Early lesion of N-nitroso-N-ethylurea-induced hamster neurofibromatosis model.

To investigate histogenesis of neurofibroma, early lesions of N-nitroso-N-ethylurea-induced neurofibroma of Syrian golden hamsters were examined. The lesions were detectable from 8 weeks of age, and a total of 14 lesions in 9 hamsters from 8 to 12 weeks of age were observed. Most of the lesions were found in the skin; they were also observed in the trigeminal nerve, cervical plexus, and abdominal sympathetic ganglion. Histological examination revealed there were two types of early lesions such as solitary and plexiform types. The former developed in the s.c. or dermal part of the skin and showed invasive growth of the surrounding tissue, while the latter originated from the large nerves such as the trigeminal nerve or cervical plexus. Growth kinetics of the early lesions was quantitated by continuous administration of bromodeoxyuridine and double immunostaining. Using these systems we observed that various kinds of cells had already participated in early lesions and later Schwann cells became a main component of the tumor. The histogenesis of neurofibroma was considered to be complexed by proliferation of several kinds of cell types at the early stage.

Dynamics of lymphocytic subpopulations in Friend leukemia virus-induced leukemia.

The in vivo roles of the immunosurveillance mechanism of the host against leukemia induced by Friend leukemia virus (FLV) were examined. The significance of T-cells in host defense against FLV-induced leukemia was indicated by the fact that thymus-deprived C57BL/6N-nu/nu mice were sensitive to FLV, although normal C57BL/6N mice were, as already reported by many authors, resistant to FLV. In relation to the role of T-cells on the onset of FLV-induced leukemia, the population dynamics of the lymphocytic subpopulations of the systemic lymphoid organs after FLV injection in FLV-resistant C57BL/6N mice were examined in comparison with the dynamics in FLV-sensitive strains, C57BL/6N-nu/nu mice and normal C3H/HeN mice. In this system, Lyt-1+2- helper T-cells in the spleen of FLV-resistant C57BL/6N mice increased in number after FLV injection. The number of immunoglobulin positive cells did not remarkably change in FLV-resistant C57BL/6N mice after FLV injection, whereas the number increased in the lymph node of FLV-sensitive C3H/HeN mice. The results indicated that a major contribution to the relative susceptibility and resistance of the host to FLV was controlled by the capacity to mobilize T-cells to the spleen in an early stage of disease, although the interaction of these T-cells with other immune cells may play an important role in mediating host resistance to FLV-induced disease.

Friend leukemia virus-induced leukemogenesis in fully H-2 incompatible C57BL/6-->C3H radiation bone marrow chimeras.

Resistance and/or susceptibility for Friend leukemia virus (FLV)-induced leukemogenesis was examined in the fully H-2 incompatible C57BL/6 (B6)-->C3H radiation bone marrow chimeras (RBMC). The results indicated that B6-->C3H chimeras never developed FLV-induced leukemias when infected with FLV 4 months after bone marrow transplantation (BMT). Spleen cells from B6-->C3H chimeras that were preimmunized with 100 Gy-irradiated FBL-3 cells (FLV-induced leukemic cell line originated from B6 mice) were shown to generate anti-FBL-3 specific T-cell proliferation as well as cytotoxic T cells. We also found that when bone marrow cells from B6 mice were mixed with those from C3H mice and then grafted into supralethally irradiated C3H mice, resulting chimeras whose peripheral blood contained less than 30% C3H-derived (susceptible) cells were refractory to FLV-induced leukemogenesis. On the other hand, when C3H mice were infected with FLV and then supralethally irradiated 5 days later and grafted with bone marrow from B6 donors, they developed leukemias which were of B6 origin. Athymic nu/nu mice of B6 background were again shown to develop leukemia following infection with FLV. Possible implication of these findings on the role of T cell-mediated immune response in resistance to FLV-induced leukemogenesis and the immunocompetent nature of fully H-2 incompatible RBMC were discussed.

Specific incorporation of 4-S-cysteinylphenol into human melanoma cells.

The incorporation of 4-S-cysteinylphenol (4-S-CP), a tyrosine analog, into malignant melanoma cells was evaluated. 4-S-CP was specifically incorporated into the melanotic melanoma cells (HMV-II), which have activity for melanin synthesis, but was scarcely incorporated into HeLA S3 or HMV-I cells, which have no activity for melanin synthesis. Electron microscopic autoradiography revealed that the intracellular localization of 4-S-CP was closely correlated with melanogenesis and that 4-S-CP served as an initial substrate for tyrosinase and was utilized in melanin synthesis. On the basis of these findings we hypothesize that tyrosinase is required for intracellular incorporation of 4-S-CP. This specific incorporation of 4-S-CP into melanoma cells should be useful in the development of an effective procedure for chemotherapy of malignant melanomas and in analysis of melanin synthesis.

The cytotoxicity of cysteinylcatechols and related compounds to human melanoma cells in vitro.

L-3,4-Dihydroxyphenylalanine (L-dopa) and its structural analogs are known to be potently cytotoxic to melanoma cells. We examined the effects of cysteinylcatechols and related compounds, which were newly synthesized as cysteinyl derivatives of L-dopa, on the growth of human melanoma cells in vitro, and their actions were compared with those of L-dopa. 4-S- and 3-S-Cysteinylcatechols showed significantly more potent cytotoxicity to melanoma cells than did L-dopa, and 2-S-cysteinylhydroquinone was next to the catechols in potency. The mechanism of action may involve interaction with the melanocyte-specific enzyme, tyrosinase, for which the cysteinylcatechols could become a better substrate than L-dopa itself. 4-S-Cysteaminylphenol was almost comparable to L-dopa in cytotoxicity, suggesting that this phenol might be oxidized to the corresponding catechol by tyrosinase within the melanoma cells.

Mechanism of selective toxicity of 4-S-cysteinylphenol and 4-S-cysteaminylphenol to melanocytes.

Our previous studies showed that 4-S-cysteinylphenol (4-S-CP) and 4-S-cysteaminylphenol (4-S-CAP) inhibit the growth of malignant melanoma and cause depigmentation of black skin. In this study we examined kinetic constants of CP and CAP as substrates for tyrosinases and their properties as sulphydryl scavengers. 4-S-CP and 4-S-CAP were found to be much better substrates for mushroom tyrosinase than L-tyrosine while their 2-S isomers were not the substrates. 4-S-CP and 4-S-CAP were also good substrates for mammalian tyrosinase. Upon tyrosinase oxidation the two phenols conjugated with cysteine to form the cysteinyl derivatives of the corresponding catechols via o-quinone forms. The tyrosinase oxidation product of 4-S-CP had a poor ability to conjugate with alcohol dehydrogenase, a sulphydryl enzyme, while that of 4-S-CAP had a much higher ability. These results suggest that in melanocytes these phenols are oxidised by tyrosinase to the corresponding o-quinone forms, some of which conjugate with sulphydryl enzymes through cysteine residues, thus exerting cytotoxic effects.

Expression of the proliferating cell nuclear antigen in bone marrow cells from patients with myelodysplastic syndromes and aplastic anemia.

To determine the proliferative activity of the hematopoietic cells under nonneoplastic and/or neoplastic conditions, the expression of a cell cycle-related antigen, the proliferating cell nuclear antigen (PCNA), was examined in the bone marrow trephines of 79 individuals, 12 of whom had no hematologic disorder, 32 of whom had a diagnosis of myelodysplastic syndromes (MDSs), 20 of whom suffered from aplastic anemia, and 15 of whom had a diagnosis of acute myeloid leukemia. Most of the patients with MDS had more than 15% PCNA-positive cells (23.5% +/- 1.5%) while patients with no hematologic disorder showed fewer than 15% PCNA-positive cells (11.7% +/- 0.7%). The overall ratio of the PCNA-positive cell fraction in the bone marrow was considered of prognostic value for predicting transition into overt leukemia from MDS. Aplastic anemia cases usually exhibited hypocellular bone marrow and an infrequent labeling with the anti-PCNA antibody (3.3% +/- 0.5%). However, a few aplastic anemia cases showed hypercellular bone marrow and a significantly high PCNA-positive cell ratio (32.0% +/- 4.4%). In the bone marrow of acute myeloid leukemia patients more than 20% of total nucleated cells were positive for PCNA (30.0% +/- 2.2%). The results suggest that the expression of PCNA is associated with the regulation of bone marrow cell proliferation and the bone marrow cellularity, and that these findings would serve as an early indicator of evolution of overt leukemia in MDS and also would be useful in distinguishing MDS cases from aplastic anemia cases when the bone marrow is hypocellular or normocellular.

Association of Sjögren's syndrome with pulmonary hypertension: report of two cases and review of the literature.

We report two autopsy cases of Sjögren's syndrome associated with pulmonary hypertension. The pulmonary muscular arteries of both cases showed concentric fibrocellular intimal proliferation, medial hypertrophy, and plexiform lesions. To determine the significance and pathogenesis of this rare association, we carried out morphometric and immunofluorescent studies and reviewed the seven similar cases reported in the literature. Depositions of immunoglobulin G, Clq, C3c, C4, and C5 were observed in the pulmonary arterial walls of both of our cases. Morphometric studies revealed increased medial thickness to radius ratios and intimal thickness to radius ratios of the pulmonary muscular arteries in both cases. Previously reported patients were all female, and those cases were frequently associated with Raynaud's phenomenon. This report provides additional and convincing evidence for an association of Sjögren's syndrome and plexogenic pulmonary hypertension based on a detailed study of two cases and a review of the literature. The significance and pathogenesis of this association were examined, but not clarified. However, our studies add to the accumulating data suggesting a link between autoimmune diseases and chronic pulmonary hypertension.

Idiopathic cardiomyopathy: the pathologic roles of arteriolopathy.

Structural alterations and pathologic features of arteriolopathy of the heart were studied in 12 patients with idiopathic cardiomyopathy, five with hypertrophic cardiomyopathy (HOCM), and seven with congestive cardiomyopathy (COCM). The patients, six men and six women, ranged in age from 25 to 66 years. Diagnoses of idiopathic cardiomyopathy were confirmed clinically and at autopsy. Heart weights ranged from 350 to 700 g. Dilatative hypertrophy of both ventricles was observed in four patients, dilatation of both ventricles in six patients, and concentric hypertrophy of the left ventricle in two patients. The ventricular septa ranged in thickness from 7 to 27 mm, and the septal:free wall ratios ranged form 1.0 to 1.8. The lumens of extramural coronary arteries showed less than 30 per cent narrowing. Sections of the heart and all body organs were examined. The myocardia showed microscopic areas of hypertrophy, degeneration and disorganization of muscle cells, interstitial fibrosis, and various features of arteriolopathy. Histometric studies showed that hypertrophy of muscle cells was greater in HOCM than in COCM, that the content of connective tissue in the myocardium was higher in COCM, and that interstitial fibrosis was greater in the epicardial zone than in the endocardial zone in COCM but was greater in the endocardial zone in HOCM. Of the arteriolar changes, those of smooth muscle cells and internal elastic lamina were observed most frequently; no arteriolar narrowing or stenosis was observed in either HOCM or COCM. The correlation between the frequency of arteriolopathy (loss of smooth muscle cells in the media) and myocardial interstitial fibrosis was significant in COCM (P less than 0.05) but not in HOCM. No significant correlations were found between arteriolopathy and age, the duration of symptoms, the cause of death, the heart weight, the thickness of the septum or posterior free wall, the septal:free wall ratio, the presence of disorganized cells in the septum, mural thrombi, the mean muscle cell diameter, or the extent of very hypertrophic cells (P greater than 0.05) in either HOCM or COCM.

Preparation and mechanical properties of polylactic acid composites containing hydroxyapatite fibers.

Ceramic-polymer composite biomaterials were prepared by hot-pressing a mixture consisting of poly-L-lactic acid (PLA) and hydroxyapatite fibers (HAF) with dimensions of 40-150 microm length and 2-10 microm diameter, which were converted from beta-Ca(PO3)2 fibers. After PLA dissolved with methylene chloride was mixed with the fibers, the mixture was dried completely and subsequently hot-pressed uniaxially under a pressure of 40 MPa at 180 degrees C, resulting in the fabrication of the PLA/HAF composite. The modulus of elasticity was improved effectively even by introducing a small amount of HAF; almost no degradation in the bending strength was observed and the modulus of elasticity showed high values of 5-10 GPa when the fibers of 20-60 wt% were introduced. With increasing HAF content, the maximum strain decreases and the specimen is apt to show a brittle fracture; this result implies that HAF in the composites can share the applied load efficiently due to the formation of a bond between HAF and PLA.

Bioactive ceramics prepared by sintering and crystallization of calcium phosphate invert glasses.

Novel glass-ceramics were synthesized via sintering and crystallization by heating powder compacts of SiO2-free calcium phosphate invert glasses of 60CaO x 30P2O5 x 7Na2O x 3TiO2 or 60CaO x 30P2O5 x 7Na2O x 3MgO at 800-850 degrees C in air. The glass-ceramics were relatively dense materials consisting of crystalline phases such as beta-Ca3(PO4)2 and beta-Ca2P2O7 with glassy phases. The compacts were densified by the viscous flow of the glassy phases while heating. By soaking in simulated body fluid at 37 degrees C, a calcium phosphate phase was formed newly on the surface of the glass-ceramic derived from 60CaO x 30P2O5 x 7Na2O x 3TiO2 glass, while the phase was not formed on that derived from 60CaO x 30P2O5 x 7Na2O x 3MgO glass: the former was implied to show bioactivity. Composition of the glassy phase as the matrix varies with the additives such as TiO2 and MgO, and the chemical properties of the phase influence the bioactivity of the glass-ceramics. The glass-ceramic derived from 60CaO x 30P2O5 x 7Na2O x 3TiO2 glass has relatively high fracture toughness of K(IC) approximately 2 MPa m(0.5) and bending strength of 100-120 MPa.

Calcium phosphate invert glass-ceramic coatings joined by self-development of compositionally gradient layers on a titanium alloy.

A glass-ceramic layer containing beta-Ca3(PO4)2 crystals could be joined easily with a new type of titanium alloy (Ti-29Nb-13Ta-4.6Zr) consisting of a beta-titanium phase by heating the metal, on which glass powders with a composition of 60CaO x 30P2O5 7Na2O x 3TiO2 were placed, at 800 degrees C in air. Measurement of tensile bonding strength revealed that the joining between the coating layer and the substrate is very strong. Even after the large deformation (e.g., approximately 90 degrees in bending angle) of the titanium alloy, the coating layer was not peeled off from the substrate. A compositionally gradient layer in the TiO2-P2O5-Na2O-CaO system is developed automatically on the titanium alloy during the heating, resulting in the formation of the strong joining. By soaking in simulated body fluid at 37 degrees C, hydroxyapatite phase was formed newly on the surface of the coating layer.

Fatigue characteristics of bioactive glass-ceramic-coated Ti-29Nb-13Ta-4.6Zr for biomedical application.

A new surface-coating method by which CaP invert glass is used to improve the bioactivity of titanium alloys has been developed recently. In this method, the powder of CaP invert glass (CaO-P2O5-TiO2-Na2O) is coated on the surface of titanium alloy samples and heated between 1073 and 1123 K. With this treatment, a calcium phosphate layer mainly containing beta-Ca3(PO4)2 phase can be coated easily on titanium alloy samples. In the present study, the effect of this coating process on the fatigue properties of Ti-29Nb-13Ta-4.6Zr, a new metastable beta alloy for biomedical applications, has been investigated. The fatigue endurance limit of the coated alloy was found to be about 15% higher than that of uncoated alloy, as a result of the formation of a hard (alpha + beta) layer and a small amount of the omega phase during the coating process. The coating exhibits excellent adhesion to the substrate during the tensile and fatigue tests. Subsequent ageing at 673 K for 259.2 ks greatly improves the fatigue resistance of the coated alloy due to isothermal omega phase precipitation, and does not have obvious detrimental effect on the coating properties.

Responses of a murine B16 melanoma to pharmacotherapy studied and compared with different assay systems.

The responses of B16 melanoma in C57BL mice to cytotoxic agents, cyclophosphamide, doxorubicin, and dactinomycin, were studied by different methods both in vivo and in vitro. Either treated tumors were allowed to grow in vivo or cells were isolated and cultured in vitro afterwards. The tumor cells were also treated in vitro and assayed in vitro. For cyclophosphamide, a fairly good correlation was found among the dose responses measured: the tumor growth delay, incidence of giant cells, decrease in mitotic index, clonogenic cell survival of tumor cells treated in vivo and the survival of cells treated in vitro. For doxorubicin and dactinomycin, on the other hand, the tumor growth delay was not marked, the appearance of giant cells and decrease in mitotic index were minimal, and almost no decrease was found in the clonogenic cell survival for tumor treated in vivo, although the cells responded well to in vitro exposure. The results indicate substantial differences in the expression of damage according to the conditions of tumor cell growth and assay after treatment with different agents.

In vivo antimelanoma effects of 4-S-cysteaminylphenol, a newly synthesized therapeutic agent specific to melanoma.

Antimelanoma effects of 4-S-cysteaminylphenol (4-S-CAP), which is a newly synthesized melanin precursor, as a chemotherapeutic agent specific to malignant melanoma were determined in an in vivo system using mouse B16 melanoma. The intraperitoneal injection of 4-S-CAP induced a slight delay in the growth period of subcutaneous melanoma of C57BL/6 mice. Survival times of mice after treatment with 4-S-CAP were a little longer than those of control mice (P < 0.05), although all mice died from the local growth of tumours. The viable cell ratio of in vivo subcutaneous tumour cells reduced to 52.8% within 24 h after treatment with 4-S-CAP, but the ratio had recovered to the control level 72 h after treatment (> 90%). Similarly, the proliferating-cell-nuclear-antigen-positive cell ratio of melanotic melanoma had reduced 24 h after treatment and recovered within 72 h after treatment, while 4-S-CAP had no effect on amelanotic tumours. The formation of lung colonies by intravenous inoculation of malignant melanoma cells was compared between mice with intraperitoneal injection of 4-S-CAP and phosphate-buffered saline only. The 4-S-CAP-treated mice had significantly fewer lung colonies compared with the control mice (P < 0.01). The results indicate that the agent, 4-S-CAP, would have a therapeutic effect on malignant melanoma for a short time in vivo and therefore the agent can be effective against a small number of tumour cells, such as lung colonies, although it had little effect on the local tumours.

Effects of Friend leukemia virus (FLV) inoculation in F1 mice and differentiation of FLV-induced leukemia.

Effects of Friend leukemia virus (FLV) inoculation in F1 specific pathogen free (SPF) mice were examined. Resistance to FLV was dominantly inherited both in F1 hybrid mice (BDF1) (FLV-resistant & FLV-sensitive with polycythemia) and F1 hybrid mice (B6C3F1) (FLV-resistant & FLV-sensitive with anemia). But the population dynamics of the nucleated cell components of F1 mice after FLV inoculation differed from those of FLV-resistant inbred mice. A small number of mature erythroblasts appeared in the peripheral blood of BDF1 mice. In B6C3F1 mice, erythroblastosis with splenomegaly and polycythemia occurred. However, all of these findings in BDF1 and B6C3F1 mice regressed spontaneously. In F1 mice, FLV induced an intermediate reactive pattern of the two patterns that had been induced in the parental strains. The results indicate that FLV may induce leukemia with various degrees of differentiation, according to the genetic difference of the host.

Portal vein resection with a new antithrombogenic catheter.

Curative resection of pancreatic and hepatobiliary tumors is rarely possible because of local invasion, especially into the portal vein. We developed a new antithrombogenic catheter using a heparinized hydrophilic polymer to allow portal vein bypass during resection of tumors invading the portal vein. Pancreatectomy or hepatectomy accompanied by portal vein resection was performed for pancreatic or hepatobiliary cancer, with an intraoperative shunt from the superior mesenteric vein to the femoral vein or from the superior mesenteric vein to the intrahepatic portal vein through the umbilical vein or the hepatic hilar portal vein. Use of the shunt prevented stasis in the superior mesenteric vein and hepatic ischemia even during prolonged occlusion of the portal vein, and portal vein resection was performed in 81 patients with hepatobiliary and pancreatic disease with greater safety and ease.

Digital angiotomosynthesis for preoperative evaluation of cerebral arteriovenous malformations and giant aneurysms.

PURPOSE: To evaluate the clinical utility of the digital angiotomosynthesis technique for giving additional information regarding critical anatomy of cerebrovascular lesions before surgical intervention. METHOD: Seven arteriovenous malformations and three giant aneurysms were examined with digital angiotomosynthesis; these images were compared with conventional angiograms. RESULTS: 1) Detailed recognition of three-dimensional vascular structures of the arteriovenous malformation and giant aneurysm was facilitated by the cine mode of digital angiograms and angiotomograms. 2) Reconstructed angiotomograms could show clear separation of overlapping vessels and demonstrate fine vasculature. 3) Fine feeders, which were difficult to trace on the conventional angiogram, were more easily recognized in all cases of arteriovenous malformation. 4) Small arteries passing in close proximity to the arteriovenous malformation nidus were identifiable. 5) Fine arterial branches, being obscured by big shadows of giant aneurysms on the conventional angiograms, were well identified. 6) The anatomic relationship of bone structures to the giant aneurysm was clearly shown. CONCLUSIONS: Digital angiotomosynthesis is helpful for recognizing the three-dimensional and detailed vascular anatomy of arteriovenous malformations and giant aneurysms and provides neurosurgeons with useful information for preoperative evaluation.

Polymerase chain reaction heteroduplex polymorphism analysis by entangled solution capillary electrophoresis.

Heteroduplex DNA polymorphism analysis (HPA) makes use of conformational polymorphisms to alter electrophoretic mobility of fragments and can be used to detect non-restrictable loci. We have developed a novel application of entangled solution capillary electrophoresis (ESCE) to separate heteroduplex and homoduplex DNA molecules. The addition of ethidium bromide and glycerol to the free solution sieving buffer resulted in the improved peak resolution and good reproducibility. Reannealed polymerase chain reaction products could be used directly for mutation screening and with fully automated ESCE the entire HPA may be completed in less than 30 min including sample handling. This technology could provide a rapid and highly efficient way for screening rare mutations among large numbers of individuals.

High resolution ultrasound of the chest wall.

To study the detailed normal ultrasonic anatomy of the pleura and chest wall, high resolution (7.5 MHz) ultrasonograms were obtained from cadaver chest wall specimens and compared with thin section computed tomograms and anatomical specimens. Ultrasonograms show three layers of the intercostal muscles (internal, external and innermost), covered by the "echogenic pleural line." The "echogenic pleural line" is caused by composite echoes from the inner parietal pleura, and the outer endothoracic fascia, with the fatty tissue covering both sides of the fascia, which are located deep to the chest wall muscles. On ultrasonograms, the subpleural fat tissue, when abundant, appeared as an apron-like structure hanging down from the inner surface of the rib (subpleural fat pad), or diffuse fat accumulation mimicking the pleural thickening.