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A multimeric peptide, Ac-Y16, consisting of 16 YIGSR sequences from laminin was evaluated for its effect on experimental metastasis, angiogenesis and tumour growth of HT1080 human fibrosarcoma cells. Co-injection of 0.5 mg per mouse of Ac-Y16 i.v. with HT 1080 cells inhibited lung colonisation by 100%, whereas 0.5 mg per mouse of monomeric Ac-YIGSR-NH2(AcY1) inhibited by 94%. Ac-Y16 did not show any direct cytotoxicity in tumour cells in vivo. The effect of the peptides on angiogenesis and tumour growth respectively were evaluated by counting areas of neovessels and weighing tumours after the s.c. implantation of HT1080 cells with basement membrane extracts and the peptide into nude mice. Co-injection of 0.5 mg per mouse of AC-Y16 s.c. with HT1080 cells inhibited angiogenesis and tumour growth by 92% (P0.05) respectively. It can be inferred from these data that anti-tumour effects of Ac-Y16 are likely to result from anti-angiogenesis. Intraperitoneal administration of Ac-Y16 was also effective in inhibiting angiogenesis, tumour growth and lung colonisation of HT1080 cells. It was concluded that the multimeric YIGSR-containing peptide, Ac-Y16, inhibits angiogenesis, tumour growth and experimental metastasis more than the monomeric form and that it is active when administered i.p., iv. and s.c. Images Figure 3

Several difficult problems arise in the treatment of necrotic bone disorders such as femoral head necrosis or Kienböck's disease for which no definitive treatment as yet exists. The recent literature has recommended vascularized bone transfers as suitable for these disorders, but there have been few experimental studies done and resulting effects are still currently obscure. Using a rabbit model, the effects of vascularized bone graft on the surrounding necrotic bone were analyzed. Results suggest that the periosteal muscle cuff remains as fibrous or fatty tissue and interferes with the bony union between the graft and the host bone; the graft was found to be gradually resorbed. The main function of the procedure appears to be the provision of vessels into avascular bone.

The laser speckle method is a new form of flowmetry that can obtain a two-dimensional distribution of blood flow in tissue. This method is a noncontact, simple, and rapid technique that may aid in the diagnosis of osteonecrosis. We investigated whether the subchondral bone blood flow within the femoral condyles of rabbits could be measured by the laser speckle method. The hydrogen washout method was chosen as a comparison technique because of its ability to allow repetitive measurements of blood flow in various conditions in one rabbit and because of its reliability, which already has been established. We simultaneously measured the bone blood flow in 20 femoral condyles of 10 rabbits with the laser speckle and hydrogen washout methods and found a significant correlation between the blood flow levels with use of these two methods. For the clinical application of the laser speckle method, we also investigated the influence of cartilage thickness on the measurements and the depth in the bone to which blood flow could be measured with this method. A cartilage thickness of 0.2 mm did not influence the measurement of the bone blood flow, and the depth in the bone to which the laser speckle method could be used was approximately 2 mm.

Human macrophage inflammatory protein-1 alpha (hMIP-1 alpha), also known as LD78, is a member of the chemokine/ intercrine family and an inhibitor of the proliferation of the hematopoietic stem cells in vitro. Using a specific monoclonal antibody, we observed significant localization of hMIP-1 alpha in eosinophilic myelocytes in human bone marrow. We further examined the expression of hMIP-1 alpha mRNA in human bone tissue by in situ hybridization. A high level of hMIP-1 alpha mRNA expression was detected in eosinophilic myelocytes in bone marrow, confirming these cells as the site of hMIP-1 alpha synthesis. hMIP-1 alpha mRNA expression was also detected in osteoblasts in the bone-remodeling sites, and osteoclasts were frequently observed in the vicinity of these osteoblasts. hMIP-1 alpha was also able to induce osteoclastogenesis on calcified matrices in the absence of any other osteotropic hormones. These results strongly suggest that hMIP-1 alpha is involved not only in the regulation of hematopoiesis but also in the modulation of bone remodeling.

We describe a quantitative assay for assessing tumor angiogenesis in vivo using basement membrane extracts (Matrigel). Nude mice were injected s.c. with liquid Matrigel mixed with HT1080 human fibrosarcoma cells. Since Matrigel rapidly forms a solid gel at body temperature, the gel containing tumor cells can be removed immediately and then processed for histological studies. Tumor angiogenesis was monitored quantitatively by measuring both the number and the total area of neovessels present in the gels using an image analyzer, which could be achieved approximately 72 hr later. Furthermore, HT1080 cell-conditioned medium, which may contain various tumor-derived factors, promoted the basement membrane degradation, migration, proliferation and tube formation of endothelial cells in vitro, as did Matrigel, although to a lesser extent. In addition, Northern blot analysis demonstrated that the amount of vascular endothelial growth factor (VEGF) mRNA in HT1080 cells was much higher than that in human fibroblasts or NIH3T3 cells. Our results suggest that angiogenesis observed in our assay may be due to the synergic effects of tumor angiogenic factors such as VEGF, and Matrigel. The advantages of our assay are: 1) it is possible to assess early angiogenesis quantitatively; and 2) this assay may be applicable for screening anti-angiogenic therapeutic agents to be used against human neoplasms.

Skeletal immature rabbits were used to study the pathogenesis of Osteochondritis Dissecans (OCD). Both histological studies and a radiographical examination were utilized after sagittal and coronal surgical chondral fractures were made in the femoral condyles cartilage. Serial microangiographies were performed in rabbits between 0 and 84 days after the chondral fractures were made. Analyses of the histology and microradiography findings suggest in either a coronal or sagittal direction, that avascular lesions like an experimental OCD occur as a sequence of chondral injury. A fracture in a wide pedicle of a stable cartilaginous flap with abundant cartilage canals heals in the usual way. However, a fracture in an unstable fragment with a small isthmus devoid of cartilage canals and of nutritious vessels, probably doesn't heal completely and a fragment closely resembling OCD is instead formed. An experimental OCD depends on the slender hinge of the flap and on the lack of stability in a rabbit's non-ossified epiphyseal cartilage. The damage to the cartilage canals and the rupture of vessels in the canals by a chondral fracture and the disturbance in the revascularization in the healing process by abnormal mechanical forces are thus most likely considered to be the main factor for OCD production.

Tissue inhibitors of metalloproteinases (TIMPs) play an important role in regulating the activity of matrix metalloproteinases (MMPs). Tumor cell invasion and metastasis closely correlate with the activities of two members of MMPs, MMP2 and MMP9, both of which degrade type IV collagen in basement membranes. We herein report that the treatment of HT1080 cells with 8-bromo-cAMP and other chemicals that activate cyclic adenylase activity induces the expression of TIMP1 and TIMP2 both at the mRNA and the protein levels and that this induction of TIMPs correlates with suppression of invasive phenotypes of HT1080 cells. Treatment with various cAMP-elevating reagents induced the expression of TIMPs and MMP2 in HT1080 cells, whereas the expression of MMP9 was not significantly affected. The protein amounts of TIMP1, TIMP2, and MMP2 secreted into the medium from HT1080 cells treated with 1 mM 8-bromo-cAMP were 7.9-, 9.3-, and 8.5-fold higher than those secreted from untreated cells, respectively. Induction of these mRNAs by 8-bromo-cAMP was blocked by HA1004, a protein kinase A inhibitor, but not by calphostin C, a protein kinase C inhibitor. Cycloheximide abolished the induction of TIMPs and MMP2 mRNAs by 8-bromo-cAMP, indicating that the induction depends on a newly synthesized protein(s) whose expression may be regulated by cAMP. Type IV collagenolytic activity and the invasiveness of HT1080 cells, both of which were suppressed by 8-bromo-cAMP, were efficiently restored when the cells were exposed to anti-TIMP antibodies, demonstrating the importance of the increased levels of TIMP1 and TIMP2 proteins for the cAMP-mediated suppression of both type IV collagenolytic activity and the invasiveness of HT1080 cells.

The histogenetic and biologic features of peripheral neuroepithelioma (PN) are still a matter of controversy. More detailed analyses might be facilitated by permanent PN cell lines. However, there have been only a few reports on the establishment of a PN cell line.We established and characterized a PN cell line and further carried out experiments for potentiality of drug-induced neural differentiation.The cultured cells were spindle shaped with slender cytoplasmic processes. The histologic features of transplanted tumors in nude mice were essentially the same as those of the original tumor. Immunohistochemically, the neuroectodermal characteristics of the cell line were demonstrated by positive stain for neuron specific enolase, glial fibrillary acidic protein, S-100 protein, neurofilament, and beta 2-microglobulin. Ultrastructurally, microtubules and dense core granules were observed in the cultured cells. Northern blot analyses revealed that c-myc was overexpressed in the cell line, whereas N-myc was not. The cell line showed neural differentiation after treatment with cAMP elevating reagents and nerve growth factor (NGF). The synergistic effects of cAMP analog and NGF on the neurite outgrowth were also observed. Furthermore, a cAMP-dependent protein kinase (PKA) inhibitor strongly blocked the action of cAMP analog, although it was not able to inhibit the same action of NGF. These findings suggested that the neurite outgrowth induced by the cAMP analog requires activation of PKA, whereas the same actions of NGF do not mediate such a pathway involving PKA activation.This is, to our knowledge, the first NGF responsive cell line derived from PN. This cell line might be valuable for further investigations of signal transduction pathways induced by cAMP analogs and NGF, for more precise analyses of the biological characteristics of PN, and finally, for the identification of differences between PN and several related tumors.

We have used an extract of basement membranes which can be reconstituted into a biologically active gel matrix composed predominantly of collagen IV, laminin, nidogen, and heparin sulfate proteoglycan, in order to study the mechanisms involved in tumor cell invasion. When layered onto a porous filter in a Boyden chamber, the gel forms a barrier to the passage of normal cells. Malignant cells are able to cross this layer when the conditioned medium of NIH 3T3 cells is used as a chemoattractant to stimulate cell migration. A variety of human tumor cells have thus been studied in this system and we find a high correlation between their invasiveness in vitro and their malignant behavior as exhibited in vivo. We have used this in vitro invasion assay to test for factors which might inhibit tumor cell invasion. Collagenase IV is produced by malignant cells and is thought to be required for invasion. Indeed, inhibitors of this enzyme have demonstrated reduced tumor cell invasiveness. One site of five amino acids, on the B1 chain, which has been shown to promote cell adhesion, migration and binding to laminin receptor, was found to inhibit the invasion of tumor cells. In addition, factors which elevated cAMP levels were also able to suppress the invasiveness of tumor cells. These data suggest that the assay system described herein can be successfully utilized to study the invasive activity of tumor cells and those factors that may inhibit the spread of malignant cells.

The role of the cartilage canals of the epiphysis was investigated by studying the process of initial enchondral osteogenesis of the secondary ossification center using decapitated femoral heads of one day old Japanese rabbits. Specimens of decapitated femoral heads returned to the original acetabulum demonstrated granulation tissue invasion at ten days after birth, followed by the normal process of enchondral osteogenesis of the secondary ossification centers. In contrast, specimens of heads inserted in the peritoneal cavity to prevent invasion of granulation tissue showed no enchondral osteogenesis of the secondary ossification center, and the major portion of the decapitated femoral head was replaced by necrotic chondrocytes and empty lacunae. The results of this study confirm that the cartilage canal is important not only in the nutrition of the chondroepiphysis but also in the initial enchondral osteogenesis of the secondary ossification center.

Using a whole-limb graft model in rats, morphologic changes and variations in the expression of Ia antigen on epidermal cells were investigated in the allografted skin during acute rejection. BN right limbs were transplanted to F344 recipients. Skin tissues were excised during acute rejection on days 1, 3, 5, 7, and 9 after the transplantation. Sections were examined for Ia antigen expression using immunohistologic techniques, and in situ quantification of Ia antigen was made using an immunogold method. Epidermal keratinocytes expressed Ia antigen before the grafts were rejected and the amount of Ia antigen expression increased and exceeded the amount of Ia antigen of Langerhans cells during the course of rejection. The progressive increase in class II antigen expression on EKs correlated with the appearance and relative accumulation of dermal lymphocytic cells. On the other hand, Ia antigen was not expressed on vascular endothelial cells during rejection. Our results suggest that the Ia-positive keratinocytes can serve as target cells in skin rejection of limb allografts. The immunogold technique we used seems most pertinent for a quantitative examination of cell-surface antigens in situ.

The authors studied how bone reacts to hydroxyapatite (HAP) and alumina (Al 2 O 3 ) in osteoporosis experimentally induced in a rat animal model by resection of the sciatic nerve. Sixteen and 32 weeks after resection of the sciatic nerve, implants of HAP and Al 2 O 3 were inserted into rat tibias. The reaction of bone to HAP was compared with its reaction to Al 2 O 3 , which is bioinert, as a control. The results were analyzed using the affinity index (the length of bone directly opposed to the implant without an intervening fibrous membrane/total length of the bone-implant interface × 100%). In the rats left for 16 weeks after resection of the sciatic nerve the index values 24 weeks after insertion of HAP and Al 2 O 3 were 96.9 ± 2.4% (N = 8) and 75.2 ± 22.2% (N = 8), respectively. The index value of HAP was 97.2 ± 2.6% (N = 12) and of Al 2 O 3 was 86.9 ± 15.4% (N = 12) at 24 weeks after insertion, when the rats were first left for 32 weeks, after sciatic nerve resection. Bone was demonstrated to have a superior affinity to HAP, compared with Al 2 O 3 ( P

The effect of the new immunosuppressive agent FK-506 on rat limb allograft was investigated across the BN-to-F344 histocompatibility barrier. A 14-day course of FK-treatment at doses of 1 mg/kg per day or more, begun on the day of operation, significantly increased the period of graft survival. Additional studies demonstrated that single treatment with FK only on the day of operation prolonged the graft survival in a dose-dependent manner. These results stress the profound immunosuppressive effect of FK and suggest the possible future application of composite tissue allograft in humans.

To observe the natural history of chondral injury and clarify the genesis of osteochondritis dissecans, various types of experimental osteochondral or chondral injuries were made on the articular surface of femoral condyles of adult and young rabbits. Pathological features resembling osteochondritis dissecans were frequently observed in young rabbits, especially in the groups in which the injured part was most unstable (58% in rabbits aged from 7 to 14 days, 24% in those aged from 3 to 7 weeks). Therefore, a neglected chondral injury in early childhood and following ossification disturbances may relate to this disease.

lambda DNA (a double-stranded DNA) was exposed to several adriamycin-mediated active oxygen generating systems (O2- and H2O2 generating, OH generating, and perferryl ion complex generating), extracted, and analyzed by gel electrophoresis on agarose gel. Only the DNA exposed to and subsequently isolated from the adriamycin-mediated OH generating system contained many DNA fragments of low molecular weight, indicating the breakage of DNA strands. Such a breakage was strikingly inhibited by catalase or 50 mM sodium benzoate, but not by superoxide dismutase. The local OH concentration near the DNA strand was considered to be important for DNA strand cleavage.

Administration of 2-acetylaminofluorene to rats for 12 weeks induces hyperplastic nodules (HPNs) and later well-differentiated hepatocellular carcinomas (HCCs) in the liver. Total cellular proteins from normal liver, HPN, and HCC were analyzed by two-dimensional gel electrophoresis with a high resolution. Several hundred polypeptides were well resolved as seen by Coomassie blue staining, forming a reproducible and characteristic pattern for each tissue. The polypeptide patterns were very similar among normal liver, HPN, and HCC. Especially the proteins of HPN and HCC were almost indistinguishable. These neoplastic lesions, however, were clearly different from control liver in that a new spot p35-6.6 (designated by molecular weight X 10(-3) and pl) appeared, and five polypeptides, p57-6.9, p57-6.7, p26-6.9, p26-6.6, p26-6.4, increased dramatically in amount as compared with normal liver. These last three spots were found to be a new type of glutathione S-transferase as judged from the specific binding to the antibody. The same changes in polypeptide pattern were found in HCCs induced by other chemical carcinogens, diethylnitrosamine and 3'-methyl-4-dimethylaminoazobenzene, but not in regenerating and neonatal livers. Fetal liver showed a rather different pattern than adult liver, but only p26-6.6 was increased among the spots characteristic of HPN and HCC. Protein phosphorylation was also examined for these cells by incubating tissue slices with 32PO4. After alkali treatment of the gels to eliminate serines phosphorylation, several dozens of phosphoproteins were clearly detected. The patterns of the labeled spots were again very similar among control liver, HPN, and HCC. Only the intensity of a spot designated p57-6.6 increased markedly in both HPN and HCC. This spot was further resolved by an expanded pH gradient into four distinct spots, the major one of which contained phosphothreonine. Similar changes in phosphorylation were noted in hepatomas induced by diethylnitrosamine and 3'-methyl-4-dimethylaminoazobenzene but not in regenerating, fetal, and neonatal livers. These changes are discussed in terms of gene expression relevant to malignant transformation of hepatic cells.

The cytoplasmic components of chondrocytes from the various zones of the rat costochondral junction, grown in vitro, were quantitated from electron micrographs using a point-counting technique, and the results were compared statistically with the cytoplasmic components of chondrocytes in the corresponding zones of the junction in vivo. Detectable changes occurred in vitro as early as after six hours of culture. The major changes that occurred in vitro were: an increase in the amount of lipid bodies in chondrocytes in the hypertrophic zone and in the bottom portion of the proliferative zone, failure of the chondrocytes in the bottom portion of the hypertrophic zone to increase in size, retention of intactness of the cell membrane of chondrocytes at the base of the hypertrophic zone, a decrease in the number of mitochondria in chondrocytes in the hypertrophic zone and in the middle and bottom portions of the proliferative zone, and a decrease in the number of holes in the cytoplasm of chondrocytes in the bottom one-half of the hypertrophic zone. In vitro hypertrophic chondrocytes of the growth plate demonstrated cellular changes associated with aging, while in vito the hypertrophic chondrocytes demonstrated changes associated with degeneration. The final result in both cases was cell death.