Arthroscopic reduction and fixation of avulsion fracture of the tibial attachment of the anterior cruciate ligament.

In treating avulsion fracture of the tibial attachment of the anterior cruciate ligament, surgical reduction and fixation of fractured bone is necessary for patients who have a wide displacement of bone fragment (i.e., types III and IV in the Meyers classification). Our arthroscopic technique allows the creation of bone tunnels on the medial and lateral sides of the bone fragment from the medial side of the tibial tubercle without using special equipment. At surgery, fixation wire is prepared into a loop, pulled into the joint space, and the loop is opened within the joint. This makes intra-articular manipulation easy, and the bone can be reduced more accurately. This arthroscopic technique decreases surgical invasion of the joint, allows good postoperative range of motion without problems, and is useful in preventing extension limitation due to dislocation of the anterior portion of the fragment.

Reconstruction of the anterior cruciate ligament using a double bundle.

We developed a reconstruction technique for the anterior cruciate ligament using a double bundle that is the combination of bone-tendon-bone (BTB) from the patellar tendon and semitendinosus tendon (ST). BTB is fixed in the tunnels produced on the isometric points on the tibia and femur. ST is fixed on the tibial tunnel through the same route as the BTB, but on the femur, through the over-the-top route, which is located posterolateral to the femoral tunnel. Use of this double bundle realizes a physiologically more durable reconstruction because BTB corresponds to the anteromedial bundle of the ACL and ST corresponds to the posterolateral bundle, and these produce 2 different tension patterns within the bundle. This technique is also applicable to revision surgeries for patients with an extended bone loss on the tibia.

Fluorovisualization effect of acridine orange on mouse osteosarcoma.

If the localization of musculoskeletal sarcomas could be visualized during surgery, it would be possible to completely resect the tumor with minimum damage to normal tissues and the patients could retain a functional limb. Therefore, we conducted the present study to clarify the usefulness of acridine orange (AO) for fluorovisualization of tumors using a mouse osteosarcoma model. At 2 hours after injection of 10 mg/kg AO to mice inoculated with MOS mouse osteosarcoma cells, fluorovisualization of mouse osteosarcoma reached the maximum level. Even a 1-mm-diameter lesion of pulmonary metastasis was visualized. The results suggested that AO may be useful for specific fluorovisualization of human osteosarcomas during surgery.

Total tumor cell elimination with minimum damage to normal tissues in musculoskeletal sarcomas following photodynamic therapy with acridine orange.

Acridine orange (AO) has unique biological actions enabling tumor visualization (fluorovisualization) and a strong cytocidal effect (photodynamic therapy: AO-PDT) under illumination with blue light. Accordingly, in this study, we attempted to develop a new surgical technique for total tumor cell elimination using these photodynamic reactions with AO in a mouse osteosarcoma model. The results showed that local tumor recurrence was significantly inhibited (23%) in the group treated with curettage under fluorovisualization and AO-PDT, compared to that (80%) in the control group treated with curettage alone under ordinary light. Therefore, we concluded that the combination of curettage under fluorovisualization and AO-PDT may be useful for total tumor cell elimination with minimum damage to normal tissue in musculoskeletal sarcomas.

Case report: natural development of osteosarcoma from precancerous lesion.

We encountered a very rare case that suggested the natural development of osteosarcoma from a precancerous lesion. The patient presented with a huge osteosarcoma in the distal femur on the initial consultation to our hospital. He had undergone X-ray examination twice previously, due to a knee injury. The findings of the lesion detected by the first X-ray examination were similar to a fibrous cortical defect (FCD), differing from those of an osteosarcoma lesion detected by second and last X-ray examinations. We retrospectively estimated the growth rate of the FCD-like and osteosarcoma lesions and found that FCD-like lesion was not osteosarcoma, but might have been a precancerous lesion. We also speculated that this osteosarcoma lesion might have appeared 18 months before the patient consulted our hospital.

Osteoblastic differentiation and P-glycoprotein multidrug resistance in a murine osteosarcoma model.

A recent study of multidrug resistance (MDR) 1 gene transfected osteosarcoma cells found a cause-effect relationship between increased expression of P-glycoprotein (P-gp) and a low aggressive phenotype. However, several experimental and clinical studies have observed contradictory findings in that P-gp expression has been associated with tumour progression. In the present study, we characterized P-gp-positive and P-gp-negative single-cell clones of a murine osteosarcoma, to further investigate the relationship between P-gp expression and changes in cell phenotype. Although these clones were all selected by doxorubicin (DOX) exposure, they were heterogeneous with respect to MDR1 gene expression. The P-gp-positive clones revealed MDR phenotype, whereas the P-gp-negative clones showed no resistance to drugs. Morphological and functional analysis showed that both the P-gp-positive and P-gp-negative clones were more differentiated than the parent cells in terms of enhanced activity of cellular alkaline phosphatase, an increase in well-organized actin stress fibres and enhanced osteogenic activity. Moreover, these subclones all displayed a decrease in malignant potential such as oncogenic activity, tumour growth rate and metastatic ability, regardless of their P-gp status. These results indicate that the observed osteoblastic differentiation and less aggressive phenotype in DOX-selected osteosarcoma cells may not only be explained by the direct effect of P-gp, and accordingly, consideration of the effect of DOX, as well as P-gp, appears to be important.

A new arthroscopic method for reconstructing the anterior and posterior cruciate ligaments using a single-incision technique: simultaneous grafting of the autogenous semitendinosus and patellar tendons.

We established a simultaneous reconstruction method for ruptured anterior and posterior cruciate ligaments (ACL, PCL) using a single-incision technique. Residual PCL was used to determine the position of bone tunnel for ACL reconstruction. The bone tunnel position on the tibia for PCL reconstruction was arthroscopically confirmed by conducting through debridement from the posteromedial portal. Reconstruction substitutes were patellar-tendon bone-tendon-bone for ACL, and semitendinosus tendon for PCL. In the fixation procedure, the PCL substitute was fixed using the Endobutton (Smith & Nephew, Andover, MA) and a ceramic button, and the ACL substitute was fixed with an interference screw. During the surgery, radiographic monitoring and the PCL guide system were not required.

Mechanism of tumoricidal activity of OK-432-specific L3T4+ Lyt2- T-cells.

OK-432, a streptococcal preparation, has been used in the treatment of malignant diseases. We have found that OK-432 can act as an antigen and have established an OK-432-specific L3T4+ Lyt2- T-cell line (OK2) and a clone (OK2.21) from OK-432-immunized BALB/c mice (Iad) as antitumor effector cells. OK2 proliferated and secreted interleukin 2, but only when OK-432 was presented by Iad-positive antigen-presenting cells. Despite its helper phenotype and function, OK2 could kill OK-432-pulsed Iad-positive B-lymphoma cells. This killing was inhibited only by cold specific target cells (cold-target inhibition). OK-432 induced the cytotoxicity of OK2 as a specific antigen, not as a nonspecific immunostimulator. OK2 and OK2.21 also killed Ia-negative bystander target cells only in the presence of OK-432-pulsed Iad-positive cells (bystander killing). Double-chamber experiments suggested that the bystander killing was mediated by a short-acting soluble cytolytic factor. Finally, the OK-432-specific T-cells selectively killed tumor cells, suggesting that these T-cells play an important role in the immune surveillance against malignancy.

Enhanced production of interleukin-1 and tumor necrosis factor alpha by cultured peripheral blood monocytes from patients with scleroderma.

Cultured peripheral blood mononuclear cells from 20 patients with scleroderma were shown to produce high levels of interleukin-1 alpha, interleukin-1 beta, and tumor necrosis factor alpha. There were positive correlations between the production of these 3 molecules. Purified monocytes from these patients produced much more tumor necrosis factor alpha than did those from normal subjects, even without lipopolysaccharide stimulation. These results show the hyperactivity of monocytes from scleroderma patients.

[A case of polymyositis associated with chronic active hepatitis].

A case of polymyositis associated with chronic active hepatitis was reported. A 53-year-old man, who had no previous history of blood transfusion nor hepatitis, noticed proximal dominant muscle weakness on January 29, 1985. He was admitted to Kyoto National Hospital on February 7, and laboratory studies disclosed the elevation of serum enzyme levels; creatine kinase (CK) 9845 IU/L (normal 54-263), glutamate oxaloacetate transaminase (GOT) 834 IU/L (9-31), glutamate pyruvate transaminase (GPT) 491 IU/L (4-34), lactate dehydrogenase (LDH) 2135 IU/L (248-464). Also serum gamma globulin was high (1.8 g/dl) and LE-like cell was found. The diagnosis of polymyositis was made and prednisolone therapy (60 mg/day) was started on February 23. The elevated serum enzymes decreased gradually, but severe muscle weakness persisted for about one month. On April 3, he was admitted to our hospital. Physical examination revealed moderate proximal dominant muscle weakness without skin eruption, jaundice or hepatosplenomegaly. The serum enzymes were still high; CK 1826, GOT 173, GPT 232 (GOT less than GPT), LDH 1548. However, alkaline phosphatase (ALP) and bilirubin were normal. Hepatitis B surface antigen (HBsAg) was not detected. Antinuclear antibody was positive. The electromyogram study showed myopathic change, and the muscle biopsy demonstrated myopathic change and cell infiltration, compatible with polymyositis. These results suggested liver dysfunction associated with polymyositis. Prednisolone therapy was continued and muscle weakness decreased. From December, 1985, serum enzymes (CK, GOT, GPT, LDH) elevated again and muscle weakness also slightly increased. Anti-smooth muscle antibody was positive. It was suggested that both polymyositis and liver dysfunction deteriorated.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible different mechanisms of B cell activation in systemic lupus erythematosus and rheumatoid arthritis: opposite expression of low-affinity receptors for IgE (CD23) on their peripheral B cells.

To clarify the differential state of B cell activation in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), we investigated the expression of low-affinity receptor for IgE (Fc epsilon RII; CD23) on their peripheral B cells by a cytofluorometry using H107 (CD23) and Leu-16 (CD20) monoclonal antibodies. The percentage of CD23-negative B cells in total lymphocytes was significantly greater in both groups of patients than in normal subjects, suggesting the hyperactivity of late-phase B cells in both diseases. However, the increase of CD23-negative B cells in RA was brought about by the increased number of total B cells, although that in SLE was mainly based on the relative decrease of CD23-positive B cells. The number of IgD-positive B cells was decreased, and the number of colony-forming B cells was markedly increased in SLE patients. These observations indicate that a B cell abnormality is mainly qualitative in SLE but quantitative in RA.

Biological response modifier as antigen: OK432-specific T-cell clone as an anti-tumor effector cell.

OK432, a Streptococcus preparation, is a biological response modifier that has been used in the treatment of malignant diseases. A cloned Thy 1.2+, L3T4+, Lyt 2.2- T cell (OK2.21), specific for OK432 and restricted to I-Ed, was isolated from BALB/c mice immunized with OK432. OK2.21 not only secreted interleukin 2 but also killed Iad-bearing B-lymphoma cells in an antigen-specific manner. The clone also killed Ia-negative bystander tumor targets, but only in the presence of both OK432 and antigen-presenting cells. Despite the cytotoxicity against tumor cells, OK2.21 did not kill OK432-pulsed normal spleen cells. Injection of this clone together with OK432 to tumor-bearing BALB/c mice prolonged their survival, suggesting that this clone acts as an anti-tumor effector (or effector-inducer) cell in the therapy with OK432.

Enhanced production of interleukin-2 in patients with progressive systemic sclerosis. Hyperactivity of CD4-positive T cells?

A significant enhancement of interleukin-2 production was observed in phytohemagglutinin-stimulated peripheral lymphocytes from patients with progressive systemic sclerosis (PSS). Their Leu-3a:Leu-2a ratio was also significantly higher than normal. Moreover, Leu-3a+ cells freshly purified from PSS patients' blood produced much more interleukin-2 than did cells from normal subjects. These results suggest that T cell hyperactivity, especially among Leu-3a+ T cells, occurs in patients with PSS.

Systemic lupus erythematosus sera antilymphocyte reactivity: detection of antibodies to Tac-antigen positive T cell lines.

Sera obtained from patients with systemic lupus erythematosus (SLE) were tested for their reactivity to cell lines derived from cutaneous T-cell lymphoma (CTCL), or adult T cell leukaemia (ATL), and with other cell lines, by indirect immunofluorescence method. Approximately one half of SLE sera reacted with the surface antigens of HUT-102 cells, a cell line from CTCL, which constitutively expresses Tac antigen. The titre tended to be higher in the active than in the inactive stage. These positive sera also reacted with other neoplastic or normal T cell lines having Tac antigen. SLE sera reacting with HUT-102 surface antigens were further examined for their reactivities to Tac antigen, the putative IL-2 receptor, using HUT-102 or ATL-2. Pretreatment with anti-Tac monoclonal antibody partially blocked the reactivities to HUT-102 surface antigens in nine of 15 SLE sera tested. The binding of 125I-labelled anti-Tac monoclonal antibody was displaced by the addition of sera from six of 15 SLE patients. In addition, nine of the 15 SLE sera could inhibit the binding of 125I-labelled IL-2 to ATL-2 cells. These results suggested that some of SLE sera contained antibodies against the IL-2 receptor.

Tac antigen-positive T cells activated in autologous mixed lymphocyte reaction regulate the generation of killer T cells against hapten-modified autologous cells.

T cells that proliferate in the autologous mixed lymphocyte reaction (auto-MLR) have been shown to acquire some suppressor or regulatory activities. In the present study, we examined the suppressive effects of T cells activated in the auto-MLR on the induction of hapten-specific cytotoxic T cells. NRFT (depletion of ARFT from UT) were used as the responder cells of TNP-MLR. After primary and secondary TNP-MLR, the cells were harvested and tested for their cytotoxic activities against TNP-modified autologous cells by 51Cr-release assay. When UT cells cultured for 1 wk in auto-MLR were added to primary TNP-MLR at the beginning of culture, the cytotoxic activity tested at the end of the culture was suppressed from 15.6% +/- 2.7 to 5.8% +/- 1.1 (percent cytotoxicity, mean +/- SE). However, these auto-MLR-activated UT cells had little suppressive activity against cytotoxic T cells when they were added to the final assay of TNP-CTR. Suppressive activities of these cells on the generation of cytotoxic T cells during secondary TNP-MLR were also tested. The addition of auto-MLR-activated UT cells to the secondary TNP-MLR at the beginning of the culture reduced the cytotoxic activities of NRFT from 23.8% +/- 2.3 to 9.7% +/- 1.7 after secondary TNP-MLR. Allo-activated T cells, PHA blasts, and fresh autologous T cells were used as the controls, but none of the cells had suppressive effects on the generation of CTL. Characteristics of these suppressor cells were examined. Auto-MLR-activated cells from ARFT fractions exhibit very powerful suppressor activity. Treatment of the auto-MLR-activated T cells with mitomycin C eliminated their suppressive effects on the generation of CTL; 21.2% +/- 6.3 of UT cells became anti-Tac positive after 1 wk of auto-MLR. Treatment of auto-MLR-activated UT cells with anti-Tac antibody plus complement eliminated their suppressive activities on the induction of CTL. Thus, T cells stimulated in auto-MLR were shown to have suppressive effects on the induction of cytotoxic T cells against TNP-modified autologous cells. These cells were mitomycin C sensitive. Because anti-Tac antibody is reactive to activated T cells, activation of T cells during auto-MLR was thought to be necessary for the acquisition of the suppressive activity.

Quinacrine inhibits the primary but not secondary proliferative response of human cytotoxic T cells to allogeneic non-T cell antigens.

Quinacrine inhibited the generation of cytotoxic T lymphocytes from human peripheral blood T cells stimulated by allogeneic non-T cells as measured by [3H]thymidine incorporation as well as by cytolytic reactions against 51Cr-labeled allogeneic lymphocytes. These observations were further supported by the finding that quinacrine inhibited the expression of Tac antigen, the receptor for growth factor(s). Because other phospholipase A2 inhibitors such as tetracaine and p-bromophenacyl bromide could inhibit these reactions, the inhibition of cytotoxic T lymphocyte responses by quinacrine appeared to be attributable to the inhibition of phospholipase A2 in these cells. In keeping with this interpretation, arachidonate release from phospholipids in cytotoxic T cells stimulated by allogeneic non-T cells was inhibited by quinacrine. In contrast, the pretreatment of T cells with quinacrine did not result in the inhibition of their secondary proliferative response to the same stimulation with allogeneic non-T cells. These results, taken together, suggest that quinacrine does not inhibit the recognition of antigens by cytotoxic T cells, while it blocks the mitogenic response of T cells to allogeneic antigens.

Serum immunosuppression test as a new tool for immunodiagnosis of lung cancer.

The immunosuppressive activity of serum from lung cancer patients on the anti-sheep red blood cell plaque-forming cell response in mouse spleens was evaluated by an in vivo technique in 64 patients with lung cancer and in a control group composed of 53 patients with a variety of nonmalignant disorders and 15 healthy adults. When immunization with sheep red blood cells was done on Day 5 after serum injection, the frequency of immunosuppression by serum from lung cancer patients [50 of 64 (78%)] was significantly higher than with serum from patients with nonmalignant diseases [3 of 53 (6%); p less than 0.025] or from healthy adults [1 of 15 (7%); p less than 0.005]. In serum from lung cancer patients, there was no correlation between the serum immunosuppressive activity and histological classification. However, sera from Stage II patients showed a relatively high frequency of suppression [25 of 27 (93%)] compared with sera from the Stage I group [7 of 14 (50%); p less than 0.005]. In experiments examining the transfer of plaque-forming cell-suppressive activity, tests using 10(7) thymus cells and more than 10(5) spleen cells from mice given injections of lung cancer serum indicated that suppressive action is mediated by the generation of suppressor cells by lung cancer serum. The clinical use of this test in diagnosis and immunological studies of lung cancer is discussed.