mRNA and BMP-2/4 were detected in cells appearing in OA tissues. BMP-2 was localized in cells of degenerating cartilage as well as osteophytic tissue. Given the negative localization of BMP-2 in normal adult articular cartilage, BMP-2 might be involved in the regenerating and anabolic activities of OA cells, which respond to cartilage damage occurring in osteoarthritis.

Abstract: OBJECTIVE: We recently identified a new protein, pituitary gland specific factor 1a (PGSF1a), that is specifically transcribed in the pituitary gland. In our investigation of anti-PGSF1a antibody for pituitary diseases, we examined it in patients with RA and other autoimmune diseases. We unexpectedly discovered the frequent existence of anti-PGSF1a antibody in patients with RA. We therefore examined the prevalence of this antibody to understand its clinical significance in RA. METHODS: Anti-PGSF1a antibody was detected by radioligand assay using recombinant (35)S-labelled PGSF1a protein. Antibody activity is expressed as an index that was obtained by comparison with normal pooled serum. RESULTS: RA patients had a significantly higher mean anti-PGSF1a antibody index (n=46, 1.28+/-0.38, P < 0.001) than healthy controls (n=36, 1.04+/-0.13). Indices greater than the cut-off value (mean+2 S.D. of healthy controls) were found in 43.5% (20/46) and 10.0% (2/20) of patients with RA and osteoarthritis, respectively. There was no correlation between the activities of anti-PGSF1a antibodies and titres of rheumatoid factor (RF) or serum C-reactive protein concentrations, but RA patients with more erosive disease had a higher mean anti-PGSF1a antibody index. Four of eight sera samples obtained from RF-negative RA patients were positive for anti-PGSF1a antibodies. CONCLUSION: Anti-PGSF1a antibody is a useful new marker for the diagnosis of RA, especially for RF-negative RA, and may relate to clinical manifestations of RA.

Abstract: Localization and expression of mRNAs for sonic hedgehog (Shh) at a fracture site in the early phase postfracture were investigated by in situ hybridization and reverse transcription and polymerase chain reaction (RT-PCR). A closed fracture was made in the midshaft of the right tibia of 5-week-old ICR mice, and fractured sites were harvested prefracture (day 0) and on days 2 and 12. In situ hybridization revealed that transcripts for Shh were not detected on day 0, but they were detected in proliferating callus-forming cells in the periosteum and the surrounding tissue, and in the medullary cavity prior to apparent new cartilage and bone formation. Gli 1 (a signaling mediator for Shh) and bone morphogenetic protein-4 transcripts were colocalized with those for Shh transcripts on day 2. The RT-PCR showed that Shh mRNA was detected in the PCR product from day 2, but not from days 0 and 12. These findings are the first description about the activation of Shh gene in the early postfracture reaction.

Abstract: The aim of this study was to elucidate the utility and limitation of gadopentetate (Gd)-enhanced MRI as a method for evaluating the anterior cruciate ligament (ACL) in the rheumatoid arthritis (RA) knee, using both surgical macro findings and histological findings to ascertain the pathological condition of the affected knee. Thirty-six knees of 25 RA patients were studied in this study. Four imaging protocols were employed: protocol A, T1-weighted and T2-weighted sagittal images; protocol B, T1-weighted sagittal image, after infusion of Gd-DTPA (0.2 mmol/kg, i.v.); protocol C, T1-weighted angled coronal image, parallel to the ACL; and protocol D, T1-weighted angled coronal image, parallel to the ACL, after infusion of Gd-DTPA. Sagittal image was determined as previously described. Angle coronal image was newly determined as coronal image parallel to the ACL. Surgical and MRI findings of the ACL were classified into four types: Type I (normal group) indicated that the thickness of the ACL was almost normal, adequate tension was maintained (surgical findings),and the ACL had thick and a more complex appearance with a homogeneous signal intensity and well-defined borders (MRI findings). Type II (degenerated group): the ACL had degenerated and tension was reduced (surgical findings), and the ACL had thin and a more complex appearance with a less homogeneous signal intensity and less well-defined borders. This appearance was more evident on Type II than Type I (MRI findings). Type III (ruptured group): the parenchyma of the ACL remained but lacked continuity (surgical findings), and the ACL appeared as partial lack of low signal intensity (MRI findings). Type IV (absent group): the parenchyma of the ACL was practically absent (surgical findings), and the ACL appeared as complete lack of signal low signal intensity (MRI findings). The concordance rate between surgical and MRI findings was investigated. Moreover, we investigated the extent to which histological changes of the ACL could be discriminated using MRI. In RA knees, the overall concordance rate between surgical and MRI findings was 41.7% under imaging protocol A. The overall rate improved up to 69.4% under imaging protocol B. But the overall rate dropped to 36.1% under imaging protocol C. The overall rate improved up to 83.3% under imaging protocol D. Especially, significant differences between imaging protocols A and B ( p<0.05), and imaging protocols C and D ( p<0.01), with respect to ACL degenerated group, were recognized. But significant differences between imaging protocols A and C, and imaging protocols B and D, with respect to ACL degenerated group, were not recognized. The concordance rate between histological and MRI findings was 41.7% in ACL normal group, and 61.5% in ACL degenerated group. The concordance rate between surgical and MRI findings was 100% in ACL normal group, and 78.9% in ACL degenerated group. There was a significant difference in the concordance rates between histological, surgical, and MRI findings in normal group ( p<0.05). The results of this study suggested that with Gd-enhanced MRI, the degree of synovial proliferation around the ACL and the degree of degradation of the ACL in the RA knee can be evaluated more accurately than with conventional MRI; however, in RA knees with severe synovial proliferation, it may be difficult to discriminate between the invasive synovium going into the ligament from synovium surrounding the ligament. This may be a limitation of Gd-enhanced MRI at present. In the clinical setting, the present imaging technique does allow the ligament to be evaluated to a certain degree, and may prove useful in the evaluation of temporal changes in the RA knee.

Abstract: We investigated the effects of treatment with anti-parathyroid hormone-related protein (1-34) monoclonal murine antibody (anti-PTHrP MoAb) on apoptosis and the differentiation of chondrosarcoma HTB-94 cells. Treatment with anti-PTHrP MoAb accelerated apoptosis of HTB-94 cells in a dose-dependent manner, and anti-PTHrP MoAb also promoted the chondrogenic differentiation of HTB-94 cells. The induction of apoptosis by anti-PTHrP MoAb via imbalance of Bcl-2/Bax ratio and activation of caspase-3 may provide a mechanistic explanation for its potential antitumor effects. Our results suggest the possibility that anti-PTHrP MoAb may be beneficial as a new treatment for chondrosarcoma.

Abstract: Recent progress in molecular biology has provided application of gene transfer methods in arthritis. Two clinical trails using ex vivo retrovirus mediated delivery of interleukin -1 receptor antagonist gene for rheumatoid arthritis has begun in USA and Germany. However, there are still many issues to be elucidated; one is the development of gene delivery system, and the other is the selection of therapeutic gene. Arthritis is nonlethal disease, and safety is one of the important issues. Currently viral mediated vectors are major even in clinical trials however, non viral efficient gene transfer system should be developed in future. Recently the application of DNA technologies, such as antisense oligonucleotide (ODN) strategies to regulate the transcription of disease-related genes in vivo, has significant therapeutic potential. Transfection of cis-clement double-stranded oligonucleotides (decoy ODN) for nuclear factor kappaB binding site has been reported as a new powerful tool in arthritis. The concept of regulation the disease related gene expression at the level of transcriptional factor may be more therapeutic effects compared with monotherapy in arthritis.

Abstract: OBJECTIVE: FR167653 is a potent inhibitor of p38 mitogen-activated protein kinase (MAPK) and inhibits tumor necrosis factor alpha (TNFalpha) and interleukin-1 beta (IL-1 beta) production in inflammatory cells. In this study we investigated the effect of FR167653 on collagen-induced arthritis (CIA). METHODS: Rats with CIA were subcutaneously injected with FR167653 (32 mg/kg/day) starting on the day of the booster injection (day 7) in the prophylactic treatment group and after the onset of arthritis (day 21) in the therapeutic treatment group. Hind-paw swelling, body weight, radiographic and histologic scores, and osteoclast number were evaluated. Cytokine levels in the serum and tissue were assessed by enzyme-linked immunosorbent assays. Flow cytometric analysis of T lymphocytes from bone marrow was performed. The effect of FR167653 on in vitro osteoclast formation induced by soluble receptor activator of nuclear factor kappa B ligand (sRANKL) and TNFalpha was examined. RESULTS: Swelling of hind paws and loss of weight occurred in the CIA rats, but this was not evident in the prophylactic treatment group. Therapeutic treatment also significantly reduced paw swelling. The mean radiographic and histologic scores as well as the osteoclast numbers were significantly lower in the treatment group than in the CIA rats without treatment. FR167653 treatment reduced the serum levels of TNFalpha and IL-1 beta, lowered the IL-1 beta concentration in the ankle joints, and decreased the CD4-,CD8a+ T cell population in bone marrow. Furthermore, FR167653 inhibited the osteoclast-like cell differentiation induced by both sRANKL and TNFalpha in vitro. CONCLUSION: FR167653 prevents the onset of arthritis in a prophylactic treatment model and suppresses the progression of joint destruction in a therapeutic treatment model, suggesting that p38 MAPK is a potential therapeutic target for rheumatoid arthritis.

Abstract: Recent progress in molecular biology has provided new techniques with which to inhibit target gene expression. In particular, the application of DNA technologies, such as antisense oligonucleotide (ODN) strategies to regulate the transcription of disease-related genes in vivo, have significant therapeutic potential. Recently, transfection of cis-element double-stranded oligonucleotides (decoy ODNs) has been reported as a new powerful tool in a new class of anti-gene strategies for gene therapy. Transfection of double-stranded ODN corresponding to the cis sequence will result in attenuation of the authentic cis-trans interaction, leading to removal of trans-factors from the endogenous cis-elements with subsequent modulation of gene expression.

Abstract: Localization and expression of cartilage-derived morphogenetic protein-1 in tissues of torn rotator cuff tendons were examined by in situ hybridization and immunohistochemical analysis. Histologic findings of torn rotator cuff tendons showed that active cells synthesizing the alpha-1 chain of collagen Type I messenger ribonucleic acid were localized predominantly in the torn edge and in the bursa side rather than in the joint side, and scarcely localized in a site distant from the torn edge. Cartilage-derived morphogenetic protein-1 had a similar distribution as the alpha-1 chain of collagen Type I. The current findings provide the first observational evidence that cartilage-derived morphogenetic protein-1 was activated specifically at the site of the torn rotator cuff tendon. The current findings suggest that the cells in the torn rotator cuffs are capable of synthesizing cartilage-derived morphogenetic protein-1, one of the known essential factors for tendon formation.

Abstract: Calcium hydroxyapatite ceramics (CHA) are nontoxic materials, provoke little reaction from tissues, and by virtue of these properties represent a good starting point for creating bone substitutes. Although several porous CHAs have been used clinically, there have been few reports that CHA is fully replaced by newly formed bone, which may be due to its structure and the limited connectivity between pores. We recently developed a fully interconnected porous CHA (IP-CHA) by adopting a "foam-gel" technique. Structural analysis by scanning electron microscopy revealed that IP-CHA had spherical pores of uniform size that were interconnected by window-like holes. The surface of the wall structure was smooth, and hydroxyapatite particles were bound tightly to one another. Most of the interpore connections of IP-CHA ranged from 10 to 80 microm in diameter (average, 40 microm). When the cylindrical IP-CHA (diameter, 6 mm; height, 15 mm) was implanted into a rabbit femoral condyle, bone, and bone marrow with abundant vessels formed deep in the pores through the interpore connections. Within a period of 6 weeks, new bone had formed and penetrated to a distance of 3 mm from the surface of the IP-CHA implant. Furthermore, a compression test at 9 weeks revealed that the implanted IP-CHA steadily increased in strength to more than double the value of the initial test. These results indicate that the IP-CHA may have clinical utility as a superior bone substitute.

Abstract: OBJECTIVE: To investigate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) at sites of joint destruction in rheumatoid arthritis (RA) and to correlate it with the production of matrix metalloproteinases (MMPs). METHODS: Reverse transcription-polymerase chain reaction was performed to study the existence of EMMPRIN in synovial tissue derived from RA and osteoarthritis (OA) patients. In situ hybridization with a human complementary DNA specific for EMMPRIN and immunohistochemistry were performed to characterize the EMMPRIN-expressing cells at sites of joint destruction, including bone. Northern blot analysis was performed to detect the level of expression of EMMPRIN messenger RNA (mRNA) in synovial tissue. The production of MMP-1 and MMP-3 by synovial tissue from RA patients was examined by enzyme-linked immunosorbent assay. RESULTS: Expression of EMMPRIN mRNA was detected in synovium from 9 of 11 patients with RA and 1 of 5 patients with OA. The presence of mRNA encoding EMMPRIN was recognized in the invasive synovium at sites of joint destruction in RA but not OA. Fibroblast-like synovial cells and granulocytes were demonstrated to express EMMPRIN mRNA. MMP-1 and MMP-3 production by synovial tissue was correlated with levels of expression of EMMPRIN mRNA, as detected by Northern blotting. CONCLUSION: The expression of EMMPRIN stimulates the production of MMP-1 and MMP-3 in the synovial tissue of affected joints in RA. The results of this study suggest that EMMPRIN may be one of the important factors in progressive joint destruction in RA.

Abstract: OBJECT: Little is known about the molecular mechanisms underlying the process of spondylosis. The authors determined the extent of genetic localization of major regulators of chondrogenesis such as Indian hedgehog (Ihh) and parathyroid hormone (PTH)-related peptide (PTHrP) and their receptors during the development of spondylosis in their previously established experimental mouse model. METHODS: Experimental spondylosis was induced in 5-week-old ICR mice. The cervical spines were chronologically harvested, and histological sections were prepared. Messenger (m) RNA for PTHrP, Ihh, PTH receptor (PTHR; a receptor for PTHrP), patched (Ptc; a receptor for Ihh), bone morphogenetic protein (BMP)-6, and collagen type X (COL10; a marker for mature chondrocyte) was localized in the tissue sections by performing in situ hybridization. In the early stage, mRNA for COL10, Ihh, and BMP-6 was absent; however, mRNA for PTHrP, PTHR, and Ptc was detected in the anterior margin of the cervical discs. In the late stage, evidence of COL10 mRNA began to be detected, and transcripts for Ihh, PTHrP, and BMP-6 were localized in hypertrophic chondrocytes adjacent to the bone-forming area in osteophyte. Messenger RNA for Ptc and PTHR continued to localize at this stage. In control mice, expression of these genes was absent. CONCLUSIONS: The localization of PTHrP, Ihh, BMP-6, and the receptors PTHR and Ptc demonstrated in the present experimental model indicates the possible involvement of molecular signaling by PTHrP (through the PTHR), Ihh (through the Ptc), and BMP-6 in the regulation of chondrocyte maturation leading to endochondral ossification in spondylosis.

Abstract: Abnormalities of the epiphyseal growth plate that occur in collagen-induced arthritis (CIA) were studied. CIA was induced in 6-week-old Lewis rats by immunization with type II collagen. Radiographic examination revealed the early closure of the epiphyseal growth plate with growth retardation of the femur and tibia. Histological evaluation confirmed the early closure of the epiphyseal growth plate accompanied by decreased intensity of safranin-O staining indicating decreased amounts of proteoglycans in the extracellular matrix (ECM) of the cartilage. Immunohistochemical methods showed that the number of chondrocytes expressing matrix metalloproteinase (MMP)-3 and/or vascular endothelial growth factor (VEGF) increased in the growth plates of CIA rats. This study confirmed that disturbances of long bone growth with early closure of the epiphyseal growth plates occur in CIA. There appeared to be overexpression of MMP-3, which may be involved with proteoglycan degradation. Additionally, VEGF, which is associated with cartilage ossification and angiogenesis, might also play a role in this event. Further clarification of the mechanism of the growth disturbance in CIA may yield clinical benefits, especially in prevention of the premature closure of growth plate that is seen in juvenile rheumatoid arthritis and other diseases.

Abstract: Recent studies have suggested the involvement of bone marrow in the pathogenesis of rheumatoid arthritis (RA), in which proliferation of monocyte-lineage cells (MLC) as well as local B cell activation in the synovium play an important role. Here, we show that bone marrow-derived MLC have the capacity to activate human peripheral blood IgD- B cells. Bone marrow CD34+ cells from RA patients that had been stimulated with stem cell factor and GM-CSF for 3-4 weeks (>90% CD14+ HLA-DR+ cells, <0.5% CD19+ B cells, and <0.5% CD3+ T cells; MLC) induced the production of IgG much more effectively than that of IgM by highly purified B cells from healthy donors in the presence of IL-2 and IL-10. CD34+ cells from cord blood or from bone marrow of osteoarthritis patients also displayed the capacity to induce IgG production. The induction of IgG production by the bone marrow-derived MLC was markedly decreased when they were separated from B cells by a membrane filter. The bone marrow-derived MLC interacted preferentially with IgD- B cells to induce IgG production. These results indicate that upon stimulation with stem cell factor and GM-CSF, CD34+ progenitor cells differentiate into MLC that activate preferentially IgD- B cells through direct cellular interactions to produce IgG. Therefore, the data suggest that the accelerated recruitment of MLC from the bone marrow to the synovium might play a role in the local B cell activation in RA.

Abstract: Periprosthetic fracture of the tibial plateau associated with osteolysis resulting from mechanical failure of the rotating patellar component after total knee arthroplasty with the New Jersey Low-Contact-Stress (LCS) knee (DePuy, Warsaw, IN) has not been reported previously. A 67-year-old woman with rheumatoid arthritis of the left knee had a LCS prosthesis implanted without cement, using a rotating patellar component. Seven years later, a fracture of the lateral tibial plateau occurred owing to an osteolytic defect with no traumatic accident. The rotating patellar bearing over-rotated and locked; consequently, wear occurred between the patellar metal tray and the femoral component. Immunohistochemistry revealed CD68-positive macrophages in the osteolytic region and phagocytosis of metal particles. The osteolytic region was filled with autogenous bone, and all components were exchanged and cemented. The patient's condition became satisfactory with relief of pain.

Abstract: Nurse-like stromal cell lines from the synovial tissue of patients with rheumatoid arthritis (RA-SNC) produce, on coculture with lymphocytes, large amounts of proinflammatory cytokines. In the present paper, we analyze the molecular events necessary for the induction of cytokine release from RA-SNC cells, and particularly the roles played by cell adhesion and the transmigration (also known as pseudoemperipolesis) of lymphocytes. For this purpose, the effects of various mAbs on the binding and transmigration of a human B-cell line, MC/car, were examined using a cloned RA-SNC line, RA-SNC77. To analyze the role of lymphocyte binding and transmigration on upregulated cytokine production by the RA-SNC77 cells, we used C3 exoenzyme-treated MC/car cells, which could bind to RA-SNC77 cells but could not transmigrate. Treatment with anti-CD29 or anti-CD49d mAb significantly reduced binding and transmigration of the MC/car cells. In contrast, the neutralizing anti-CD106/vascular cell adhesion molecule 1 mAb did not show any inhibitory effect. Likewise, none of the neutralizing mAbs against CD11a, CD18, CD44, CD49e, or CD54 showed significant effects. Binding of C3-treated or untreated MC/car cells to RA-SNC77 cells induced comparable levels of IL-6 and IL-8 production. In addition, the enhanced cytokine production by RA-SNC77 cells required direct lymphocyte contact via a very late antigen-4 (VLA-4)-independent adhesion pathway. These results indicate that, although both the VLA-4-dependent/vascular cell adhesion molecule 1-independent and the VLA4-independent adhesion pathways are involved in MC/car binding and subsequent transmigration, only the VLA4-independent adhesion pathway is necessary and sufficient for the enhanced proinflammatory cytokine production by RA-SNC77 cells. The transmigration process, which is dependent on Rho-GTPase, is not a prerequisite for this phenomenon.

Abstract: OBJECT: Because little is known about the molecular mechanisms underlying the process of spondylosis, the authors examined the extent of genetic localization of several members of bone morphogenetic protein (BMP) and BMP receptors in chondrogenesis during the process of inducing spondylosis in their previously established experimental mice model. METHODS: Experimental spondylosis was induced in 5-week-old ICR mice. The cervical spine was harvested chronologically, and histological sections were prepared. Messenger RNA for BMP-4, growth and differentiation (GDF)-5, BMP-6, and BMP receptors (ALK-3, -6, and BMP-RII) was localized in the tissue sections by in situ hybridization. In the early stage, BMP-4-derived mRNA was localized mainly in cells in the anterior margin of the cervical discs, together with ALK-6 and BMP-RII mRNA. No GDF-5 and BMP-6 mRNA was detected at this stage. In the late stage, cells positive for BMP-4 decreased, whereas GDF-5 and BMP-6 mRNA were localized in cells undergoing chondrogenesis. The ALK-3 mRNA began to appear in this stage, as did ALK-6 and BMP-RII. CONCLUSIONS: The localization of transcripts for BMP-4, -6, and GDF-5 as well as BMP receptors shown during the present experimental model indicate the possible involvement of molecular signaling by these BMPs in the chondrogenic progress in spondylosis.

Abstract: In iliac bone marrow the absolute number of mononuclear cells (MNCs) was increased in RA patients compared with the non-RA controls. In CD8 positive cell and myeloid cell fractions, significant differences were recognized between RA patients and non-RA controls. The presence of abnormal myeloid lineage cells in epiphyseal bone marrow adjacent to joints affected with severe RA was shown. Stroma cell lines from RA bone marrow with nursing activity were established and shown to play a pivotal role in the pathogenesis in RA bone marrow. Histologic study also shows that subchondral region expressing tissue-damaging proteinases plays an important role in joint destruction in RA.

Abstract: OBJECTIVE: We previously described abnormalities in the bone marrow of patients with rheumatoid arthritis (RA), but were able to shed little light on the pathogenic roles of inflammatory cytokines and proteinases in joint destruction in the subchondral region in RA. This is the first report to describe the co-localization of cytokines and proteinases in this area. METHODS: Decalcified paraffin-embedded sections from 10 patients with RA and five patients with osteoarthritis (OA) were examined for the immunolocalization of cathepsins B, K and L and the localization of messenger RNAs for interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha) and matrix metalloproteinase 9 (MMP-9). The cells were double-stained with anti-CD68 or anti-prolyl 4-hydroxylase (PH) antibody. RESULTS: An immunohistochemical study confirmed the expression of cathepsins B and L by CD68-positive mononuclear cells at the sites of significant cartilage and bone erosion from the subchondral region in all RA specimens. Osteoclast-like cells showed intense staining for cathepsin K and MMP-9. Osteoblast-like cells strongly expressed MMP-9. Analysis of serial sections revealed that expression of the IL-1beta and TNF-alpha genes occurred near that of the cathepsins and MMP-9 in the subchondral region. CONCLUSION: We conclude that inflammatory cytokines and tissue-damaging proteinases play important roles in joint destruction in the subchondral region in RA.

Abstract: A unique feature in inflammatory tissue of rheumatoid arthritis (RA) is the formation of ectopic lymphoid aggregates with germinal center (GC)-like structures that can be considered to contribute to the pathogenesis of RA, because local production of the autoantibody, rheumatoid factor, is thought to be a causative factor in tissue damage. However, the factors governing the formation of GC in RA are presently unknown. To begin to address this, the expression of B cell attracting chemokine (BCA-1) (CXCL13), a potent chemoattractant of B cells, was examined in the synovium of patients with RA or with osteoarthritis (OA). Expression of BCA-1 mRNA was detected in all RA samples, but in only one of five OA samples. Lymphoid follicles were observed in four of seven RA samples and in two of eight OA samples, and in most of them BCA-1 protein was detected in GC. BCA-1 was not detected in tissues lacking lymphoid follicles. Notably, BCA-1 was detected predominantly in follicular dendritic cells in GC. CD20-positive B cells were aggregated in regions of BCA-1 expression, but not T cells or macrophages. These data suggest that BCA-1 produced by follicular dendritic cells may attract B cells and contribute to the formation of GC-like structures in chronic arthritis.

Abstract: To assess the role of bone marrow in the pathogenesis of rheumatoid arthritis (RA), we examined the capacity of CD34(+) cells from bone marrow to generate fibroblast-like type B synoviocytes. CD34(+) cells from the bone marrow of 22 RA patients differentiated into cells with fibroblast-like morphology, which expressed prolyl 4-hydroxylase, in the presence of stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha), much more effectively than CD34(+) cells from bone marrow of 15 control subjects (10 patients with osteoarthritis and 5 healthy individuals). The generation of fibroblast-like cells was not at all observed in cultures with SCF, GM-CSF, and interleukin 4 (IL-4) with or without TNF-alpha. Generation of fibroblast-like cells was correlated with matrix metalloproteinase (MMP)-1 levels in culture supernatants. Thus, MMP-1 levels were significantly higher in TNF-alpha-stimulated cultures of bone marrow CD34(+) cells from patients with RA than in those from the control group. These results indicate that bone marrow CD34(+) cells from patients with RA have abnormal capacities to respond to TNF-alpha and to differentiate into fibroblast-like cells producing MMP-1, suggesting that bone marrow CD34(+) progenitor cells might generate type B synoviocytes and thus could play an important role in the pathogenesis of RA.

Abstract: This study describes the distributions of bone morphogenetic protein (BMP)-2 as well as mRNAs for BMP receptor type IB (BMPRIB). collagen types II (Col II) and III (Col III) in a growing "cartilage cap" of osteochondroma. In situ hybridization and immunohistochemical study were performed using histological sections obtained during surgery. BMP-2 was detected in mesenchymal cells in the outer fibrous layer and chondrocytes in the inner cartilaginous matrix, positive for Col III and Col II, respectively. BMPRIB mRNA was distributed in chondrocytes. This is the first study to provide observational evidence of the involvement of BMP-2 signaling in the pathogenesis of cartilage cap of osteochondroma. and suggests the role of BMP-2 in the growth of cartilage cap in osteochondroma.

Abstract: Calcifying tendinitis of rotator cuff tendons is a common and painful condition caused by ectopic calcification in humans. To examine the involvement of osteopontin (OPN), a potent regulator of calcium deposition on connective tissues, localization and expression of OPN protein and messenger (m)RNA were investigated in human tissue samples of calcified rotator cuff tendons. Immunohistochemistry demonstrated that OPN was localized in cells surrounding the calcified area. OPN was localized in two distinct cell types, i.e., fibroblast-like cells negative for CD68 and tartrate-resistant acid phosphatase (TRAP) and multinucleated macrophages positive for CD68 and TRAP. In situ hybridization revealed that the mRNA expression of OPN in these cells coincided with the immunohistochemistry results, and these results were supported by reverse transcriptase polymerase chain reaction analysis using human OPN-specific oligonucleotides. Cells located away from the calcified area did not express OPN. The present findings indicate the involvement of OPN in the process of calcification of rotator cuff tendons and suggest that OPN plays a role in such painful disorders through the actions of at least two cell types.

Abstract: Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (Ihh), and patched (Ptc; a receptor for Ihh) were immunolocalized in tissue undergoing endochondral ossification in the human. PTHrP, Ihh, and Ptc were immunolocalized in prehypertrophic and hypertrophic chondrocytes in mature cartilage matrix. PTHrP and Ptc were immunostained in proliferating chondrocytes and perichondrial cells, whereas Ihh was not. PTHrP, Ihh, and Ptc showed positive immunostaining in osteoblasts in the bone-forming area. In the bone resorption site, PTHrP was immunolocalized in osteoclasts, whereas Ihh and Ptc were not. The present findings indicated that PTHrP, Ihh, and Ptc were associated with the process of endochondral ossification, and suggested the possible involvement of Ihh and PTHrP signaling in the regulation of proliferation and hypertrophy of chondrocytes in human chondrogenesis.

Abstract: Bone resorption in the joints is the characteristic finding in patients with rheumatoid arthritis (RA). Osteoclast-like cells are present in the synovial tissues and invade the bone of patients with RA. The characteristics of these cells are not completely known. In the work reported here, we generated these cells from peripheral-blood monocytes from healthy individuals. The monocytes were co-cultured with nurse-like cells from synovial tissues of patients with RA (RA-NLCs). Within 5 weeks of culture, the monocytes were activated and differentiated into mononuclear cells positive for CD14 and tartrate-resistant acid phosphatase (TRAP). These mononuclear cells then differentiated into multinucleated giant bone-resorbing cells after stimulation with IL-3, IL-5, IL-7, and/or granulocyte-macrophage-colony-stimulating factor. TRAP-positive cells with similar characteristics were found in synovial fluid from patients with RA. These results indicate that multinucleated giant bone-resorbing cells are generated from monocytes in two steps: first, RA-NLCs induce monocytes to differentiate into TRAP-positive mononuclear cells, which are then induced by cytokines to differentiate into multinucleated giant bone-resorbing cells.

Abstract: IL-6 and tumor necrosis factor (TNF)-alpha have been proven to play an important role in the development of rheumatoid arthritis (RA). It is well known that TNF-alpha induces IL-6 production from synovial cells as well as their proliferation. The effect of IL-6 on synovial cells, however, is not clear. An in vitrostudy was performed to determine the effect of IL-6 on the proliferation of synovial cells. Fibroblastic synovial cells isolated from the synovial tissues of eight RA patients were employed after the third to sixth passages. IL-6 in the presence of soluble IL-6 receptor (sIL-6R) inhibited the proliferation of synovial cells in a dose-dependent manner in seven cases without increasing the number of necrotic or apoptotic cells, while TNF-alpha increased synovial cell proliferation in all cases. The inhibitory effect of IL-6 was observed only in the presence of sIL-6R although small amounts of IL-6R were detected in these cells by RT-PCR analysis. However, anti-IL-6R or anti-gp130 mAb treatment increased spontaneous growth of synovial cells in all eight cases, suggesting that endogenous IL-6 and a small amount of IL-6R expressed in synovial cells suppressed their growth without exogenous IL-6 or sIL-6R. In addition, the IL-6-sIL-6R complex reduced the TNF-alpha-induced proliferation of synovial cells while TNF-alpha induced their IL-6 production. These data suggest that IL-6 may act as a negative feedback factor for TNF-alpha-induced synovial cell growth.

Abstract: We show that bone marrow (BM) CD34+ progenitor cells from rheumatoid arthritis (RA) patients have the capacity to support spontaneous transformation of peripheral blood B cells. CD34+ cells purified from BM blood from eight RA patients and eight osteoarthritis (OA) patients were expanded with granulocyte/macrophage colony stimulating factor (GM-CSF) for 4-6 weeks. GM-CSF-stimulated BM CD34+ cells from three of eight RA patients, but none from seven OA patients, gave rise to spontaneous transformation of highly purified B cells of Epstein-Barr virus (EBV)-seronegative healthy donors. GM-CSF-stimulated BM CD34+ cells from four of six RA patients and from one of four OA patients also supported the spontaneous transformation of peripheral blood B cells from EBV-seropositive healthy donors. All the transformed B cell lines were positive for EBV-DNA as determined by PCR. Neither GM-CSF-stimulated BM CD34+ cells alone nor highly purified B cells alone gave rise to spontaneously transformed B cell lines. These results suggest that the capacity of BM CD34+ cells to support survival of B cells might contribute to the pathogenesis of RA by sustaining abnormal B cell responses.

Abstract: OBJECTIVE: To investigate foot deformities in rheumatoid arthritis (RA) in relation to the disease severity. METHODS: Radiographs of 100 weight bearing feet of 50 patients who had had RA for >10 years (mean 13.5 years) were studied. The patients were classified into 2 study groups according to the severity of disease. We measured hallux valgus angle (HVA), intermetatarsal angle between first and 2nd (M1/2), and intermetatarsal angle between first and 5th (M1/5) on anteroposterior (AP) radiographs, as well as calcaneal pitch (CP) and first metatarsal pitch (MP) on lateral radiographs. The differences in these angles between the 2 groups (Inter-group study) and the correlations among angles within each group (Intra-group study) were examined. RESULTS: Inter-group study showed significant differences between the 2 groups for all variables. Intra-group study, on the other hand, showed no correlation between variables of the 2 deformities, i.e., splaying of forefoot (M1/2 and M1/5) and flattening of longitudinal arch (CP and MP). Only HVA correlated with the splaying (M1/2 and M1/5) in both study groups. CONCLUSION: Disease severity is related to the progression of foot deformities in RA, but the flattening and the splaying are not correlated with each other. We believe that foot deformities should be treated properly and early, especially for patients who are expected to have severe disease.

Abstract: The authors evaluated a modified Lapidus technique for 21 rheumatoid hallux valgus deformities. The technique corrects the deformity by performing arthrodesis of the first tarsometatarsal joint and preservation of the first metatarsophalangeal (MTP) joint. The authors clinically studied patients' subjective improvement of pain and footwear comfort, as well as their satisfaction with the outcome of the surgery. The study also analyzed radiographic changes of the hallux valgus angle (HVA) and two intermetatarsal angles, one between the first and the second (M1/2) and the other between the first and the fifth (M1/5). They were measured before the surgery, 3 weeks after the surgery, and at the last follow-up. Pain relief was great or moderate in 17 feet and footwear comfort was improved in 16 feet. Fifteen patients were satisfied or satisfied with some reservations. The average HVA significantly decreased from 44.1 degrees preoperatively to 10.6 postoperatively and significantly increased again to 29.1 at the last follow-up. The average M1/2 and M1/5 significantly decreased postoperatively (from 13 to 8.3 and from 32.2 to 21.1, respectively), and the reduction of M1/2 remained at the last follow-up (8.7), while M1/5 significantly increased again (28.3). This modified Lapidus technique is a useful method for rheumatoid hallux valgus deformity, which can preserve the first MTP joint.

Abstract: OBJECTIVE: Numerous cytokines are expressed in lesions of synovial hyperplasia of patients with rheumatoid arthritis (RA), and their pathophysiological contributions have been the subject of speculation. These genes are regulated by the transcription factor NFkappaB which in turn is activated by tumour necrosis factor-alpha (TNF-alpha) and cytokines. In this study we examined the inhibition of the production of pro-inflammatory cytokines, adhesion molecule and matrix metalloproteinase (MMP) from synovial tissue of patients with RA by the introduction of synthetic double-stranded DNA with high affinity for the NFkappaB binding site. METHOD: NFkappaB decoy oligonucleotides (ODN) were introduced with the aid of the haemagglutinating virus of Japan (HVJ)-liposome method into synovial tissue or synovial cells derived from patients with RA. The levels of interleukin-1beta (IL-1beta), IL-6, TNF-alpha, intercellular adhesion molecule-1 (ICAM-1) and MMP-1 were determined by means of enzyme-linked immunosorbent assay (ELISA) and Northern blotting analysis. A cell counting kit was used to study the effect of NFkappaB decoy ODN on synovial cell proliferation. RESULTS: The production of these mediators was significantly inhibited by the introduction of NFkappaB decoy ODN compared with the effect of scrambled decoy ODN. Transfection of NFkappaB decoy ODN resulted in a significant inhibition of synovial cell proliferation as compared with that of scrambled decoy ODN. CONCLUSION: The results demonstrated in this study suggest the potential usefulness of NFkappaB decoy ODN for gene therapy of inflammatory synovitis of RA.

Abstract: OBJECTIVES: Calcified tendinitis of the shoulder joint is a common painful condition. Resorption of the calcium deposits is one of the key events in the pathogenesis of this disease. The aim of this study was to examine whether the multinucleated giant cells that appear in this condition have osteoclast phenotypes. METHODS: Immunohistochemical and RNA in situ hybridization analysis of cathepsin K, a marker for osteoclasts, was performed in human surgical samples. RESULTS: The multinucleated cells located near the calcium deposits were positive for cathepsin K protein and mRNA. Reverse transcription-polymerase chain reaction using human cathepsin K-specific oligonucleotide primers confirmed that synthesis of cathepsin K mRNA occurs in the tissues of calcified rotator cuffs. CONCLUSION: The multinucleated giant cells which appear in the resorption area of calcium deposits in calcified tendinitis have the osteoclast phenotype.

Abstract: Cathepsins D, K, and L were immunolocalized in tissue undergoing endochondral ossification in the human. Cathepsins D, K, and L were localized in osteoclasts and chondroclasts attached to bone matrix and cartilage matrix, respectively. Cathepsins D and L were immunostained in chondrocytes. Immunolocalization of cathepsin D was limited to hypertrophic chondrocytes adjacent to the osteochondral junction. In contrast, cathepsin L was immunolocalized in both proliferating and hypertrophic chondrocytes. In the bone marrow space, cathepsins D, K, and L were localized in multinucleated cells. Cathepsin D was diffusely detected in mononuclear bone marrow cells which were negative for cathepsins K and L. The present findings indicated that cathepsins K, D, and L were associated with the process of endochondral ossification in the human, and suggested that these cathepsins share roles in bone and cartilage turnover in the human.

Abstract: BACKGROUND: Manipulation of ligament healing has been a major focus of orthopaedic research. In recent years, gene transfer to healing ligament appears to be a feasible method for manipulating the healing process. In this study, we investigated the feasibility of gene transfer to healing rat patellar ligament by intra-arterial delivery. METHODS: An attempt was made to transfer a reporter gene (Escherichia coli, beta-galactosidase gene) to healing rat patellar ligament using the haemagglutinating virus of Japan (HVJ) liposome-mediated gene transfer method. Three days after cutting the patellar tendons of 25 14-week-old male Wistar rats, HVJ-liposome complexes containing beta-galactosidase (beta-gal) cDNA were injected into the femoral artery of 15 Wistar rats as the experimental group. HVJ liposomes without DNA were injected into the femoral artery of 10 Wistar rats as the control group. Three rats from the experimental group and two control rats were killed 3, 7, 14, 28 and 56 days after the injection. RESULTS: After X-gal staining, the rate of transfection in the experimental group (mean +/- SEM) was found to be 12.1% +/- 0.590%, 8.7% +/- 0.217%, 10.2% +/- 0.227%, 3.2% +/- 0.247% and 0.7% +/- 0.060% at post-injection days 3, 7, 14, 28 and 56 respectively. In control sections the number of blue-stained cells were very few at any point. CONCLUSION: We succeeded in introducing a reporter gene into healing rat patellar ligament by infra-arterial delivery of HVJ-liposome complexes. This method appears to have the potential to be applicable for soft-tissue healing studies and also healing studies of other tissues and organs.

Abstract: OBJECTIVE: To investigate the features of synovial stromal cells established from patients with rheumatoid arthritis (RA), and to define these cells as nurse cells. METHODS: Synovial nurse-like stromal cell lines (RA-SNCs) were established from patients with RA. These cell lines were examined for morphology, pseudoemperipolesis activity, cell surface markers, and cytokine production. The interaction between these RA-SNCs and a synovial tissue B cell clone was also examined. RESULTS: RA-SNCs had nurse cell activity. They spontaneously produced interleukin-6 (IL-6), IL-8, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Furthermore, they produced IL-1beta and tumor necrosis factor alpha and expressed higher levels of the other cytokines after coculture with the B cell clone. Proliferation and Ig production by the B cell clone were dependent on direct contact with RA-SNCs. CONCLUSION: These results indicate that the RA-SNCs were nurse cells. The findings suggest that RA-SNCs may play an important role in the pathogenesis of RA by producing large amounts of cytokines and maintaining infiltrating lymphocytes.

Abstract:

Abstract: An adequate supply of peripheral blood monocytes, granulocytes, and platelets is necessary for an optimal inflammatory process. We have previously demonstrated that the generation of CD14(+) monocyte lineage cells from the bone marrow is accelerated in patients with rheumatoid arthritis (RA). The current studies examined the influences of methotrexate (MTX), a potent disease modifying antirheumatic drug (DMARD), on the capacity of bone marrow progenitor cells to generate CD14(+) cells in patients with RA, in order to delineate its mechanism of action. CD14(-) cells purified from bone marrow specimens of 14 patients with active RA were cultured in the presence or the absence of pharmacologically attainable concentrations of MTX (0.2 microM). After incubation for 14 days, the cells were analyzed by flow cytometry for expression of CD14 and HLA-DR. The generation of CD14(+) cells from RA bone marrow CD14(-) progenitor cells was significantly suppressed by MTX. However, the expression of HLA-DR on bone marrow-derived CD14(+) cells was not significantly influenced by MTX. There was no significant difference in the effect of MTX on the generation of CD14(+) cells between patients with prednisolone and those without prednisolone. The production of IL-12 in bone marrow cell cultures was not inhibited, but was rather enhanced, by MTX, suggesting that the suppression of the generation of CD14(+) cells might not be due to the inhibition of cytokine production. The results are consistent with the hypothesis that one of the effects of DMARDs may involve the interference with monocyte differentiation in the bone marrow. Moreover, the data suggest that the generation of CD14(+) cells and the expression of HLA-DR on such marrow-derived CD14(+) cells are regulated by different mechanisms.

Abstract: OBJECTIVE: To investigate the microenvironment of bone marrow (BM) of patients with rheumatoid arthritis (RA). METHODS: Nurse cell-like BM stromal cell lines were established from BM mononuclear cells of patients with RA. We examined the various characteristics of these cell lines, including morphology, pseudoemperipolesis activity, cell surface markers, cytokine production and hyaluronan (HA) production. RESULTS: These RA BM nurse cell-like lines (RA-BMNC) were of mesenchymal origin and positive for CD44, CD54 and HLA-DR. They were defined as nurse cells because of pseudoemperipolesis activity that allowed lymphocytes to migrate underneath. RA-BMNC lines produced HA and multiple cytokines including interleukin (IL)-6, IL-7, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF). HA production by BM stromal cells was correlated with pseudoemperipolesis activity. RA-BMNC produced significantly higher levels of IL-6, IL-8 and GM-CSF by co-culture with lymphocytes. The cells also produced IL-1beta, G-CSF and tumour necrosis factor only when co-cultured with lymphocytes. The RA-BMNC maintained the growth of CD14+ myeloid cells unique to severe RA. CONCLUSION: The present results both indicate that RA-BMNC are nurse cells and suggest that they may play an important role in the pathogenesis of RA.

Abstract: OBJECTIVE: In both rheumatoid arthritis and collagen-induced arthritis (CIA), the nuclear factor kappaB (NF-kappaB) transcription factor plays a pivotal role in the coordinated transactivation of many cytokines related to pathogenesis. This study investigated whether synthetic double-stranded DNA that show a high affinity for NF-kappaB could be introduced in vivo as "decoy" cis elements to bind the transcription factor and block the activation of such proinflammatory cytokine genes as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha), and thus suppress the severity of joint destruction. METHODS: NF-kappaB decoy oligonucleotides (ODN) were introduced by an intraarticular injection into the bilateral hind ankle joints of CIA rats using the hemagglutinating virus of Japan (HVJ)-liposome method. Joint destruction was evaluated by histology and radiography. IL-1 and TNFalpha levels were assessed by enzyme-linked immunosorbent assay and Northern blot analysis. RESULTS: Using the HVJ-liposome method, the presence of fluorescein isothiocyanate-labeled ODN in the synovium was confirmed until 28 days after intraarticular injection. In vivo transfection of NF-kappaB decoy ODN by an intraarticular injection into CIA rats decreased the severity of hind-paw swelling. Histologic and radiographic studies showed a marked suppression of joint destruction treated by NF-kappaB decoy ODN transfection. This treatment method also suppressed the production of IL-1 and TNFalpha in the synovium of arthritic joints. CONCLUSION: The present results demonstrate that administration of NF-kappaB decoy ODN in arthritic joints of rats with CIA led to an amelioration of arthritis. These findings suggest that intraarticular transfection of NF-kappaB decoy ODN may provide a useful therapeutic approach for the treatment of inflammatory arthritis.

Abstract: Polymorphisms in the 5'-flanking promoter/enhancer region of the tumor necrosis factor (TNF)-a gene were examined to study the genetic background of rheumatoid arthritis (RA). Four variant alleles, -1,031C/ -863A, -1,031C/-238A, -857T and -308A, were identified and examined in 387 RA patients and 575 healthy Japanese controls. The frequency of the -857T allele in RA patients was significantly higher than that in the controls. However, the HLA-DRB1 analysis in the same subjects showed that the DRB1*0405 allele, which is in linkage disequilibrium with the -857T, was more strongly associated with the disease susceptibility than the -857T allele. These results suggest that the susceptible gene to RA is more closely linked to the HLA-DRB1 locus than to the TNF-alpha locus.

Abstract: In situ hybridization histochemistry is the sole tool available for detecting the localization and expression of specific RNA on histological sections under various in vivo conditions. For this paper, we examined the effect of microwave exposure on the time needed for decalcification of skeletal tissues and on the preservation of sensitivity for hybridization signals. Our data show that the use of microwave decalcification reduces the decalcification period while preserving intense hybridization signals for mouse alpha1 chain of procollagen type I mRNA in osteogenic cells in bone. The use of microwave treatment to decalcify skeletal tissues may prevent delay in obtaining experimental results or the loss of signals during in situ hybridization.

Abstract: OBJECTIVE. To investigate the rheumatoid arthritis (RA)-specific autoantigen(s) recognized by CD4+ T cells in patients with RA. METHODS. CD4+,CD45RO+ T cell clones were established from the joints of RA patients, and were examined for their proliferative response to synovial cells. RESULTS. Eight of 146 T cell clones responded to RA synovial cells in a DR-restricted manner. These T cell clones recognized solubilized antigens extracted from RA synovial cells in the presence of DR-matched antigen-presenting cells, but did not respond to those extracted from non-RA synovial cells. The antigens had a molecular weight of 50/25 kd. Five of the 8 T cell clones used T cell receptor BV6, and the remaining clones used BV12.2. CONCLUSION. The antigens recognized by joint-infiltrating CD4+ T cells are present exclusively in RA synovial cells. The expression of these antigens by synovial cells may trigger the autoreactivity of T cells in RA joints.

Abstract: OBJECTIVE: To investigate the expression and function of Fas ligand (FasL),which can be in a membrane-bound or soluble form, in the joints of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: The concentration of soluble FasL (sFasL) in serum and synovial fluid (SF) from 24 OA and 38 RA patients was measured using an enzyme-linked immunosorbent assay. The expression of FasL on SF lymphocytes (SFL) and peripheral blood lymphocytes (PBL) was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. A cytotoxic killing assay of membrane-bound FasL and purified sFasL against cultured synovial cells was also performed. RESULTS: Soluble FasL was detected in the SF of patients with RA and OA, but not in their serum. The concentration of SF sFasL was remarkably higher in patients with severe RA than in patients with mild RA or with OA. RT-PCR showed that SFL, but not PBL, from RA patients expressed messenger RNA for FasL. Membrane-bound FasL induced apoptosis in cultured synovial cells from the RA and OA patients, but naturally processed human sFasL did not. CONCLUSION: SFL from RA patients expressed FasL, and cleaved sFasL accumulated in the SF of inflamed joints. The different killing activity of membrane-bound FasL and sFasL against synovial cells may regulate Fas-mediated apoptosis in synovial cells.

Abstract: OBJECTIVE: To elucidate possible roles of several transcription factors in the pathogenesis of rheumatoid arthritis (RA), the transcription factor expression in RA synovial tissue and their contribution to RA synovial cell functions were studied. METHODS: Single cell suspension of dissociated synovial tissue was cultured to induce in vitro tissue outgrowth of RA synovial cells. Transcription factors were immunohistochemically identified in RA synovial tissue obtained by joint surgery and in the in vitro tissue outgrowth, and confirmed by western blotting and gel shift assays. RESULTS: Immunohistochemical examination of RA synovial tissue revealed simultaneous expression of various transcription factors (NF-kappa B, c-Jun (a component of AP-1), cAMP responsive element binding protein (CREB), and OCT-1). The same set of transcription factors was expressed in the in vitro tissue outgrowth of RA patients. The early passage RA synovial cells were treated with interleukin 1 beta (IL1 beta) and confirmed translocation of transcription factors into the nucleus by western blotting, and their DNA binding activity by gel shift assays. CONCLUSION: This study emphasises the importance of the simultaneous expression of several transcription factors for the hyperactivity of RA synovial cells that leads to tissue outgrowth.

Abstract: We describe a case of rheumatoid arthritis (RA) with collapse of the L3 lumbar vertebra for which surgery was performed. The pathogenesis of lumbar lesions affected by RA is discussed and the literature reviewed.

Abstract: The process of cartilage-to-bone transition (CBT) is a key event for the achievement of rigid bone healing during fracture repair. Since mineralization of cartilaginous matrix is a prerequisite for the initiation of CBT, the genetic localization of mineralization-related bone matrix proteins in CBT was examined in this study. An in situ hybridization method used on decalcified sections with digoxigenin-11-UTP labelled probes identified the cellular localizations of these genes in CBT. Cessation of osteonectin mRNA together with induction of osteopontin mRNA in chondrocyte maturation was observed during the process of CBT in the fracture callus on day 12 after fracture; osteocalcin mRNA was absent in chondrocytes of the CBT area. Induction of osteopontin mRNA in maturated chondrocytes was followed by the expression of mRNAs for osteonectin, osteopontin and osteocalcin in osteogenic cells in the ossification front of CBT. The data suggest that the switch from osteonectin to osteopontin mRNA expression in chondrocyte maturation is one of the key events during CBT. Transcriptional disorders of the expression of these molecules may be linked to the failure of fracture repair, i.e. delayed or prevented hypertrophic osteosynthesis.

Abstract: OBJECTIVE: We previously reported the accumulation of abnormal myeloid cell populations reacting with CD14 (MY4) monoclonal antibody in the iliac and epiphyseal bone marrow of patients with severe rheumatoid arthritis (RA). Therefore, we investigated in vitro production and modulation of CD14+ myeloid cells from iliac bone marrow cells. METHODS: Mononuclear cells were prepared from iliac bone marrow aspirates from patients with RA. The presence of unusual myeloid cells was assessed by 2 color flow cytometry of cells cultured under various conditions. RESULTS: Cultured iliac bone marrow cells of patients with severe RA produced 14.7% of CD14+ CD15+ cells on average. Cultures derived from healthy donors and from patients with a milder form of RA produced fewer CD14+ CD15+ cells (< 10%). The production of CD14+ CD15+ cells was enhanced by granulocyte macrophage colony stimulating factor and interleukin 1beta, but inhibited by T lymphocytes. CONCLUSION: Production and modulation of CD14+ myeloid cells were observed in iliac bone marrow of patients with severe RA.

Abstract: OBJECTIVE: To establish a system for efficient, direct in vivo gene transfer into joints. METHODS: A hemagglutinating virus of Japan (HVJ; Sendai virus)-liposome suspension containing SV40 large T antigen (SVT) gene was injected intraarticularly into knee joints of 6-week-old female Lewis rats. Rats were killed at various times for immunohistochemical analysis of the expression of SVT gene. RESULTS: The expression of SVT gene was detected immunohistochemically in chondrocytes in the superficial and middle zones of articular cartilage in the knee joints. The average percentage of SVT-positive cells was estimated to be approximately 30% on days 3, 7, 14, and 21 after transfection. Moreover, no pathologic change caused by HVJ-liposome injection was observed in the joints. CONCLUSION: The transfection frequency and stability of expression recognized in this study indicate the possibility of a strategy for treatment of joint disorders, including arthritis, using direct gene transfer.

Abstract: OBJECTIVE: 2-Acetylthiomethyl-3-(4-methylbenzoyl) propionic acid, KE298, a derivative or propionic acid developed in Japan has been shown to be effective for suppressing disease activity of rheumatoid arthritis (RA) in clinical trials in Japan. It is thus a candidate as a new disease modifying antirheumatic drug (DMARD). We analyzed effects of KE298 on synovial fibroblast-like cells in patients with RA to obtain insight into the clinical application of this medication. METHODS: RA synovial fibroblast-like cells were co-cultured with KE298 at 10(-4)-10(-5) M in the presence or absence of tumor necrosis factor-alpha 2 ng/ml, and their subsequent proliferative responses and proinflammatory cytokine and matrix metalloproteinase (MMP) production at the mRNA and protein levels were measured. Effects of KE298 on MMP-1 gene transcription and AP-1 transcription factor expression of RA synovial cells were studied by chloramphenicol acetyltransferase assay and gel shift assay, respectively. RESULTS: KE298 inhibited proliferation of RA synovial cells, proinflammatory cytokine production, and MMP-1 production mainly by reducing their transcription via downmodulation of AP-1 transcription factor. CONCLUSION: KE298 inhibits aberrant synovial cell functions of patients with RA by downregulating gene transcription, suggesting clinical application and usefulness of this new DMARD.

Abstract: An adequate supply of peripheral blood monocytes, granulocytes, and platelets is necessary for an optimal inflammatory process. We have previously demonstrated that the generation of CD14(+) monocyte-lineage cells from the bone marrow is accelerated in patients with rheumatoid arthritis (RA). The current studies examined the influences of gold sodium thiomalate (GST) and bucillamine (BUC), two potent disease-modifying antirheumatic drugs (DMARDs), on the capacity of bone marrow progenitor cells to generate CD14(+) cells in patients with RA, in order to delineate their mechanisms of action. CD14(-) cells purified from bone marrow specimens of 13 patients with active RA who were not taking DMARDs were cultured in the presence or absence of pharmacologically attainable concentrations of GST (25 microM) or intramolecular disulfide form of bucillamine (BUC-ID, 3 microM), a major metabolite of BUC. After incubation for 14 days, the cells were analyzed by flow cytometry for expression of CD14, HLA-DR, and CD54. The generation of CD14(+) cells from RA bone marrow CD14(-) progenitor cells was significantly suppressed by GST, but not by BUC-ID. The expression of HLA-DR on the bone marrow-derived CD14(+) cells was also significantly inhibited by GST, but not by BUC-ID. Of note, neither GST nor BUC-ID influenced the expression of CD54 on the bone marrow-derived CD14(+) cells, indicating that the expression of HLA-DR and CD54 on the bone marrow-derived CD14(+) cells is regulated by different mechanisms. The results are consistent with the hypothesis that one of the effects of DMARDs may involve the interference with monocyte differentiation in the bone marrow. Moreover, the data emphasize that in contrast with BUC, GST is a potent inhibitor of monopoiesis in RA patients.

Abstract: We investigated the in vivo introduction of a reporter gene into healing rat patellar ligaments using the hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer method. The mid-portion of the medial half of the patellar ligament was cut transversely with a scalpel in 14-wk-old male Wistar rats. A HVJ-liposome suspension containing beta-galactosidase (beta-gal) cDNA was injected directly into the injured site and pooled in the fascial pocket covering the injured site 3 d postoperatively. Thereafter, beta-gal-labeled cells were observed in the wound site accounting for 3% of the wound cells on the first day, 2% on the third, 7% on the seventh, 6% on the 14th, 2% on the 28th, and 0.2% on the 56th day after injection. The beta-gal-labeled cells were initially localized in and adjacent to the wound site, but they were observed spreading into the ligament substance away from the wound on the seventh day after injection. On day 28, beta-gal-labeled cells were observed throughout the length of the ligament substance. With double-labeling for marker antigens for monocyte/macrophage (ED-1) and for collagen I aminopropeptide (pN collagen I), it was revealed that fibroblastic (pN collagen I-positive) cells accounted for 63% and monocyte/macrophage lineage cells for 32% of the beta-gal-labeled cells in the day 7 wound. On day 28, they formed 58 and 35% of the beta-gal-labeled cells in the wound, respectively. Thus, we succeeded in introducing the beta-gal gene into healing rat patellar ligament. Moreover, labeling of the transfected cells made it possible to identify a biological event, namely that the cells in and around the wound site infiltrate into the uninjured ligament substance and come to populate the whole length of the ligament substance as repair progresses. These results suggest that ligament healing may involve not only the repair of the wound site itself but also extensive cellular infiltration of ligament substance adjacent to the wound.

Abstract: OBJECTIVE: To observe the migration of bone marrow stromal cells into the joint cavity and the contribution of such cells to synovial proliferation in rats with collagen induced arthritis (CIA). METHODS: After bone marrow stromal cells preliminarily labeled with fluorescent dye or 3H thymidine accumulated in the bone marrow of splenectomized rats by intraperitoneal injection, the migration of labeled stromal cells in rats with CIA was analyzed by liquid scintillation counting, autoradiography, and fluorography. RESULTS: In splenectomized control rats, labeled bone marrow stromal cells were mostly found in the bone marrow and not in the synovium. Over 2 weeks after immunization, labeled stromal cells were microscopically found migrating directly into the joint cavity through the area between the articular margin and the synovial insertion (the bare zone). Labeled stromal cells were mainly found in the sublining layers of proliferating synovial tissue. CONCLUSION: At the onset of CIA, bone marrow stromal cells migrated from the bone marrow into the affected joint cavity and seemed to contribute to synovial proliferation.

Abstract: OBJECTIVE: To examine the capacity of bone marrow progenitor cells to generate CD14+ cells, in order to assess the role of bone marrow in the pathogenesis of rheumatoid arthritis (RA). METHODS: CD14- cells purified from bone marrow specimens of 11 patients with active RA and 8 control patients (osteoarthritis or trauma) were cultured in the presence or absence of granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 pg/ml). After incubation for various lengths of time, the cells were analyzed by flow cytometry for expression of CD14 and HLA-DR. RESULTS: The spontaneous generation of CD14+ cells from bone marrow CD14- progenitor cells was accelerated in RA patients compared with control patients. Moreover, the expression of HLA-DR on the bone marrow-derived CD14+ cells was also accelerated in RA patients compared with controls. GM-CSF significantly enhanced the generation of CD14+ cells, as well as the expression of HLA-DR, on CD14+ cells of control patients, but not those of RA patients. GM-CSF levels in the culture supernatants of bone marrow CD14- cells were not significantly different between RA patients and control patients (undetectable in most cases). CONCLUSION: These observations strongly support the hypothesis that the accelerated generation of CD14+ cells from bone marrow progenitor cells and the accelerated maturation of such CD14+ cells into HLA-DR+ cells play an important role in the pathogenesis of RA. Moreover, the data suggest a functional alteration of RA bone marrow CD14- cells in their responsiveness to GM-CSF.

Abstract:

Abstract: OBJECTIVE. Our previous study showed the presence of abnormal myeloid lineage cells in the epiphyseal bone marrow adjacent to joints affected with severe rheumatoid arthritis (RA). Now, we investigated whether there were any changes of other marrow cell populations related to RA, and whether there were any pathologically characteristic changes in the iliac bone marrow, which is one of the major systemic hematopoietic organs. METHODS. 2-Color flow cytometry was carried out to analyze the phenotypes of mononuclear cells (MNC) fractions in bone marrow aspirates and venous blood from 56 patients with RA and 7 non-RA controls. RESULTS. The absolute number of MNC in the iliac bone marrow was increased by 3-fold in the RA patients compared with the non-RA controls. In contrast, no significant increase of MNC was observed in the tibial epiphyseal bone marrow or peripheral blood. The ratio of each MNC fraction in the iliac bone marrow did not differ significantly between the RA patients and the non-RA controls. In lymphocyte subsets, the percentage of HLA-DR+CD8+ cells to all CD8 cells in the iliac bone marrow increased significantly in the RA patients compared with the non-RA controls. Abnormal myeloid cells (MX-GA+MY4+ cells), specific to severe RA, were found to be more concentrated in the iliac bone marrow than in the tibial epiphyseal bone marrow. CONCLUSION. Characteristic pathologic changes of the iliac bone marrow suggest an important role of systemic bone marrow in the progression of RA.

Abstract: OBJECTIVE. Characteristic cellular changes have previously been reported in the bone marrow of patients with rheumatoid arthritis (RA). We investigated the levels of various cytokines in RA bone marrow. METHODS. We studied 25 patients with RA (22 women and 3 men) and 10 trauma patients (7 women and 3 men) as non-RA controls. Twelve kinds of cytokines [interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor, tumor necrosis factor (TNF)-alpha, and TNF-beta] were assayed by ELISA in iliac bone marrow serum (BMS), tibial BMS, and peripheral blood serum. RESULTS. Markedly elevated levels of IL-6 and IL-8 were detected in iliac BMS, and much lower levels were found in tibial bone marrow and peripheral blood serum. The levels of IL-6 and IL-8 in iliac BMS showed a close relationship to the extent of synovial proliferation. CONCLUSION. Iliac bone marrow may be an important site for the production or accumulation of IL-6 and IL-8 in RA, and these cytokines may influence synovial proliferation in patients with polyarthritis.

Abstract:

Abstract:

Abstract: As reported by us, a new myeloid cell population with an oncofetal membrane marker, dimeric Lex (di-Lex; III3FucV3 FucnLc6), was found in the epiphyseal bone marrow adjacent to the involved joints of patients with severe rheumatoid arthritis (RA). Patients with RA received intradermal (id) injections of di-Lex incorporated in liposome or of high molecular weight glycoprotein, or tumor associated carbohydrate antigen (TCA), containing the same carbohydrate epitope as di-Lex. The epiphyseal myeloid cells were reduced or sometimes eliminated during id injection. In random trials of id injection, observation under clinical and laboratory conditions showed improvement in 63% (17/27) of the patients treated for 6 months with appropriate doses of di-Lex (III3FucnLc4), and in 72% (31/43) of those treated with an identical protocol for TCA. However, id injection with monomeric Lex had no effect.