Biomedical subjects
Publications and source records attributed to M Azuma.
The technique of generating 3-D images from CT and MR data has made progress. Manipulation of the images has become simpler, the processing time shorter, and the quality better. Clinical application of this technique has been performed for images of brain (5, 7, 8) and bone (1, 2, 6), but only a few studies have been done of abdominal organs (3, 4).
The membrane antigen B7/BB1 (refs 1, 2) is expressed on activated B cells, macrophages and dendritic cells, and binds to a counter-receptor, CD28, expressed on T lymphocytes and thymocytes. Interaction between CD28 and B7 results in potent costimulation of T-cell activation initiated through the CD3/T-cell receptor complex. Discrepancies between results with anti-CD28 and anti-B7 antibodies have suggested the existence of a second ligand for CD28 and CTLA-4 (refs 3, 6-8). We have generated a monoclonal antibody, IT2, that reacts with a 70K glycoprotein (B70). B70 complementary DNA was cloned from a B-lymphoblastoid cell line library and encodes a new protein of the immunoglobulin superfamily with limited homology to B7. B70 is expressed on resting monocytes and dendritic cells and on activated, but not resting, T, NK and B lymphocytes. IT2 substantially inhibited the binding of a CTLA4-immunoglobulin fusion protein to human B-lymphoblastoid cell lines and, together with anti-B7 antibody, completely blocked CTLA-4 binding. Further IT2 efficiently inhibited primary allogeneic mixed lymphocyte responses. These findings indicate that B70 is a second ligand for CD28 and CTLA-4 and may play an important role for costimulation of T cells in a primary immune response.
We demonstrate the role in tumor-associated angiogenesis of factors released into conditioned medium (CM) from in vitro human salivary gland cell clones with biological phenotypes ranging from non-metastasizing to metastasizing. A non-metastasizing human salivary gland adenocarcinoma cell clone HSGc and its subclone with metastatic potential (Gc2-100 cl-1) were employed. We also used metastasizing cell clones obtained by explant cultures of organs of Gc2-100 cl-1 tumor-bearing nude mouse. The proliferation and migration of bovine aortic endothelial (BAE) cells were significantly stimulated by the addition of CM obtained from Gc2-100 cl-1 and metastasizing cell clones, while CM from HSGc was ineffective. When the effect on protease secretion by BAE cells was examined, CM from Gc2-100 cl-1 and metastasizing cell clones inhibited the secretion of type IV collagenases by BAE cells much more than did CM from HSGc. These findings, therefore, may imply that Gc2-100 cl-1 and metastasizing cell clones secrete angiogenic factors that stimulate not only the proliferation and migration of endothelial cells but also the formation of basement membrane components necessary for the reconstruction of new blood vessels at migrated sites of endothelial cells by preventing the degradation of basement membrane component, type IV collagen.
An in vitro system has been established in which conversion from non-metastasizing to metastasizing adenocarcinoma cells can be induced, and subsequently subjected to analysis of the expression of proteases and tissue inhibitor of metalloproteinases-1 (TIMP-1). A human salivary-gland adenocarcinoma cell clone HSGc, with no metastatic ability, was exposed to N-methyl-N-nitrosourea (MNU). Following exposure to MNU, cells with altered morphology were cloned. Upon s.c. inoculation into nude mice, MNU-treated HSGc clones formed metastatic foci in various organs, and then 5 metastasizing clones were isolated. Evaluation of expression of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), metalloproteinases and TIMP-1 was performed by means of enzyme immunoassay, zymogram, or immunoblot. MNU-treated HSGc and metastasizing clones were found to secrete high levels of tPA, while HSGc produced undetectable levels of this enzyme. Expression of uPA was not observed in any of the cell clones. When the secretion of gelatinolytic enzymes was examined, metastasizing clones produced higher levels of 57- and 32-kDa, but not of 92- or 72-kDa gelatinases, as compared to HSGc cells. Although TIMP-1 was detected in all cell clones, metastasizing clones secreted less TIMP-1 than HSGc cells; in addition, one metastasizing clone produced TIMP-1 with a molecular weight distinct from that of 28-kDa TIMP-1. Our results suggest that the acquisition of metastatic ability by human salivary-gland tumor cells is closely associated with increased secretion of several metalloproteinases as well as decreased or altered TIMP-1 expression.
Small, resting human peripheral blood T cells are able to mediate anti-CD3 redirected lysis against murine P815 cells transfected with human B7, a ligand of CD28. We demonstrate that cytotoxicity is mediated by preexisting cytotoxic effectors within the small, resting "memory" T cell population and by the de novo generation of additional CTL within both the "memory" and "virgin" T subsets. This conclusion is based on analysis of the kinetics of the response and the effects of metabolic inhibitors on the generation of CTL function. Memory CD45RO+ T cells demonstrated cytotoxicity within 4 h of coculture with anti-CD3 mAb and B7+ P815 cells and cytolysis was only partially prevented by inhibitors of protein synthesis. By contrast, virgin CD45RO- T cells demonstrated anti-CD3-induced lysis against B7+ P815 targets only after 6 or 8 h of coculture and cytotoxicity was completely prevented by inhibiting protein synthesis. Induction of cytotoxicity was B7 dependent in that parental P815 cells and P815 cells transfected with CD72 and vascular cell adhesion molecule-1, ligands for T cell-associated membrane receptors CD5 and very late activation antigen-4, respectively, did not initiate cytotoxicity. Our studies also revealed cooperation between the CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 pathways in the generation of CTL from small, resting T cells. However, after CTL generation, the CD28-B7 interaction was not required for cytotoxic effector cell function. These observations may have important physiologic implications because this would permit activated CTL to lyse targets in vivo that do not express B7, after the CTL were generated by APC that do express B7 or possibly other costimulatory molecules.
B7/BB1 is a membrane differentiation antigen expressed on activated B cells, macrophages, and dendritic cells that binds to a counter-receptor, CD28, expressed on T lymphocytes and thymocytes. Interaction between CD28 and B7 results in potent costimulation of T cell activation initiated via the CD3/T cell receptor complex. We now report that B7 is also expressed on activated human peripheral blood T cells, CD4 T cell clones, CD8 T cell clones, and natural killer cell clones. B7 appears relatively late after T cell activation, can be detected on both CD4 and CD8 T cell subsets, and is present on antigen-specific, major histocompatibility complex-restricted CD4 and CD8 T cell clones. Expression of B7 on activated T cells was confirmed by immunoprecipitation from 125I-labeled activated T cells and by detection of B7 transcripts. A B7+ CD4+ T cell clone was able to stimulate a primary allogeneic mixed lymphocyte response using small, resting peripheral blood T cells as responders. The alloantigen-induced proliferative response and cytokine production was partially inhibited by anti-B7 monoclonal antibody. Since activated T cells can coexpress both CD28 and its counter-receptor, B7, this suggests that activated T cells may be capable of autocrine costimulation via the CD28 activation pathway.
A subset of CD8+ alpha beta-TCR/CD3+ T lymphocytes in adult human peripheral blood lacks expression of CD28, a membrane receptor for the B7/BB1 B cell differentiation Ag that is involved in T cell activation. CD28-8+ T cells were not observed in the thymus and were present at only low frequency in cord blood, suggesting that these cells may represent a type of "memory" population. Consistent with this interpretation, CD28-8+ T cells were morphologically large, granular lymphocytes and expressed CD54 (intercellular adhesion molecule-1), CD58 (lymphocyte function-associated Ag-3 (LFA)), and high levels of CD11a (LFA-1), but did not express Ag associated with acute activation (e.g., HLA-DR, CD25, CD69). Freshly isolated CD28-8+ T lymphocytes mediated potent anti-CD3 redirected cytotoxicity against FcR-bearing targets, demonstrating that the CD3/TCR complex is functional and that these cells possess cytolytic activity. However, the anti-CD3-induced proliferative response of CD28-8+ T cells was substantially less than CD28+8+ T cells and this deficiency could not be overcome by addition of exogenous IL-2. A large panel of T cell clones were produced from the CD28+8+ and CD28-8+ T cell populations by single cell sorting using a flow cytometer. CD28 expression was stable on clones derived from CD28+8+ T lymphocytes, whereas CD28 expression was quite variable and apparently reexpressed on some clones generated from the CD28-8+ T cell population. As with the freshly isolated cells, CD28-8+ T cell clones were cytotoxic, but anergic to anti-CD3- induced proliferation and could not be costimulated using B7 or CD58 (LFA-3) murine L cell transfectants. These results indicate that CD28- and CD28+ T cells may play different roles in an immune response.
OBJECTIVE: The purpose of this study is to clarify the most useful technique of MR imaging in demonstrating the collateral venous pathways due to portal hypertension. SUBJECTS: Twenty-eight cirrhotic patients with collateral venous pathways due to portal hypertension and three patients without them were studied. We evaluated the detectability of collateral vessels of flow-enhanced transverse MR images by time of flight effect, projection MR angiography using the maximum intensity projection technique and three-dimensional (3D) computer graphic images, by using the arterial portography as the standard. RESULTS: Flow-enhanced transverse MR images showed 98% of the collateral vessels detected by angiography, however, the connection of each vessel was obscure. Fully 81% of the collateral vessels were seen by projection MR angiography, and 98% of them by the 3D computer graphic images. CONCLUSION: The detectability of collateral vessels of 3D computer graphic imaging was better than that of the projection MR angiography and was as accurate as that of transverse MR imaging. 3D Computer graphic images provided complex anatomical information in a more easily understood fashion than either transverse MR images or projection MR angiography.
To clarify co-carcinogenic effects of chrysotile (Chry) and amosite (Amo) asbestos with benzo(a)pyrene (Bap), 0.2 mg UICC (International Union against Cancer) standard reference sample of asbestos and 0.4 mg Bap were applied intratracheally once a week for 6 weeks. Eighteen and 24 months after the last instillation the number of tumors was examined. The Chry + Bap group yielded 37 tumors including 16 carcinomas in 12 animals, and the Amo + Bap group yielded 30 tumors including 11 carcinomas in 12 animals. Tumor-bearing animals were 100% in the Chry + Bap group and 92% in the Amo + Bap group, and carcinoma-bearing animals were 83% and 67%, respectively. The animals injected with Chry, Amo, and Bap alone developed no tumors. The number of tumors and carcinomas and the frequency of the tumor- or carcinoma-bearing animals in Chry + Bap and Amo + Bap were significantly higher than those of the groups injected independently. The number of tumors or the frequency of tumor-bearing animals was higher in Chry + Bap than in Amo + Bap; however, these differences were not significant. These results indicate that both Chry and Amo play an important role in the genesis of bronchogenic carcinoma.
We investigated the effects of Dermatophagoides farinae (Df) and interleukin (IL)-2 on the release of eosinophil colony-stimulating factor (Eo-CSF) activity from mononuclear cells (MNC) and lymphocytes of patients with bronchial asthma (BA) who were sensitive to Df to clarify its relationship with IL-5 and granulocyte/macrophage colony-stimulating factor (GM-CSF). MNC and T cells of patients cultured with IL-2 and Df released Eo-CSF activity. These Eo-CSF activities were partially inhibited by anti-IL-5 and anti-GM-CSF antibodies. In 11 of 15 cases studied, MNC from patients produced GM-CSF in response to IL-2. In four of 15 cases studied, MNC from patients produced GM-CSF in response to Df. On culture with IL-2 or Df, the releases of IL-5 into the medium by MNC from individual patients varied. The results indicate that in BA responsiveness of lymphocytes to Df is increased, and suggest that IL-5 and GM-CSF produced by T cells play a role in the induction of eosinophilia and the pathogenesis of BA.
We compared the inotropic and electrophysiologic effects of propofol and thiamylal in isolated papillary muscles of the guinea pig and rat. Propofol applied in clinical 10% intralipid emulsion showed concentration-dependent negative inotropic effects, accompanied by decreased action potential duration, in the guinea pig. Intralipid alone had no effect. Although thiamylal showed a concentration-dependent depression similar to propofol in the guinea pig, depolarization of resting membrane potential was seen at 0.1 and 0.3 mM, and a slight prolongation of action potential duration at 90% repolarization at 0.1 mM, and then a decrease of action potential duration at 0.3 mM. In rat papillary muscles, propofol did not produce any depression of contractile force, whereas thiamylal produced a concentration-dependent negative inotropic effect. The important findings observed in the action potential in rat papillary muscles were the modest shortening of action potential duration after propofol application, and the significant decrease of resting membrane potential and the significant prolongation of action potential duration caused by thiamylal. Both propofol and thiamylal depressed slow action potentials and contractile force in guinea pig papillary muscles depolarized by 25 mM K+ solution. In conclusion, the negative inotropic effects of propofol and thiamylal might be caused by inhibition of trans-sarcolemmal Ca2+ influx accompanied by shortening of action potential duration in guinea pig papillary muscles. The action potential of thiamylal might be affected by the suppression of K+ current in guinea pig and rat papillary muscles that was never observed in the propofol-treated tissue.
Injection of estrogen into male Xenopus laevis induced the appearance of retinals (retinal and 3-dehydroretinal) and a considerable increase in the amount of retinols (retinol and 3-dehydroretinol) in the blood plasma. These retinoids were mainly in the all-trans form. Without estrogen injection, retinols were normally found in the blood plasma of both males and females, but only trace amounts of retinals were detected and these were restricted to the plasma of females. The proteins in the blood plasma of estrogen-injected males were separated into two fractions. One fraction included vitellogenin, the precursor of egg yolk proteins, and the other contained some plasma proteins other than vitellogenin. Retinals were detected in the former and retinols in the latter. It is suggested that retinals are bound to vitellogenin and are taken up into oocytes in the process of vitellogenesis.
The purpose of these experiments was to examine the relationship between oxidation cataract and proteolysis in cultured rat lens. Hydrogen peroxide cataract showed insolubilization of protein, loss of 31 kDa beta B1-crystallin polypeptide, decreases in soluble calpain, and increases in insoluble calpain. This suggested that calpain may be activated in hydrogen peroxide treated lenses, since beta B1 is a known calpain substrate, and calpain undergoes autolysis and degradation when activated. Furthermore, the cysteine protease inhibitor E64 was partially effective in preventing development of H2O2-cataract. E64 also prevented the loss of the 31 kDa beta B1-crystallin polypeptide and decreased the loss of calpain in the lens. These results suggested that development of hydrogen peroxide induced cataract in rat lenses was associated with activation of calpain.
BACKGROUND: Based on morphologic and functional differences mainly, 4 distinct types of cells are recognized in human salivary glands. For a better understanding of cellular proliferation and differentiation of human salivary glands as well as carcinogenesis of salivary gland neoplasms, we attempted to establish normal human salivary gland cells in an in vitro system. EXPERIMENTAL DESIGN: Primary cultured human salivary gland cells were transfected with origin-defective mutant DNA of SV40. After 2 to 3 weeks of transfection, slowly expanding colonies consisting of small compact cells emerged, whereas mock-transfected cells did not grow any more and eventually entered crisis, followed by cell death. RESULTS: Using limited dilution technique, we isolated 4 cell clones with distinct morphology from a single colony. Morphologic observation of cells cultured on plastic dishes precisely revealed the characteristics of constituent cells of salivary glands; i.e., three cell clones showing cuboidal (NS-SV-DC), spindle (NS-SV-MC), and flattened (NS-SV-SC) morphology were similar to duct, myoepithelial, and squamous cells, respectively. A remaining cell clone showing polygonal in shape with numerous secretory granules (NS-SV-AC) resembled acinar cells. Characterization of cell clones by the ultrastructural examination and the search for specific antigens showed the similarity of NS-SV-DC, NS-SV-MC, NS-SV-AC, and NS-SV-SC to duct, myoepithelial, acinar, and squamous cells, respectively. Integration and expression of SV40 DNA were confirmed by Southern blot and indirect immunofluorescence staining. Anchorage-independent growth in soft-agar and tumorigenicity in nude mice were not recognized in all cell clones. CONCLUSIONS: These results demonstrate that establishment of cell clones with duct-, myoepithelial-, acinar-, or squamous phenotype was accomplished in the in vitro system, and that based on the evaluation of colony-forming ability in soft-agar and tumorigenicity in nude mice, these cell clones are considered to be non-neoplastic.
Recent dynamic advances of molecular biology have been discovering the specific virus functions and replication strategies that differ greatly from their host cells. These studies made it possible to design the antiviral agents which block virus replication specifically. In this paper, we review the recent approach for development of anti-HIV agents and the problems of anti-herpes compounds applied in clinical treatment.
The purposes of this experiment were: (1), to compare effect of three E64 derivatives, E64, E64c and E64d in preventing nuclear opacity and proteolysis in calcium ionophore-induced cataract and (2), to measure the accumulation of E64 derivatives in the cultured lenses. In vitro E64 and E64c strongly inhibited purified calpain II from porcine heart, while E64d showed weaker inhibition than E64 and E64c. In cultured lenses, all three E64 derivatives reduced nuclear opacity by calcium ionophore A23187 in a concentration-dependent manner, and E64d, the ethyl-ester of E64c, was the most effective. When lenses were cultured in E64d for 2 h, the resulting concentration of E64 derivative in the lens was markedly higher than during culture in E64 or E64c. All three E64 derivatives prevented proteolysis of crystallins seen in A23187 cataract. The stronger effect of E64d against A23187 cataract was likely due to an earlier penetration into the lens, conversion to E64c and inhibition of activated calpain.
NK cells and certain CTL can recognize and lyse targets without restriction by the MHC. NK cells do not express CD3/TCR complexes and the membrane receptors participating in MHC-unrestricted cytotoxicity are largely unknown. We demonstrate that YT2C2, a human NK leukemia cell line, expresses the CD28 differentiation Ag and can spontaneously lyse both murine and human cell lines expressing B7, a B cell- activation Ag that is a ligand for CD28. The participation of CD28/B7 interactions in MHC-unrestricted cytotoxicity mediated by YT2C2 cells was demonstrated by correlation of target sensitivity with levels of B7 expression, inhibition of cytotoxicity by anti-CD28 or anti-B7 mAb, and by making both murine and human cell lines susceptible to YT2C2-mediated lysis by genetic transfection with expression vectors containing B7 cDNA. However, CD28/B7 interactions alone were insufficient to initiate cytotoxicity. mAb inhibition experiments and selection of CD54- (intercellular adhesion molecule-1) deficient B cell targets indicated that CD11a/18 (lymphocyte function-associated Ag-1) also cooperated in CD28/B7-dependent cytotoxicity. The requirement for both CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interactions in YT2C2-mediated MHC-unrestricted cytotoxicity was confirmed by demonstrating that efficient lysis of murine L cells required cotransfection with both B7 and intercellular adhesion molecule-1. These findings support the concept that MHC-unrestricted cytotoxicity may not be due to a unique receptor, but may result from interactions between an appropriate array of "adhesion" molecules with their ligands.
Lenses cultured in diamide first developed outer cortical opacities followed by nuclear cataract. Lens hydration and total calcium were markedly increased by diamide. Proteolysis of crystallins were observed in nuclear cataract lenses. Calpain in the soluble fraction of lenses cultured with diamide was decreased, while calpain in the insoluble fraction was increased. Co-culture with E64d, an inhibitor of cysteine protease such as calpain, especially prevented nuclear opacities and proteolysis of crystallins, indicating that calpain was involved in cataract formation by diamide.