RESUMEN
This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A-derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5'- and 3'-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds.
Asunto(s)
Antivirales/farmacología , Línea Celular/virología , Virus del Dengue/genética , Proteínas Fluorescentes Verdes/genética , Animales , Antígenos Virales/genética , Cricetinae , Evaluación Preclínica de Medicamentos , Flavonas/farmacología , Virus de la Fiebre Aftosa/genética , Genes Reporteros/genética , Virus de la Hepatitis Delta/genética , Proteínas Inmediatas-Precoces/genética , Lactamas/farmacología , Mupirocina/análogos & derivados , Mupirocina/farmacología , Plásmidos/genética , ARN Catalítico/genética , ARN Viral/genética , Ensayo de Placa Viral , Proteínas Virales/genéticaRESUMEN
BACKGROUND: RCAS1 (receptor binding cancer antigen expressed on SiSo cells) is a tumour associated antigen. It is involved in immune evasion by tumour cells, by binding to receptors on cells involved in the immune response, such as T cells and natural killer cells, and inducing apoptosis. High expression of RCAS1 has been demonstrated immunohistochemically in tumours of the cervix, breast, lung, and stomach; however, the expression of RCAS1 has never been investigated in colorectal cancer. AIMS: To investigate the expression of RCAS1 in colorectal cancer and identify at which stages of colorectal carcinogenesis it is expressed. METHODS: Sixty surgically resected colorectal cancer specimens obtained from Rajavithi Hospital, Bangkok, Thailand were studied. RCAS1 expression was detected immunohistochemically using monoclonal anti-RCAS1 antibody. RCAS1 mRNA expression was also investigated by reverse transcription polymerase chain reaction in the freshly isolated tissues, and serum RCAS1 was measured by enzyme linked immunosorbent assay. RESULTS: Staining for the RCAS1 protein was intense in high stages of colorectal cancer, but weak in normal tissues. The RCAS1 mRNA results correlated with the immunohistochemistry results. Positive serum RCAS1 concentrations were found in 10 of 18 patients with stage II disease and 12 of 32 with stage III and IV, but not in patients with stage I disease. All lymph node and liver metastases showed high expression of RCAS1 protein. CONCLUSIONS: RCAS1 appears to be upregulated in high stages of colorectal cancer, both in the serum and the tissue. RCAS1 expression might be a useful additional criterion for staging this cancer.
Asunto(s)
Antígenos de Neoplasias/análisis , Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/química , Análisis de Varianza , Distribución de Chi-Cuadrado , Neoplasias Colorrectales/patología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
To address origins of glomerular endothelial and mesangial cells in embryonic mammalian kidneys, we established interspecies grafts between rats and mice, in which fetal kidneys were implanted into the anterior eye chamber of adult hosts. After 5-7 days, hosts bearing grafts received intravenous injections with species-specific monoclonal antibodies (MAbs) to matrix components. In all cases, glomerular basement membranes and mesangial matrices labeled solely for donor-derived matrix. Additionally, microvessel extracellular matrices within grafts were usually of donor origin. To examine directly the origin of glomerular endothelial and mesangial cells, we grafted embryonic gestational days 11-12 (E11-12) kidneys from normal mice into anterior eye chambers of host reverse-orientation splice acceptor 26 mice, which are transgenic animals that express beta-galactosidase in every cell. When grafts were developed for beta-galactosidase activity, host cells were seen in peripheral vessels, but the majority of glomerular endothelial cells were of donor, not host, origin. Where host-derived-endothelial cells were found in glomeruli, donor endothelial cells were present as well. Mesangial cells were always of donor origin. When E11 mouse kidneys were labeled with the endothelial cell-specific Bandeiraea simplicifolia isolectin B4, we determined that endothelial cells are present from the inception of metanephrogenesis. Together, the evidence shows that cells of endogenous kidney origin were almost entirely responsible for development of the glomerular microvasculature in oculo. External vessels from the host, although important for graft maintenance, were not major contributors to the glomerulus.
Asunto(s)
Trasplante de Tejido Fetal , Mesangio Glomerular/citología , Glomérulos Renales/citología , Trasplante de Riñón , Lectinas de Plantas , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Línea Celular , Matriz Extracelular/fisiología , Lectinas , Ratones , Ratones Endogámicos , Ratas , Ratas Sprague-Dawley , Circulación Renal , Especificidad de la Especie , Trasplante Heterólogo , Urotelio/citologíaRESUMEN
We used antibodies against mouse Englebreth-Holm-Swarm (EHS) tumor laminin to screen a newborn rat kidney lambda gt11 expression library and isolated three overlapping cDNA clones, termed 2b-11 (401 bp), 10-b7 (779 bp), and 2a (2,095 bp). DNA sequence analysis identified these cDNAs as encoding much of the carboxy terminal domain I/II of laminin gamma 1 chain (formerly referred to as B2e), and 1436 bp of the 3' untranslated region. In situ hybridization of fetal (E15) rat sections localized laminin gamma 1 chain mRNA primarily to meninges of the brain, auditory and peripheral nerve fibers, gastrointestinal system, and developing lung airway epithelium. Intense hybridization was also found in early nephric structures and glomeruli of fetal kidneys. In kidneys of three-day-old rats, hybridization persisted over early nephric figures, developing glomeruli, and collecting ducts, but considerably less hybridization was seen over tubules. On Northern blots of neonatal kidney RNA, the three cDNA clones hybridized to two species of 7.5 and 5.5 kb, suggesting that developing rat kidney laminin gamma 1 mRNAs are processed using two different polyadenylation signals.
Asunto(s)
Riñón/metabolismo , Laminina/biosíntesis , Laminina/genética , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Northern Blotting , Clonación Molecular , ADN Complementario/genética , Feto , Biblioteca de Genes , Hibridación in Situ , Riñón/crecimiento & desarrollo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
We studied the immunohistochemical and ultrastructural distribution of laminin in ovaries of immature and mature rats. When sections from 1-8-week-old rat ovaries were labeled directly with conjugates of affinity purified anti-laminin IgG-horseradish peroxidase (HRP), the antibodies bound to all ovarian basement membranes including those surrounding follicles in different stages of maturation. In addition, intracellular labeling was seen in granulosa and theca cells of follicles undergoing rapid development (preantral and antral stages) and in basement membrane-like structures of the Call-Exner bodies. Intracellular laminin was generally not detected, however, in any cells of primordial or atretic follicles. Tissue processed for immunoelectron microscopy 1 hour after the intravenous injection of anti-laminin IgG-HRP showed binding of antibody in linear patterns along endothelial and follicular epithelial basement membranes. Discontinuous strands of laminin-positive, extracellular matrices were also seen between theca cells of all follicles. In addition, injected anti-laminin IgG labeled perisinusoidal basement membranes located within corpora luteae and patches of basement membrane material between granulosa lutein cells. When ovaries were examined 5 d after the intravenous injections of anti-laminin IgG-HRP, uneven or segmented labeling was found in subepithelial basement membranes surrounding developing follicles. Our results therefore indicate that granulosa and theca cells participate directly in basement membrane laminin biosynthesis and suggest that this new laminin is spliced into existing basement membranes during follicular growth.
Asunto(s)
Laminina/análisis , Folículo Ovárico/química , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Femenino , Peroxidasa de Rábano Silvestre , Inmunoglobulina G , Inmunohistoquímica/métodos , Laminina/biosíntesis , Microscopía Inmunoelectrónica , Folículo Ovárico/metabolismo , Folículo Ovárico/ultraestructura , Ratas , Ratas EndogámicasRESUMEN
Although some progress has been made in recent years, there are truly large gaps in our basic knowledge on how the TBM is assembled during development. Some of the new evidence presented here indicates that both the tubular epithelium and interstitial fibroblasts participate in TBM protein biosynthesis during nephrogenesis. In addition, newly assembled segments of TBM are spliced or inserted into existing TBM during tubule expansion and elongation. A similar splicing mechanism has been described previously in the GBM, endocrine organs, and intestinal villi, and this mechanism therefore probably represents a fundamental process of basement membrane formation. A major unresolved question at present, however, is how this mechanism operates at the molecular level. Does the newly formed basement membrane contain identical components as that already present? Since an enzymatic process is likely occurring in the insertion of new matrix into old, which enzymes are involved? What is the cellular origin of these enzymes and which matrix component(s) is their substrate? Even more fundamental yet unanswered questions have to do with the mechanisms of epithelial induction, basement membrane gene activation, and tubular morphogenesis. Once the basement membrane is fully formed at the completion of nephrogenesis, what controls basement membrane turnover and how does this operate? Clearly, much additional research is necessary to address these questions. This work is needed, however, before we can fully understand the important roles basement membranes play in normal development as well as in disease.
Asunto(s)
Túbulos Renales/crecimiento & desarrollo , Animales , Membrana Basal/crecimiento & desarrollo , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Colágeno/metabolismo , Humanos , Túbulos Renales/metabolismo , Túbulos Renales/ultraestructura , Laminina/metabolismo , Microscopía Electrónica , Proteoglicanos/metabolismoRESUMEN
The distribution and synthetic rate of glomerular basement membrane components was examined in the Passive Heymann Nephritis model of experimental membranous nephropathy. The extensive tissue injury that developed included subepithelial electron-dense deposits, podocyte foot process effacement, and expansion of the glomerular basement membrane. Levels of mRNA for type IV collagen, laminin, and fibronectin from isolated glomeruli was quantitated by slot-blot analysis and showed no change in experimental animals as compared to controls at either 1 week, 3 weeks, or 3 months after disease induction. Immunoelectron microscopy with gold-labeled anti-laminin IgG revealed no difference in the number of particles bound to the glomerular basement membrane of experimental animals and controls. Immunofluorescence with both type IV collagen antisera and anti-laminin antibody showed no difference in the intensity or pattern of staining. Despite extensive glomerular damage and glomerular basement membrane thickening, no evidence was found for either an increase in the synthetic rate of type IV collagen, laminin, or fibronectin or for an accumulation of basement membrane laminin within the damaged glomeruli. Alternate processes, such as diminished density of matrix components or accumulation of other unmeasured matrix constituents, presumably account for the expansion of the glomerular basement membrane seen in experimental membranous nephropathy.
Asunto(s)
Colágeno/metabolismo , Fibronectinas/metabolismo , Glomerulonefritis/metabolismo , Glomérulos Renales/metabolismo , Laminina/metabolismo , Animales , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Northern Blotting , Femenino , Técnica del Anticuerpo Fluorescente , Glomerulonefritis/patología , Glomérulos Renales/ultraestructura , Microscopía Electrónica , Hibridación de Ácido Nucleico , Ratas , Ratas EndogámicasRESUMEN
Many earlier studies have shown that the intravenous injection into rats of sheep antibodies against rat glomerular basement membrane (GBM) induces a rapid influx of neutrophils and proteinuria (nephrotoxic nephritis or NTN). The GBM antigens recognized by nephrotoxic antibodies (NTAbs) have not been identified conclusively. Our experiments presented here, however, showed that NTAbs did not significantly reduce binding of anti-laminin IgGs to laminin-coated enzyme-linked immunosorbent assay (ELISA) plates or to the GBM in vivo, indicating little cross-reactivity between the NTAbs and laminin. To evaluate possible changes in GBM architecture during acute stages of NTN, the ultrastructural distribution of laminin was determined by postfixation, postembedding immunogold labeling, and compared between normal and nephritic rats. The density of immunoreactive GBM laminin was significantly reduced in rats with acute NTN. In addition, conjugates of anti-laminin IgG and horseradish peroxidase were intravenously injected into rats that then received injections of NTAbs. Anti-laminin peroxidase conjugates were also injected after administering NTAbs. In both cases, an overall decrease in anti-laminin peroxidase reaction product was observed as compared to normal controls. The densest labeling was seen in the lamina rara interna, especially in areas of endothelial cell detachment. Some immunoperoxidase reaction product was also bound to basal surfaces of detaching endothelial cells, demonstrating the removal of at least some laminin from the GBM. A decrease in GBM binding of intravenously injected anti-laminin IgG, both before and after injection of rats with NTAbs, was also confirmed by postembedding immunogold labeling. Furthermore, morphometry showed that the GBM was significantly wider in nephritic rats than in controls, indicating a redistribution of laminin over a greatly increased area. These immunoultrastructural findings show, therefore, that GBM architecture is altered in the early phase of NTN.
Asunto(s)
Glomérulos Renales/análisis , Laminina/análisis , Nefritis/patología , Enfermedad Aguda , Animales , Membrana Basal/análisis , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Técnicas para Inmunoenzimas , Glomérulos Renales/ultraestructura , Masculino , Microscopía Electrónica , Ratas , Ratas EndogámicasRESUMEN
Laminin biosynthesis and basement membrane assembly in anterior pituitary glands of gonadectomized rats were studied by immuno-electron microscopy and radioimmunoassay. Three weeks after gonadectomy, rats received intravenous injections of sheep anti-laminin IgG conjugated to horseradish peroxidase, and glands were fixed and processed for microscopy 1 h later. Peroxidase reaction product uniformly labeled all perivascular and glandular epithelial basement membranes. In addition, reaction product was also found in abnormally multi-layered basement membranes seen especially beneath gonadotrophs, and unusual basement membrane-like structures projecting between gonadotrophs were also labeled. Pituitary sections from gonadectomized rats labeled with pre-embedding immunoperoxidase and post-embedding immunogold techniques also localized intracellular laminin within biosynthetic organelles and "light body" vesicles of gonadotrophs. Neither abnormal basement membrane structures nor intracellular laminin were detected in pituitaries of nongonadectomized, control rats. Radioimmunoassays of pituitary homogenates showed nearly twice as much soluble laminin (approximately 15 ng/gland) in gonadectomized rats than in controls (approximately 8 ng/gland), which paralleled gland growth, but serum laminin concentrations did not differ (approximately 10 ng/ml in both groups). When anterior pituitary glands of gonadectomized rats that received injections of anti-laminin IgG-HRP were fixed 5 days after injection, lengths of unlabeled basement membrane were distributed between labeled lengths. This indicated that new basement membrane was "spliced" into old by a process similar to that seen in normal development. Supplementation of gonadectomized rats with testosterone, however, arrested laminin biosynthesis and basement membrane assembly and reversed glandular hypertrophy. These results indicate that, in an absence of sex hormone feedback, renewed synthesis of basement membrane components occurs in the anterior pituitary and is probably necessary to support the additional growth and differentiation of gonadotrophs and other pituitary cells.
Asunto(s)
Membrana Basal/metabolismo , Laminina/biosíntesis , Ovario/fisiología , Adenohipófisis/metabolismo , Testículo/fisiología , Animales , Castración , Femenino , Inmunohistoquímica , Masculino , Microscopía Electrónica , Adenohipófisis/fisiología , Adenohipófisis/ultraestructura , Ratas , Ratas EndogámicasRESUMEN
To determine whether circulating antibodies against laminin can bind in vivo to basement membranes within endocrine glands, affinity-purified sheep or rabbit anti-laminin IgG was intravenously injected into rats. One to five hours after injection, anti-laminin IgG was bound to all basement membranes of adrenal and anterior pituitary glands of mature as well as 2-day-old newborn rats as shown by immunofluorescence microscopy. After the injection of anti-laminin conjugated directly to horseradish peroxidase (HRP), HRP reaction product was also present throughout adrenal and pituitary basement membranes in mature and immature glands 1-5 h post-injection. Ultrathin Lowicryl sections from rats that received unconjugated rabbit anti-laminin IgG 1 h prior to fixation with paraformaldehyde were labeled directly with anti-rabbit IgG-colloidal gold. In these cases, gold also bound specifically over the lamina densa and lamina rara. When adrenal or pituitary glands from mature rats were examined by immunofluorescence 1 week after the injection of sheep anti-laminin IgG, the patterns and amounts of bound sheep IgG were indistinguishable from those observed 1 h after injection. In contrast, significantly less fluorescence was present in glands from 7-day-old rat pups that had received anti-laminin IgG 5 days earlier. In addition, when anti-laminin IgG-HRP was injected into newborns and glands were fixed 5 days later, lengths of labeled endothelial and epithelial basement membranes were often interspersed with unlabeled lengths in zones of cellular proliferation in the outer adrenal cortex and throughout the pituitary gland. These results indicated that unlabeled basement membranes in these regions were probably assembled after the injection of anti-laminin IgG, which would also explain diminished labeling of basement membranes in these animals. Despite the continued presence of heterologous anti-laminin IgG within endocrine basement membranes, however, rat IgG, rat C3, inflammatory cells, or histologic abnormalities were observed in neither newborn nor adult glands under the conditions examined here. Sections from rats injected with control IgG or control IgG-HRP were entirely negative by immunofluorescence, immunoperoxidase, and immunogold techniques. We therefore conclude that (1) apparently large amounts of circulating anti-laminin IgG can bind to adrenal and pituitary basement membranes, and (2) at least some of these basement membranes are assembled during development by progressive splicing of newly synthesized matrix into that already present.