Toshitaka Takagi

New bone formation around porous hydroxyapatite wedge implanted in opening wedge high tibial osteotomy in patients with osteoarthritis.

A porous hydroxyapatite (highly purified synthetic Ca10(PO4)6(OH)2) wedge was inserted into the tibia in 10 knees of 7 patients with osteoarthritis of the knee who underwent high tibial osteotomy. The interface of this HA wedge with bone was histologically examined in undecalcified specimens obtained at the time of hardware removal in all 10 knees, and total incorporation of the HA into bone was observed, with no inflammatory reaction. The proportion of pores that were filled with regenerated bone within 300 microm from the interface was 71.8 +/- 10.1% (mean +/- S.D.), and it was positively correlated with the time from implantation to biopsy. Radiodensity of the HA wedge was measured during the follow-up period, and it did not change significantly within 36 months after osteotomy. Our study concluded that bone ingrowth into the HA block slowly progressed and was correlated with the passage of time.

Zymosan enhances the immune response to DNA vaccine for human immunodeficiency virus type-1 through the activation of complement system.

In the present study, the adjuvant effect of zymosan on human immunodeficiency virus type‐1 (HIV‐1)‐specific DNA vaccine and the mechanism of this enhancement were studied in a murine model. We coinoculated zymosan with our candidate HIV‐1 specific DNA vaccine (pCMV160IIIB) into skeletal muscles of BALB/c mice. Higher levels of both humoral immune response and HIV‐specific delayed‐type hypersensitivity (DTH) response were observed when zymosan was coinoculated with pCMV160IIIB compared with that obtained using pCMV160IIIB alone. HIV‐specific cytotoxic T lymphocyte (CTL) activity was also enhanced. This enhancing activity was suppressed when coinoculated to the fifth complement (C5)‐deficient DDD and AKR mice. The enhanced activity was also suppressed when anti‐C3 antibody was inoculated to mice intramuscularly. There was significant induction of immunoglobulin G2a (IgG2a) and interferon‐γ (IFN‐γ) in pCMV160IIIB vaccine with zymosan. These results suggest that zymosan‐mediated DNA vaccination enhances helper T cell (Th) 1‐mediated immunity. The effect is suggested to be based on the consequences of its recruitment and activation of macrophages, dendritic cells or antigen‐presenting cells (APC) through complement activation, especially through the alternative pathway. Taken together, these results suggest that zymosan can be an effective immunological adjuvant in DNA vaccination against HIV‐1.

Up-regulation of CD44 in rheumatoid chondrocytes.

The adhesion molecule CD44 is thought to play an important role in the inflammatory process. To identify the expression of CD44 in articular chondrocytes in rheumatoid arthritis (RA), monoclonal anti-CD44 antibodies were immunohistochemically used to react with articular cartilage specimens of 15 patients with RA, 9 with osteoarthritis (OA), and 6 with femoral neck fracture (FF). The proportion of CD44-positive chondrocytes in RA was 93±2% (N=16), which was significantly higher than that in OA (59±7%, N=9, p<0.001) and FF (46±5%, N=6, p<0.001). Among CD44 isoforms examined, the hemopoietic form was dominant in chondrocytes in RA. Therefore, up-regulation of CD44 on chondrocytes may play a significant role in cartilage degeneration in RA.The adhesion molecule CD44 is thought to play an important role in the inflammatory process. To identify the expression of CD44 in articular chondrocytes in rheumatoid arthritis (RA), monoclonal anti-CD44 antibodies were immunohistochemically used to react with articular cartilage specimens of 15 patients with RA, 9 with osteoarthritis (OA), and 6 with femoral neck fracture (FF). The proportion of CD44-positive chondrocytes in RA was 93 +/- 2% (N=16), which was significantly higher than that in OA (59 +/- 7%, N=9, p<0.001) and FF (46 +/- 5%, N=6, p<0.001). Among CD44 isoforms examined, the hemopoietic form was dominant in chondrocytes in RA. Therefore, up-regulation of CD44 on chondrocytes may play a significant role in cartilage degeneration in RA.

Highly activated matrix metalloproteinase-2 secreted from clones of metastatic lung nodules of nude mice injected with human fibrosarcoma HT1080

The promoting effects of matrix metalloproteinase-2 (MMP-2) on lung metastasis of human fibrosarcoma cells (HT1080) were studied using nude mice. The fourth generation of HT1080 was established by consecutive clonal selection of metastatic lung nodules formed by intravenous transplantation. MMP-2 and MMP-9 in the culture supernatants of the first and fourth generation cells were analyzed by gelatin zymography and Western blotting, and quantified by scanning densitometry. In gelatin zymograms, mean ratios of values for the 59-kDa band (the active form of MMP-2) to those for the 72-kDa band (the inactive form of MMP-2) for optical density; area, and volume measured by densitometry were 1.44 +/- 0.12, 0.93 +/- 0.05, and 1.27 +/- 0.20, respectively, in the culture supernatant of fourth generation cells isolated from metastatic lung nodules. These values were significantly (P < 0.01) higher than those of first generation cells (0.70 +/- 0.04, 0.48 +/- 0.01, and 0.57 +/- 0.42). Three weeks after intravenous transplantation of HT1080 cells into nude mice, the incidence of lung metastasis and mean number and diameter of metastatic nodules formed by injection of first generation cells were 20% (2 of 10 mice), 2.9 +/- 0.2 and 2.0 +/- 0.2 mm, respectively; while they were 100%, 99.8 +/- 7.2 and 4.3 +/- 0.3 mm following injection of fourth generation cells. These findings suggest that the active MMP-2 produced by human fibrosarcoma cells is important for the cells to form lung metastatic lesions in nude mice.

Osseous tissue reaction around hydroxyapatite block implanted into proximal metaphysis of tibia of rat with collagen-induced arthritis.

This study was conducted to investigate the influence of osteoporosis on new bone formation around a hydroxyapatite (HA) block implanted into the proximal metaphysis of the tibia of rats with collagen-induced arthritis (CIA). Ten rats were immunized with an emulsion of bovine type II collagen and Freunds complete adjuvant (arthritis group). Another 10 rats, which were not immunized were used as the control group. Seventeen days after immunization, HA block was implanted into the proximal metaphysis of the tibia. Four weeks after implantation, all rats were killed. The serum level of tetrate-resistant acid phosphatase (TRAP), bone mineral density (BMD) in the proximal metaphysis of the tibia and the affinity index in the arthritis group were 28.0+/-3.5 IU/ml, 130.3+/-28.7 mg/cm2 and 77.6+/-10.8%, respectively, and those in the control group were 24.6+/-5.5 IU/ml, 175.9+/-30.5 mg/cm2 and 56.3+/-14.8%. The serum level of TRAP was higher (P < 0.05) and BMD was lower (P < 0.005) in the arthritis group. The amount of new bone formation around the HA block was larger (affinity index, P < 0.05) in the arthritis group than in the control group. These findings suggest that bone formation around HA block might be enhanced even in conditions associated with highly activated bone resorption and bone formation, such as arthritis.

Augmentation of anti-tumor effects of methotrexate by distilled water on Dunn osteosarcoma in mouse air pouch

The anti-tumor effects of hypoosmotic solution of MTX in distilled water (DW) on Dunn osteosarcoma were evaluated in mouse air pouches. Dunn osteosarcoma cell suspension (1 x 10[5] cells in 0.1 ml of medium) was inoculated into the mouse subcutaneous air pouch that had formed 7 days after the initial injection of air. Two hours after the inoculation of tumor cells, 5 ml of various concentrations of MTX (from 0 to 1 x 10[-3] M) dissolved in DW or PBS were injected into the air pouch. Five minutes later, the entire solution in the air pouch was aspirated. The mice were sacrificed 3 weeks after the inoculation of tumor cells and the air-pouch tissue was transected in the coronal plane with the largest area of tumor mass. The sections were stained with H&E and the area was measured with the NIH Image program. The largest area of tumor mass in the air pouch treated with 1 x 10(-3) M of MTX in DW was 11.8+/-3.4 mm2 (N = 5), which was significantly (P < 0.005) smaller than that in PBS (51.7+/-8.3 mm2). These findings suggested that hypoosmotic solution in DW might augment the anti-tumor effect of MTX on sarcoma cells.

Quantitative analysis of NF-κB-expression in cartilage and synovium of rat knee induced by intra-articular injection of synthetic lipid A

Abstract. Objective: NF-κB expression in cartilage and synovium of rat knee after intra-articular injection of lipid A was investigated.¶Methods: Inflammation was evaluated in histological sections stained with hematoxylin and eosin, toluidine blue and immunostained with monoclonal anti-NF-κB antibody or monoclonal anti-TNF-α antibody.¶Results: Inflammatory cell infiltration into the synovium, hyperplasia of synovial lining layers, loss of metachromasia of cartilage, TNF-α-positive cells and NF-κB-positive cells in the synovium and cartilage were observed in all 26 rat knees intra-articularly injected with lipid A. The numbers of TNF-α-positive and NF-κB-positive synovial cells and chondrocytes after the injection were significantly greater than those in knees injected with triethylamine (p < 0.05). The amount of inflammatory cell infiltration into the synovium, synovial lining layers, TNF-α-positive synovial cells and NF-κB-positive synovial cells 12 h after the injection was significantly smaller than those after 1 day (p < 0.05).¶Conclusion: Intra-articular injection of lipid A induced NF-κB expression in cartilage and synovium. Cartilage degeneration started earlier than synovitis in this model. Quantitative analysis of NF-κB in cartilage might be useful to evaluate the extreme acute phase of cartilage degeneration.