Assuntos
Receptores de Hialuronatos/química , Sinovite/metabolismo , Variação Antigênica , Sequência de Bases , Linhagem Celular , Humanos , Receptores de Hialuronatos/genética , Imuno-Histoquímica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Membrana Sinovial/química , Sinovite/genética , Sinovite/patologiaRESUMO
Thrombomodulin (TM) is an essential cofactor for the physiologic activation of the anticoagulant protein C by thrombin. We have observed that the expression of TM mRNA in response to retinoic acid was markedly increased in human U937 monoblast-like cells, and human MEG01 megakaryocyte-like cells, but not in human umbilical vein cells, murine hemangioma cells, human K562 erythroblast-like cells, and murine HSD fibroblast-like cells. TM activity in U937 cells and MEG01 cells was not detectable in untreated cells, but developed rapidly after treatment with retinoic acid. In endothelial cells there was minimal change in TM activity in response to retinoic acid treatment. We have isolated clones for the genes for murine and human TM and have identified potential retinoic acid response elements in the 5'-flanking region of the human gene. In U937 cells the increase in mRNA levels was associated with increased transcription, and transient transfection studies with reporter plasmids demonstrate functional retinoic acid response elements present in the 5'-flanking region of the gene. Deletion of, and mutations introduced into, the potential retinoic acid response element confirm the functional response in transient transfections.
Assuntos
Hominidae/genética , Regiões Promotoras Genéticas , Trombomodulina/biossíntese , Trombomodulina/genética , Tretinoína/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetinae , DNA/genética , DNA/metabolismo , Hemangioma/metabolismo , Humanos , Megacariócitos/metabolismo , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Veias Umbilicais/metabolismoRESUMO
The extracellular matrix glycoprotein tenascin is limited to the periductal matrix of normal breast tissue but is markedly increased in both malignant and fibroadenomatous proliferations. It has been hypothesized that the changes in tenascin expression in these tissues are the result of epithelial induction of tenascin expression by the underlying mesenchyme. We have used Western and Northern blotting techniques to examine tenascin expression by normal and malignant mammary epithelial cells in culture. Normal mammary epithelial cells express tenascin in culture and incorporate the protein into the underlying matrix. The SV40-transformed mammary epithelial cell line HBL100 and some established mammary carcinoma cell lines also express tenascin. In contrast to normal mammary epithelial cells, carcinoma cells incorporate very little tenascin into the underlying matrix. To examine the source of tenascin expression in vivo, we have used in situ hybridization to demonstrate that the epithelial cells are a significant source of tenascin in both normal and malignant breast tissues.
Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Moléculas de Adesão Celular Neuronais/biossíntese , Proteínas da Matriz Extracelular/biossíntese , RNA Mensageiro/biossíntese , Adenofibroma/metabolismo , Northern Blotting , Western Blotting , Mama/citologia , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Moléculas de Adesão Celular Neuronais/análise , Divisão Celular , Linhagem Celular , Linhagem Celular Transformada , Células Epiteliais , Epitélio/metabolismo , Epitélio/patologia , Proteínas da Matriz Extracelular/análise , Feminino , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , RNA Mensageiro/análise , Vírus 40 dos Símios/genética , Tenascina , Células Tumorais CultivadasRESUMO
To determine the feasibility of using the C.B-17 scid/scid (severe combined immune deficient, SCID) mouse as a recipient of human synovial xenografts, we have engrafted human synovium under the renal capsule of SCID mice, and determined synovial graft survival and histologic characteristics 4 to 7 wk after tissue implantation. Both normal and inflammatory synovial tissue grew well in SCID mice and maintained histologic and phenotypic components of the fresh synovial tissue before implantation. However, the number of T cells in synovial grafts decreased after implantation. To determine whether leukocytes could migrate to human synovial xenografts, either allogenic or autologous PBMC were injected in the peritoneum of SCID mice bearing synovial xenografts. We found that 7 days after i.p. injection of autologous or allogeneic PBMC, injected T cells had selectively migrated to human synovial grafts and to SCID mouse lymph nodes. Our data demonstrate that normal and inflammatory human synovial tissues will grow in SCID mice and serve as recipients for autologous and allogenic peripheral blood human T cells injected i.p. into engrafted mice.
Assuntos
Membrana Sinovial/transplante , Linfócitos T/imunologia , Idoso , Animais , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Movimento Celular , Modelos Animais de Doenças , Feminino , Glicoproteínas/metabolismo , Sobrevivência de Enxerto , Humanos , Linfonodos/citologia , Linfonodos/imunologia , Masculino , Metaloendopeptidases/metabolismo , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Fenótipo , RNA Mensageiro/metabolismo , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Sinovite/etiologia , Sinovite/imunologia , Sinovite/patologia , Linfócitos T/citologia , Inibidores Teciduais de Metaloproteinases , Transplante HeterólogoRESUMO
OBJECTIVE: Tenascin is an extracellular matrix glycoprotein with effects on cell adhesion, cell migration, and lymphocyte activation. We proposed to identify the expression of human tenascin messenger RNA (mRNA) and protein in inflammatory synovitis and in normal synovium, and to identify potential regulatory cytokines. METHODS: Immunohistochemistry and in situ hybridization were used to identify the expression of tenascin in synovium. Northern blot analysis of RNA and both immunoblot analysis and enzyme-linked immunosorbent assay of proteins were used to identify tenascin in synovial cell cultures. RESULTS: Tenascin was found along the synovial lining layer and in perivascular areas of normal synovium. In inflammatory synovitis, tenascin protein and mRNA expression were shown to be increased in the synovial lining layer, in perivascular areas, in lymphoid aggregates, and in areas of fibrosis. Interleukin-1, a major mediator of tissue injury in inflammatory synovitis, induced tenascin expression and deposition in primary synovial fibroblast cultures. CONCLUSION: Tenascin mRNA and protein are increased in inflammatory synovitis, and interleukin-1 is an inducer of tenascin in synovial fibroblasts. This identifies a new pathway by which interleukin-1 alters the extracellular matrix composition in synovitis. Since tenascin has effects on lymphocyte activation and cell adhesion, the induction of tenascin in inflammatory synovitis may play a role in the pathophysiology of arthritis.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Interleucina-1/fisiologia , Sinovite/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , RNA Mensageiro/metabolismo , Membrana Sinovial/metabolismo , TenascinaRESUMO
Thrombomodulin is an essential cofactor for the activation of the anticoagulant protein C by thrombin. We have identified the expression of thrombomodulin messenger RNA (mRNA) and protein in peripheral blood monocytes. While untreated monocytes expressed thrombomodulin mRNA by Northern blot analysis, lipopolysaccharide-treated cells had decreased mRNA expression. Thrombomodulin antigen was shown in the cytoplasm and on the surface of monocytes by immunohistochemical staining, and thrombomodulin activity was shown on the surface of intact monocytes. One population of synovial lining cells that normally expressed mononuclear phagocyte antigens also expressed thrombomodulin in both noninflamed osteoarthritic synovium and in inflamed rheumatoid arthritis synovium. However, these cells did not express another endothelial protein, von Willebrand factor. We conclude that both circulating and tissue mononuclear phagocytes are capable of expressing thrombomodulin.
Assuntos
Expressão Gênica , Macrófagos/metabolismo , Monócitos/metabolismo , Receptores de Superfície Celular/genética , Membrana Sinovial/citologia , Artrite Reumatoide/metabolismo , Northern Blotting , Humanos , Imuno-Histoquímica , Lipopolissacarídeos , Osteoartrite/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Trombina , Ferimentos e Lesões/metabolismoRESUMO
The expression of messenger RNA encoding neutral metalloproteinases and the tissue inhibitor of metalloproteinases (TIMP) in human arthritic synovium was evaluated in situ, using RNA probes. Interstitial collagenase and stromelysin were expressed by synovial lining cells in patients with active rheumatoid arthritis (RA). Proteinase messenger RNA was found both in cells expressing mononuclear phagocyte antigens and in cells that were negative for the antigens. TIMP was also expressed predominantly along the synovial lining layer. In highly inflammatory RA, TIMP expression appeared less intense than that of the proteases. In osteoarthritic synovium, TIMP was expressed at easily detectable levels, whereas the expression of collagenase and stromelysin was less prominent. The balance between expression of the metalloproteinases and of the metalloproteinase inhibitor in synovium appears to be altered during inflammation. These results are consistent with the notion that synovium plays different roles in the cartilage damage of RA and of osteoarthritis.
Assuntos
Artrite/metabolismo , Glicoproteínas/genética , Metaloendopeptidases/genética , Colagenase Microbiana/antagonistas & inibidores , Membrana Sinovial/metabolismo , Idoso , Artrite/patologia , Artrite/fisiopatologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Expressão Gênica , Glicoproteínas/metabolismo , Humanos , Imuno-Histoquímica , Metaloendopeptidases/metabolismo , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Membrana Sinovial/patologia , Membrana Sinovial/fisiopatologia , Inibidores Teciduais de MetaloproteinasesRESUMO
Addition of pentoxifylline to lymphocytes caused a dose-dependent decrease in PHA-induced interleukin-2 receptor (IL-2R) expression. Expression of IL-2R protein and mRNA were inhibited by 60% at a concentration of 1 mM. Pentoxifylline also inhibited release of IL-2R into the medium by 85%. Treatment with recombinant IL-2 (50 U/ml) did not abrogate the effect of pentoxifylline. In addition to inhibition of IL-2R expression, pentoxifylline also decreased the expression of transferrin receptors and class I MHC antigens. Pentoxifylline also inhibited cell proliferation. However, aphidicolin, an inhibitor of DNA polymerase alpha inhibited cell proliferation to the same extent as pentoxifylline, but had no effect on IL-2R expression, indicating that inhibition of cell proliferation does not necessarily lead to inhibition of IL-2R expression. The inhibitory effect on IL-2R expression was also noted with other methylxanthines, theophylline and isobutylmethylxanthine, and with dbcAMP and forskolin. The inhibitory activity of pentoxifylline was prevented by W-13, a calmodulin antagonist, but not by HA-1004, a cyclic AMP-dependent protein kinase inhibitor. This suggests that pentoxifylline might act in part through a Ca2+/calmodulin-dependent mechanism. Pentoxifylline and other methylxanthines may prove useful in delineating the biochemical pathways involved in induction and expression of cell surface receptors.
Assuntos
Linfócitos/efeitos dos fármacos , Pentoxifilina/farmacologia , Receptores de Interleucina-2/análise , 1-Metil-3-Isobutilxantina/farmacologia , Afidicolina , Calmodulina/fisiologia , Células Cultivadas , AMP Cíclico/fisiologia , Diterpenos/farmacologia , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , RNA Mensageiro/análise , Receptores de Interleucina-2/efeitos dos fármacos , Receptores de Interleucina-2/genética , Receptores da Transferrina/análiseRESUMO
Collagenase has been implicated as playing an important role in the connective tissue destruction that is a major feature of rheumatoid arthritis. Numerous cell types in the hyperplastic rheumatoid synovium are capable of synthesizing collagenase. Past studies have used predominantly synovial fibroblasts in culture as a model system for the regulation of collagenase production, but the major cellular source of the enzyme in vivo has not been determined. Using the techniques of in situ hybridization histochemistry and indirect immunofluorescence, we determined the cellular source of collagenase in frozen sections of human synovium. Collagenase mRNA production was localized to cells along the synovial lining layer in rheumatoid arthritis. These were identified as the macrophage-like Type A synovial lining cells by immunofluorescence with antibody LeuM3. Endothelial cells, fibroblasts, and T and B lymphocytes were devoid of detectable collagenase mRNA. Synovial tissue sections from patients with osteoarthritis and trauma did not contain detectable collagenase mRNA. These data identify the Type A macrophage-like synovial lining cell as the primary source of collagenase mRNA in vivo in the rheumatoid arthritis synovium and, potentially, as a major effector cell in the tissue destruction of the disease.
Assuntos
Artrite Reumatoide/enzimologia , Colagenase Microbiana/biossíntese , RNA Mensageiro/metabolismo , Membrana Sinovial/patologia , Anticorpos Monoclonais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Imunofluorescência , Humanos , Macrófagos/enzimologia , Hibridização de Ácido Nucleico , Sondas RNARESUMO
Interleukin-1 (IL-1) may contribute to tissue destruction in rheumatoid arthritis, in part, by inducing messenger RNA (mRNA) that encodes interstitial collagenase. In human synovial fibroblasts in vitro, IL-1 induced collagenase mRNA accumulation 6 hours after being added to the cells. High levels of mRNA remained present for at least 48 hours after treatment. The rate of transcription of collagenase in isolated nuclei peaked after approximately 6 hours of treatment with IL-1 and declined thereafter, becoming nearly undetectable by 24 hours. The persistence of mRNA, in view of the transient peak of transcription, suggested that collagenase mRNA was stable in synovial fibroblasts. The half-life of collagenase mRNA after the synoviocytes were treated with actinomycin D was approximately 27 hours, both in the presence and in the absence of IL-1. It has been noted that induction of the expression of collagenase by phorbol esters requires fos protein synthesis and is mediated through a tetradecanoyl phorbol acetate response element in the 5'-flanking region of the gene. However, we found that cycloheximide, when added to synovial fibroblast cultures up to 6 hours after treatment with IL-1, inhibited the expression of collagenase mRNA. These results suggest that fos alone is unlikely to be sufficient for collagenase expression, and that additional factors, or alternative pathways, are involved in the induction of collagenase by IL-1.
Assuntos
Interleucina-1/fisiologia , Colagenase Microbiana/metabolismo , RNA Mensageiro/metabolismo , Membrana Sinovial/enzimologia , Cicloeximida/farmacologia , Estabilidade de Medicamentos , Fibroblastos/enzimologia , Humanos , Colagenase Microbiana/genética , Proteínas/antagonistas & inibidores , RNA Mensageiro/antagonistas & inibidores , Membrana Sinovial/citologia , Fatores de Tempo , Transcrição GênicaRESUMO
Human promyelocytic leukemia cells (HL-60) differentiate along a monocytoid pathway in response to recombinant human tumor necrosis factor or recombinant human interferon gamma. Together, these agents act synergistically to induce phenotypic differentiation. Since reduced expression of mRNA for the proto-oncogene c-myc correlates with differentiation of HL-60 cells induced by other agents, we tested the abilities of tumor necrosis factor and interferon gamma to regulate expression of c-myc mRNA. Tumor necrosis factor rapidly and effectively reduced c-myc mRNA levels. In contrast, interferon gamma did not affect c-myc mRNA levels, nor did it show synergy with tumor necrosis factor in reducing c-myc. Transcription run-on studies confirmed that tumor necrosis factor caused interruption of c-myc transcription after exon 1. Phorbol diesters also caused interruption of transcription of c-myc. Thus, interruption of transcription may be a common mode of regulation of c-myc during induced differentiation of HL-60 cells.
Assuntos
Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Transcrição Gênica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Éxons , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Ésteres de Forbol/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Until now there has not been a treatise summarizing the management of oncologic emergencies in the aged. All of the well-described acute complications of malignancy occur in the elderly. Such clinical events are best labeled urgent rather than emergent, and some may be less well tolerated in the older patient because of normal physiologic changes of aging. However, all should be recognized as early as possible and can be treated, when it is appropriate to do so, as has been detailed for 14 problems discussed in this article. The authors also address two important topics, the quality of life and the use of intensive care units, both germane to the difficult question of whom and when to treat.
Assuntos
Neoplasias/complicações , Idoso , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/terapia , Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/terapia , Cuidados Críticos , Emergências , Fraturas Espontâneas/etiologia , Fraturas Espontâneas/terapia , Gastroenteropatias/etiologia , Gastroenteropatias/terapia , Doenças Hematológicas/etiologia , Doenças Hematológicas/terapia , Humanos , Hipercalcemia/etiologia , Hipercalcemia/terapiaRESUMO
The human promyelocytic leukemia cell line HL-60 is induced to differentiate along a myelocytic pathway by dibutyryl cyclic adenosine monophosphate (dbcAMP). Other cAMP analogs are ineffective as inducing agents. The effect of these compounds on expression of c-myc was investigated using a DNA probe for c-myc to detect RNA transcripts. The dose response and time to commitment for reduction in c-myc expression with dbcAMP was similar to the findings for phenotypic changes. Bromo-cyclic AMP and butyrate alone caused no changes in c-myc expression in 24 hours, but demonstrated dramatic synergism together, suggesting that butyrate contributes in part to the effects of dbcAMP. Evidence for mechanisms of action of cAMP other than activation of the cAMP-dependent protein kinase is reviewed.