Inhibition of human endothelial cell proliferation in vitro and neovascularization in vivo by D-penicillamine.

To investigate the effects of D-penicillamine (D-Pen) on angiogenesis, we have studied the effects of this drug on in vitro proliferation of human endothelial cells (EC) and in vivo corneal neovascularization. D-Pen, in the presence of copper sulfate, suppressed tritiated thymidine ([3H]TdR) incorporation into EC in a dose-dependent manner. Significant inhibition was observed with D-Pen concentrations attainable in the serum and tissues of treated patients. Neither D-Pen nor copper ion alone significantly affected [3H]TdR incorporation into EC. The inhibition by D-Pen and copper was blocked by catalase (CAT) or horseradish peroxidase but not by boiled CAT or SOD. When rabbits were daily injected intravenously with D-Pen at the per kilogram dosage administered to rheumatoid patients, neovascularization as quantitated by the proliferation of corneal new blood vessels was significantly inhibited. These results suggest that hydrogen peroxide generated by D-Pen and copper exerts a pronounced antiangiogenic effect through inhibition of EC proliferation. It is, therefore, considered that D-Pen may suppress rheumatoid synovitis by reducing the number of small blood vessels available for the emigration of chronic inflammatory cells, and the proliferation of the synovial tissue.

Immunomodulatory effects of therapeutic gold compounds. Gold sodium thiomalate inhibits the activity of T cell protein kinase C.

Previous studies have shown that the gold compounds, gold sodium thiomalate (GST) and auranofin (AUR), which are effective in the treatment of rheumatoid arthritis, inhibit functional activities of a variety of cells, but the biochemical basis of their effect is unknown. In the current studies, human T cell proliferation and interleukin 2 production by Jurkat cells were inhibited by GST or AUR at pharmacologically relevant concentrations. Because it has been documented that protein kinase C (PKC) is involved in T cell activation, the capacity of gold compounds to inhibit PKC partially purified from Jurkat cells was assayed in vitro. GST was found to inhibit PKC in a dose-dependent manner, but AUR caused no significant inhibition of PKC at pharmacologically relevant concentrations. The inhibitory effect of GST on PKC was abolished by 2-mercaptoethanol. To investigate the effect of GST on the regulation of PKC in vivo, the levels of PKC activity in Jurkat cells were examined. Cytosolic PKC activity decreased slowly in a concentration- and time-dependent manner as a result of incubation of Jurkat cells with GST. To ascertain whether GST inhibited PKC translocation and down-regulation, PKC activities associated with the membrane and cystosolic fractions were evaluated after phorbol myristate acetate (PMA) stimulation of GST incubated Jurkat cells. Translocation of PKC was markedly inhibited by pretreatment of Jurkat cells with GST for 3 d, but the capacity of PMA to down-regulate PKC activity in Jurkat cells was not altered by GST preincubation. The functional impact of GST-mediated downregulation of PKC in Jurkat cells was examined by analyzing PMA-stimulated phosphorylation of CD3. Although GST preincubated Jurkat cells exhibited an increased density of CD3, PMA-stimulated phosphorylation of the gamma chain of CD3 was markedly inhibited. Specificity for the inhibitory effect of GST on PKC was suggested by the finding that GST did not alter the mitogen-induced increases in inositol trisphosphate levels in Jurkat cells. Finally, the mechanism of the GST-induced inhibition of PKC was examined in detail, using purified PKC subspecies from rat brain. GST inhibited type II PKC more effectively than type III PKC, and also inhibited the enzymatic activity of the isolated catalytic fragment of PKC. The inhibitory effect of GST on PKC activity could not be explained by competition with phospholipid or nonspecific interference with the substrate. These data suggest that the immunomodulatory effects of GST may result from its capacity to inhibit PKC activity.

Changes in bone marrow blood flow with aging.

We investigated changes in bone marrow blood flow and histology with aging in rabbits. Bone marrow blood flow was measured by the hydrogen washout method, which was performed in the proximal tibia of Japanese white rabbits, 3-42 months of age. Bone marrow blood flow was found to decrease with age. Histological observations of the proximal tibia identified a decrease in the number of osteoblasts and hematopoietic cells, an increase of the number of empty lacunae, a reduction in bone formation rate, and a degeneration of marrow fat with aging. These results suggest that the decrease of bone marrow blood flow is related to histological changes found with aging.

An electron microscopic study of the changes observed in osteocytes under ischemic conditions.

The purpose of this study was to observe the process of ischemia in osteocytes using light and electron microscopy and to compare the changes in these ischemic osteocytes with those in other types of osteocytes (i.e., degenerative osteocytes in physiological states, steroid-induced lipid-accumulating osteocytes) that have been previously reported. Five female Japanese white rabbits were used in this study. Osteochondral chips were taken from one side of the femoral condyle, covered with Millipore filters, and then inserted into the other side of the knee joint. These tissues were examined after 12 h and after 2, 5, 8, and 14 days of ischemia under both light and electron microscopy. Under light microscopy, osteocytes and lacunae were classified into four types: normal osteocyte, pyknotic osteocyte, pale osteocyte, and empty osteocyte lacuna. The number of each type of osteocyte (or lacuna) in a settled area was counted. The ratio of normal osteocytes decreased significantly (p less than 0.001) after the second day of ischemia. Pyknotic osteocytes increased at 12 h (p less than 0.01) and 2 days (p less than 0.001) of ischemia. On the fifth day of ischemia, the percentage of pale osteocytes reached a peak. This was followed by a gradual increase in the number of empty lacunae. On the fourteenth day of ischemia, empty lacunae constituted greater than 40% of the cell types. When viewed by electron microscopy, these necrotic osteocytes were similar to the degenerative osteocytes that have been observed in physiological states and apparently different from lipid-accumulating osteocytes. The results suggested that there could be at least two types of necrotic processes in osteocytes that eventually lead to cell death.

Pathogenesis of chronic inflammation in experimental ferritin-induced arthritis. V. Electron microscopic localization of specific antibodies in the synovial membrane.

Antibody synthesis by the arthritic synovium of rabbits with antigen-induced arthritis has been demonstrated, but knowledge of the details of the specific localization of antibody in the membrane is limited. Specific antibody in the synovium of ferritin-induced arthritis was detected by electron microscopic observation of bound ferritin. The specific antibodies were localized in the cisternae of the endoplasmic reticulum and perinuclear spaces within plasma cells. In the extracellular matrix, specific antibody was found distributed as a network within collagen bundles and in the basement membrane of the subsynovial small blood vessels.

The effect of N-(2-carboxyphenyl)-4-chloroantranilic acid disodium salt (CCA) on concanavalin A-induced superoxide anion release in human mononuclear phagocytes.

Superoxide anion (O2-), generated and released by mononuclear phagocytes (M phi), plays significant roles in tissue damage accompanying inflammatory and immunological reactions. N-(2-carboxyphenyl)-4-chloroantranilic acid disodium salt (CCA), is a newly synthesized immunomodulator for which therapeutic efficacy in the treatment of rheumatoid arthritis (RA) has been reported. In this investigation, we have examined the effect of CCA on concanavalin A (Con A)-induced O2- release in human M phi. Exposure of M phi to Con A induced a large increase of O2- release. Preincubation of M phi with CCA suppressed O2- release induced by Con A almost to a control level. On the contrary, CCA itself did not have a significant effect on basal O2- release from M phi. These results indicate that the inhibition of O2- release from activated M phi may be an important factor in the anti-inflammatory effects of this drug.

Long-term results of synovectomy in rheumatoid patients.

Seventy-eight knees in fifty-five rheumatoid patients were evaluated clinically and radiographically at an average of 14.1 years (range, ten to twenty-three years) after synovectomy. The functional result was excellent in seventeen, satisfactory in thirty-one, unsatisfactory in eighteen, and poor in twelve knees. Pain and recurrent effusions were significantly reduced after synovectomy, but the range of motion of the knee remained unchanged. Radiographs demonstrated progressive changes of degenerative joint disease but apparent arrest of inflammatory disease changes in about 67 per cent of the knees. We believe that synovectomy of the knee is a good treatment alternative for selected patients with early stage-I rheumatoid arthritis that has proved resistant to standard medical therapy.

Steroid-induced accumulation of lipid in the osteocytes of the rabbit femoral head. A histochemical and electron microscopic study.

A high dose of methylprednisolone was administered intramuscularly to rabbits, and we studied the accumulation of lipid in the osteocytes of the femoral head by histochemical methods and electron microscopy. Advanced hyperlipidemia and a fatty liver were observed in four weeks. Electron microscopy was used to define the ultrastructural changes in osteocytes, which showed small lipid droplets that gradually enlarged, finally resulting in the formation of vacuolated vesicles. An increase in the size of the lipid droplets caused them to compress the nucleus to one side of the lacuna, resulting in discontinuities of the cell membrane followed by cell disintegration. These observations demonstrate the effect on osteocytes of a disturbance in lipid metabolism caused by the administration of large doses of steroids.

Fracture-separation of the distal humeral epiphysis in young children.

Fracture-separation of the distal humeral epiphysis in young children is frequently misdiagnosed as a fracture of the lateral humeral condyle or a dislocation of the elbow. The roentgenographic relationships between the ossification center of the capitulum humeri and the radius are important in distinguishing between the three conditions. Arthrographic studies in our patients showed that this is an extracapsular supracondylar fracture. In six fracture-separations treated either by open reduction and wire fixation or in overhead traction, the results were excellent.

An electron microscopic study of early pathology in chondromalacia of the patella.

For a study of the ultrastructural pathology of chondromalacia patellae, we studied biopsy specimens that had been obtained at operation in twelve young patients who had pain in the anterior aspect of the knee due to mechanical derangement and also had the pathological changes in articular cartilage that are characteristic of chondromalacia. The initial pathological finding was swelling of the superficial matrix associated with breakdown of the collagen fiber network, especially at matrix streaks in the superficial and transitional zones. Fibrillated cartilage contained surface fissures that penetrated the middle layers. Amorphous electron-dense material was found covering the internal surfaces of the fissures; this appeared to contain degraded components of the matrix. In association with these changes, there were formation of nests of cells (clusters of chondrocytes) and increased numbers of organelles in the chondrocytes. A limited repair reaction also was observed in some specimens; this was characterized by the migration of fibroblast-like cells over the surface of the cartilage. The ultrastructural observations on these specimens from patients who had chondromalacia were compatible with a pathogenesis resulting from mechanical overload.

Localization and identification of root canal bacteria in clinically asymptomatic periapical pathosis.

Twenty-one teeth with clinically asymptomatic periapical pathosis (class 3) were extracted and the isolation, identification, and localization of bacteria in the root apex were examined. Mixtures involving several bacteria were isolated from more than 60% of the cases. Scanning electron microscopy revealed bacterial masses to be associated with the apical part of the root canal, but not with the area of apical foramen or on the surface of root apex. Our results indicate that the bacteria in class 3 cases may be derived from organisms which colonized before or during endodontic treatment, but not from anachoresis. The bacteria-positive cases of asymptomatic periapical pathosis have the potential to progress to symptomatic periapical pathosis.

[Establishment and characterization of human lung cancer cell line (KUM.LK-2)].

Human lung cancer cell line, named KUM.LK-2 was established from xenograft implanted in nude mice and maintained for 43 months. And serially transplanted in nude mice. This line has the following biological characters. 1) Spontaneous lung metastases are made in nude mice inoculated subcutaneously. 2) This line can produce some kinds of proteases, like as tissue type plasminogen activator, urokinase type plasminogen activator and collagenase. 3) KUM.LK-2 has the ability of platelet aggregation.

Meniscal repair using fibrin sealant and endothelial cell growth factor. An experimental study in dogs.

The effect of fibrin sealant and endothelial cell growth factor on the healing of defects of the avascular portion of canine menisci was investigated in 30 menisci of 15 adult mongrel dogs. The defects were treated in one of three ways: Group 1, the defect was left empty; Group 2, the defect was filled with fibrin sealant; and Group 3, the defect was filled with fibrin sealant and endothelial cell growth factor. The healing process was evaluated macroscopically and histologically at intervals of 1, 2, 6, 12, and 24 weeks. The average percentage of each defect that was filled with connective tissue was 5% in Group 1, 76.6% in Group 2, and 89.4% in Group 3. In the early phase of this repair, the extrinsic pannus-like tissue that contained many capillary vessels extended from the meniscosynovial junction into the defect. Subsequently, organized fibrous connective tissue was formed, which had changed to cartilaginous tissue at 12 to 24 weeks. The combination of fibrin sealant and endothelial cell growth factor enhanced the neovascularization and formation of granulation tissue, which accounted for the increased healing level in the avascular portion of the meniscus.

Bone and joint lesions associated with pustulosis palmaris et plantaris. A clinical and histological study.

We have reviewed 41 patients with pustulotic arthro-osteopathy (PAO), all having both the typical skin rash of pustulosis palmaris et plantaris and bone lesions. The most common bones affected were the clavicle, sternum and ribs. Changes in the clavicle started, not as an enthesopathy, but with periosteal bone formation, indicative of a bone marrow disorder. About 30% of the patients also had lesions in the spine, sacroiliac region or the peripheral joints. Bone and joint lesions followed a variable and intermittent clinical course over a long period of time. Biopsies in eight cases showed similar inflammatory changes in skin, bone and synovium, with infiltration of lymphocytes and polymorphonuclear leucocytes. This suggests that there is a common pathogenesis in the three tissues.

The response of osteocytes to a lipid clearing agent in steroid-treated rabbits.

We studied the effect of a lipid clearing agent (clinofibrate) on the osteocytes of rabbits treated with corticosteroids. Thirty-one rabbits were divided into four groups: (A) steroid-treated with a normal diet, (B) steroid-treated and one a diet with added clinofibrate, (C) non-steroid-treated, on a diet with clinofibrate; and (D) non-steroid-treated on a normal diet. All the steroid-treated animals demonstrated hyperlipidaemia and fatty degeneration of the liver. Lipid-containing osteocytes were seen in the femoral heads of these animals. However, those which received clinofibrate (group B) had less severe lipidaemia, and less severe degeneration of the liver. In them, only the osteocytes around the haversian canals exhibited lipid inclusions. Clinofibrate appears to modify lipid metabolism, diminishing the steroid induced accumulation of lipids within osteocytes. This effect may protect against steroid-mediated osteonecrosis.

The osteogenetic potential of fracture haematoma. Subperiosteal and intramuscular transplantation of the haematoma.

We studied the precise role of the fracture haematoma in healing by the experimental transplantation of the haematoma at two days and four days after fracture of the rat femur to subperiosteal and intramuscular sites. We used bone marrow and peripheral blood haematomas for control experiments. The transplanted two-day fracture haematoma produced new bone by endochondral ossification at the subperiosteal site, but not at the intramuscular site. Four-day fracture haematoma produced new bone formation at both subperiosteal and intramuscular sites. These results suggest that fracture haematoma has an inherent osteogenetic potential.

Inflammatory cell changes in haversian canals. A possible cause of osteoporosis in rheumatoid arthritis.

We studied the morphology of the haversian canals in the osteopenic cortical bone of the medial femoral neck from patients with rheumatoid arthritis and compared the findings with those in patients with osteoarthritis and with uncomplicated coxa valga. In the rheumatoid bone, the diameters of the canals were larger and many more contained osteoclasts. Fewer haversian canals showed only lining cells than in the osteoarthritic or coxa valga patients. In bone from rheumatoid patients, especially in canals with osteoclasts, small blood vessels were frequently lined by tall endothelial cells with an infiltration of mononuclear cells. These morphological differences are discussed with reference to the possible mechanisms of loss of cortical bone in rheumatoid arthritis and other conditions.

Solitary paralysis of the triceps muscle due to trauma.

We report a rare solitary paralysis of the triceps muscle. The patient, an 18-year-old man who had had a work-related accident, sustained a severe contusion of the proximal posterolateral arm after being struck by a piece of lumber. This injury produced an extraordinary amount of soft-tissue swelling in the region of the trauma. After the accident it was noted that active flexion of the elbow was preserved, but active extension was absent. Three months after injury, surgical exploration of the radial nerve and its rami musculares to the three heads of the triceps muscle was undertaken. Dense adhesions were found fixing the rami musculares to the surfaces of the triceps muscle. Neurolysis of the rami musculares was performed, leading to complete recovery of the triceps muscle. The etiology of this isolated paralysis of the triceps muscle was determined to be dense adhesions compressing and compromising the function of the rami musculares to the three heads of the triceps muscle. These adhesions formed because of severe posttraumatic bleeding into the potential posterior humeral space, where the rami musculares are found.

Anatomical study on the menisco-femoral ligaments of the knee.

The menisco-femoral ligaments are important accessory ligaments of the knee. They extend from the posterior horn of the lateral meniscus to the lateral aspect of the medial femoral condyle close to the femoral attachment of the posterior cruciate ligament. Most of the authors reported that in some cases these ligaments are absent. But in the 100 knees that we carefully examined, obvious menisco-femoral ligaments were identified in all of the specimens. When the anatomical relationship between the menisco-femoral ligaments and the lateral meniscus is considered, it can be easily speculated that the menisco-femoral ligaments draw the posterior horn of the lateral meniscus in anterior, medial and superior direction during flexion of the knee joint. Thus, the congruency between the lateral meniscus and femoral condyle and the stability of the knee joint increase.

Interaction of macrophages with rheumatoid articular cartilage.

These experiments investigated the in vitro interaction of macrophages and rheumatoid articular cartilage. When rheumatoid articular cartilage samples were incubated with normal peripheral blood mononuclear cells (PBM) for 1 hour or 12 hours in vitro, there were large numbers of PBM attached to the articular surface, and some PBM invaded the cartilage during the 12 hour incubation period. Conversely, in cartilage from osteoarthritic and normal subjects in the control, only a few PBM were attached to the articular surface after incubation with PBM. Under electron microscopic observation, macrophages were tightly attached to the surface and within the eroded cartilage. Light and electron microscopic observation with immuno-peroxidase staining of the rheumatoid cartilage surfaces showed the deposition of immunoglublins adjacent to the attached macrophages, suggesting the presence of immune-complexes as chemoattractants. These results suggest that macrophages in a rheumatoid joint may play an important role in the formation of pannus, resulting in the destruction of cartilage to interact with immune-complexes trapped in the superficial region of the rheumatoid articular cartilage.