Expression of CD44 in synovium of rabbits with chronic arthritis induced by immunization with Escherichia coli.

OBJECTIVE: To investigate the expression of CD44 and its role in experimental chronic arthritis in rabbits. METHODS: Rabbits were immunized with Escherichia coli 0:14 for short (4 mo) and long (8-10 mo) periods. Immunohistochemical staining was performed on knees, using anti-CD44 antibodies. RESULTS: Lymphocyte infiltration in the synovium was found in 30.0% of rabbits after short term immunization, and the rate increased to 58.3% after longterm immunization. CD44 was present in synovial lining cells in 30.0% of rabbits after short term immunization, and it increased significantly (p < 0.05) after longterm immunization (66.7%). CD44 was also observed in lymphocytes in knee synovium after longterm immunization (25.0%). CONCLUSION: CD44 in lining cells might play a role in promoting chronic arthritis in rabbits immunized with E. coli.

Characterization of the macromolecular components of the articular cartilage surface.

The intact surface of articular cartilage is a highly organized structure composed of a variety of macromolecules. The studies reported here deal with a partial characterization of the non-covalently bound components of the outermost layer of articular cartilage. Normal bovine and human cartilage articular surfaces were extracted for 5 min with 4-M guanidine HCl solution. Analysis and quantitation of small proteoglycans in the extract were carried out by PAGE (polyacrylamide gel electrophoresis), Western blot, and radioimmunoassays. The present studies indicate that the major proteins extracted from the articular surface of bovine and human cartilage are the collagen-binding small proteoglycans designated as fibromodulin and albumin. Fibronectin, decorin, and biglycan were also detected in smaller amounts. Immunoblotting of the surface material developed with a monoclonal antibody with keratan sulfate specificity confirmed the presence of fibromodulin coinciding with the major protein band of approximately 70-100-kDa molecular mass. Gel filtration chromatography of the surface material confirmed the previous results. Additional in vitro assays showed that the collagen-binding material extracted from the cartilage surface contained the small proteoglycans. Anti-human fibromodulin antibodies bound in significantly greater amounts to the intact articular surfaces than to cut surfaces of normal human cartilage. It is concluded that small, non-aggregating proteoglycans constitute the major proteoglycan species non-covalently bound to macromolecules at the articular surface of cartilage partially responsible for the interference of anti-collagen type II antibody binding and for the inhibition of cell adhesion to the intact surface.

Inhibition of human fibroblast adhesion by cartilage surface proteoglycans.

OBJECTIVE: Recent studies from our laboratory have identified the nonaggregating, collagen-binding proteoglycans, fibromodulin (FM) and decorin, and fibronectin (Fn) and albumin, noncovalently bound at the articular surface of cartilage. The present studies were designed to investigate the interactions between these cartilage macromolecules and the underlying collagen matrix and their role as a barrier to cell adhesion in intact articular cartilage. METHODS: Cell adhesion studies were carried out with human skin fibroblasts incubated on the articular surface of bovine cartilage explants and on collagen-coated and/or Fn-coated plastic surfaces. Interactions of collagen and Fn with either FM or decorin were studied by radioimmunoassay of the same surfaces, using specific antibodies. RESULTS: The present studies show that 1) Fn is immunologically detectable at the intact articular surface of cartilage; 2) fibroblast adhesion to Fn is inhibited by cartilage surface extract proteins and by purified FM, but not by purified decorin; 3) FM has binding affinity for Fn; 4) FM interferes with the binding of a monoclonal antibody specific for the cell-binding domain of Fn; and 5) FM and decorin inhibit collagen-dependent fibroblast adhesion. CONCLUSION: These results indicate that the small proteoglycans at the normal articular surface may act as a barrier to cell adhesion. Since protective cartilage surface proteins break down readily after the induction of acute arthritis in experimental animals, and in rheumatoid cartilage specimens, it is postulated that proteolytic degradation of the surface proteoglycans may be responsible for increasing cell adhesion to, and subsequent pannus invasion of, articular cartilage in inflammatory arthritis.

Repair characteristics of the articular cartilage surface following acute inflammatory arthritis.

OBJECTIVE: To investigate the repair characteristics of the surface protein in an acute arthritis of short duration. METHODS: Knee arthritis in rabbits was induced by intraarticular (ia) injection of 20 micrograms E. coli endotoxin into one knee of 12 rabbits. The contralateral knee served as control. Four animals were killed 3, 6, and 10 days after ia injection. The articular cartilage surface was probed by quantitation of anticollagen type II (anti-CII) antibody binding. The suprapatellar bursae were washed, and the recovered cells counted. RESULTS: A significant increase in anti-CII antibody binding compared to control was measured 3 days after ia injection, coinciding with evidence of acute arthritis (injected joint: 1.1 x 10(7), control: 4.2 x 10(4) cells/joint; percent increase in anti-CII binding: 36.7 +/- 10.7; p < 0.02). Six days after ia injection, the acute arthritis showed a major decrease in intensity whereas anti-CII binding was still abnormal (injected joint: 1.7 x 10(6) cells/joint; percent increase in anti-CII binding: 24.7 +/- 19.6; p < 0.05). On Day 10, there was minimal evidence of arthritis in 3 of 4 rabbits, and anti-CII antibody binding returned to normal (injected joint: 1.9 x 10(5) cells/joint; percent anti-CII binding: -5.8 +/- 3.9). There was a strong positive correlation between individual synovial fluid cell counts and the percent increase in anti-CII binding (R = 0.72, p < 0.02). CONCLUSION: The magnitude of binding of anti-CII antibodies to the articular cartilage surface constitutes a sensitive probe for the detection of early damage following a transient inflammatory insult. Our studies indicate that after acute injury, the cartilage surface may show evidence of damage for at least 6 days when probed with anti-CII antibodies.

Experimental induction of arthritis in rats immunized with Escherichia coli 0:14 lipopolysaccharide.

OBJECTIVE: Escherichia coli 0:14 (E. coli 0:14) induces arthritis in rabbits, mice and rats. This study was designed to investigate the effects of lipopolysaccharide (LPS) on the rat arthritis model induced by systemic injections of E. coli 0:14. METHODS: The induction of arthritis in the ankles of rats immunized by subcutaneous injections with heat-killed E. coli 0:14 and its LPS was studied. The appearance and levels of serum IgM rheumatoid factor-like substance (RFLS) was also investigated. The localization of interleukin 1 (IL-1) and LPS in the ankle joints were investigated immunohistochemically. RESULTS: The induction rate of arthritis in rats immunized with LPS was the same as that in rats immunized with E. coli. LPS and IL-1 were detected in synovial cells, infiltrating cells and some cells on pannus in arthritic joints. Anti-LPS IgM levels in rats immunized with E. coli were as high as those in rats immunized with LPS. RFLS levels in rats immunized with LPS increased more gradually than those in rats immunized with E. coli. CONCLUSION: Our findings suggest that LPS induces arthritis resembling rheumatoid arthritis in rats. The detection of IL-1 in synovial cells in conjunction with LPS suggests that local stimulation of IL-1 production may play an important role in the induction of this experimental arthritis.

Binding characteristics of antitype II collagen antibody to the surface of diseased human cartilage as a probe for tissue damage.

OBJECTIVE: Studies have shown that collagen type II (CII) on the intact articular surface of cartilage is partially protected from binding to anti-CII antibodies by material proteinaceous in nature, not present in synovial fluid, synthesized by resident chondrocytes, and exquisitely sensitive to polymorphonuclear (PMN) attack and neutrophil elastase digestion. Thus, anti-CII antibody differential binding to articular cartilage surfaces before and after brief neutrophil elastase digestion may be used as a sensitive marker of cartilage damage. METHODS: We measured binding of anti-CII antibodies to the pannus-free articular surfaces of 4 normal, 11 rheumatoid (RA), and 10 osteoarthritic (OA) cartilage specimens, before and after brief digestion with PMN elastase. In addition, antibody binding was quantitated with an antiserum against a 4 M guanidine extract of human cartilage surface. RESULTS: Whereas anti-CII binding increased 59.0% +/- 2.8 after 1 h incubation of normal cartilage with elastase, both the RA and OA specimens failed to show significant increases (RA: 1.0 +/- 0.1; p < 0.001; OA: 27.2% +/- 1.6, p < 0.05). Moreover, anti-CII antibody binding to untreated cartilage specimens was highest for the RA group (Normal: 189.2.1 +/- 38.7 pg anti-lg/mg tissue; RA: 407.5 +/- 80.6, p < 0.05; OA: 243.6 +/- 50.6, NS). Concomitant binding studies with antiserum against cartilage surface material showed greater antibody binding to the articular surfaces than to the cut cartilage surfaces in normal and OA specimens. RA cartilage samples exhibited somewhat smaller antibody binding to the articular surfaces. CONCLUSION: Our studies suggest that in human inflammatory and noninflammatory arthritides the articular cartilage surface undergoes alterations that can be detected by differential binding with anti-CII antibodies, before and after brief digestion with PMN elastase.

Characteristics of anti-type II collagen antibody binding to articular cartilage.

OBJECTIVE: Previous studies suggested that it was possible to characterize the intact surface of articular cartilage by probing it with antibodies against matrix macromolecules. The present studies were undertaken to investigate type II collagen (CII) epitope availability on the intact surface of articular cartilage. METHODS: Normal bovine, rabbit, and human cartilage specimens were used to measure binding of anti-CII antibodies to the articular and cut surfaces of cartilage. Antisera were raised against the material obtained after brief extraction of the cartilage surface with 4M guanidine solution. RESULTS: Anti-CII did not bind to the intact surface of rabbit articular cartilage when compared with control rat sera, but did bind significantly to the cut surface. With normal human articular cartilage, the cut surfaces bound approximately 4 times as much anti-CII antibody as the intact articular surfaces. These findings suggested that the CII epitopes are normally protected by the superficial cartilage layer. In experiments carried out to characterize this layer, binding of anti-CII antibodies was unchanged after exhaustive washing of bovine or rabbit cartilage with phosphate buffered saline or 1M NaCl solution, whereas it was significantly increased after brief exposure to 4M guanidine solution or after incubation with neutrophil elastase. No restoration of the protection of CII epitopes in guanidine-treated cartilage could be achieved by incubation with undiluted normal bovine synovial fluid; however, 8-day culture of elastase-treated cartilage explants resulted in partial restoration of protection of the CII epitopes. Rat and rabbit antisera raised against the cartilage surface material extracted with 4M guanidine contained antibodies that bound to the cartilage surface. By Western blotting, rat antibodies identified 50-65-kd protein bands present in the guanidine extract, but not present either in the material obtained from brief digestion of cartilage with neutrophil elastase or in synovial fluid. The rabbit antisera identified a broad 70-95-kd band. Exposure of elastase-treated cartilage to the guanidine-extracted material resulted in a partial decrease of anti-CII antibody binding. CONCLUSION: These results suggest that CII on intact cartilage is protected from antibody binding, and that the protective material is at least partly proteinaceous in nature, is unlikely to be derived from synovial fluid, is noncovalently bound to the underlying intercellular matrix, and is synthesized by resident chondrocytes. Further characterization of the protective material may provide important information on the mechanisms of early cartilage damage in inflammatory arthritis.

[Immunohistochemical localization of interleukin-1 and lipopolysaccharide (LPS) of experimental arthritis in the ankles of rats immunized with LPS extracted from Escherichia coli].

Sprague Dawley (SD) rats were immunized by subcutaneous injections with heat-killed E. coli 0:14 and lipopolysaccharide (LPS) extracted from E. coli for 15, 29 and 39 weeks which induced arthritis in the ankle. Localization of interleukin-1 (IL-1) and LPS in the ankle joints were investigated immunohistochemically. Serum IgM rheumatoid factor-like substance (RFLS) and anti-LPS IgM were detected by enzyme-linked immunosorbent assay (ELISA). Rats immunized with LPS for 39 weeks developed synovial lining cell hyperplasia in 25 of 40 ankles and lymphoid cell infiltration in 25 and pannus formation in 23, the rates of which were significantly higher than those of control and rats immunized with LPS for 15 and 29 weeks. The induction rate of arthritis in rats immunized with LPS was the same as that in rats immunized with E. coli. LPS and IL-1 were located in synovial cells and pannus in arthritic joints. Changes of RFLS level in rats immunized with LPS were elevated more gradually than those in rats immunized with E. coli. These findings suggest that LPS could stimulate IL-1 and RFLS production and may induce arthritis in rats resembling rheumatoid arthritis.

[Comparison of wound healing of linear incisions in the rabbit cornea produced by the newly developed excimer laser, a metal blade, and a diamond blade].

We developed a new delivery system for corneal ablation with the 193 nm argon-fluoride excimer laser. The laser was used to make linear incisions in the rabbit cornea, and wound healing of the incisions was compared with the healing of incisions made with metal and diamond blades. The morphology of incisions made with excimer laser radiation (193 nm) was compared with the morphology of incisions produced by diamond and metal blades, and the corneal wound was examined by light and electron microscopy. The surface of the cornea at the site of the laser wound was smooth; the laser wound was wider than the blade wounds immediately after surgery. Two weeks after surgery, all wounds had healed equally. One month after surgery, there were fewer fibroblastic keratocytes in the laser wound, and the epithelial layer at the wound site was thinner than in the case of the blade wounds. These results suggest that wound healing of laser incisions is more rapid than healing of wounds created with metal or diamond blades.

[Effects on the retina and choroid of transpupillary diode laser photocoagulation].

A transpupillary retinal photocoagulator utilizing GaAlAs diode laser which emits an 810 nm beam has been constructed. It has a continuous-wave mode with a maximum output of 400 mW at the cornea. Animal experiments with monkeys were carried out successfully at various intensities to produce controlled coagulations. Observation by ophthalmoscopy, light and electron microscopes at different time intervals proved diode laser photocoagulation produced more intensive effects in choroid than krypton red laser (647 nm). However, due to the greater absorption of the choroid, there is a tendency to over-coagulation unless conditions are carefully set. No serious side effects in the retina and choroid were observed during the experiments.

[Clinical results of total knee arthroplasty using Kinematic type in patients with rheumatoid arthritis under the age of 50 years].

We reviewed 24 total knee arthroplasties using Kinematic type (anteriorly joined type or posterior cruciate retention type) in 18 rheumatoid patients who were under the age of 50 years. Thirteen knees with Kinematic type total knee arthroplasty in 10 rheumatoid patients who were over 70 years of age were also reviewed. Using rheumatoid arthritic knee rating score of J.O.A. (Japan Orthopedic Association), the clinical results of young patients were evaluated and were compared with those of elderly patients. The average total score of young patients and that of older patients was almost equally increased after arthroplasty. Postoperatively in older patients, pain score was elevated more remarkably than in young patients. However, increase in score of quadriceps muscle strength, walking ability and climbing stairs ability were less in older patients than that in young patients. Range of motion was not improved in both young and older patients. However, flexion contracture was improved remarkably in both groups.

High tibial osteotomy with fixation by a blade plate for medial compartment osteoarthritis of the knee.

Surgical techniques were described on tibial osteotomy above the tuberosity, the fragments of which were fixed with a Koshino blade plate. Postoperative clinical results on 176 knees with medial compartment osteoarthritis of 138 patients with an average age of 62.1 years were excellent in 110 and good in 55 knees with an average of 5.5 years' follow-up. The best knee score was obtained in the knee with postoperative limb alignment of 6 to 15 degree valgus angulation (standing). The results were satisfactory, especially in postoperative management, which was much easier with the Koshino blade plate fixation than with other methods. The sinking of the osteotomy site was checked through observation of screw movement in the slot of the plate during postoperative management in 53 selected knees, and the averaged distance of sinking was 3.9 +/- 2.5 mm. The time of mechanical bone union judged by cessation of sinking averaged 12.9 +/- 6.2 weeks after osteotomy.

Krypton and argon laser photocoagulation effects in subretinal hemorrhage.

Previous studies suggested that krypton laser photocoagulation was more effective in the treatment of macular diseases than argon laser. Furthermore, it could perform photocoagulation more effectively in some lesions with subretinal hemorrhage, because the krypton laser beam was poorly absorbed by hemoglobin. In the present experiment, hemorrhagic retinal detachment was produced in monkey eyes with Q-switched Nd-YAG laser, and 4 weeks later photocoagulation was performed with krypton and argon lasers to compare the differences in the effects of these two lasers. When the subretinal hemorrhage and a heavy coagulation effect was produced in the detached retina, but no coagulation effects were observed in the choroid. Krypton laser beam could go through the hemorrhage and certain coagulation effects were observed in the choroid and the detached retina. It is suggested that krypton laser photocoagulation is more effective in the lesions behind subretinal hemorrhages than photocoagulation with argon laser.

Wide field fluorescein angiography by use of contact lens.

A system has been developed to obtain fluorescein angiographs with a field measuring to 90 degrees. This involves a contact lens and a new fundus camera with a basic field of 60 degrees. The contact lens, however, can be used with any fundus camera and will result in an increase in the area of the photographs of over 2X. There are numerous clinical applications for high resolution, high contrast, wide field fluorescein angiography.