RESUMEN
Laminins, the main components of basement membranes, are heterotrimers consisting of alpha, beta, and gamma polypeptide chains linked together by disulfide bonds. Laminins-1 and -2 are both composed of beta1 and gamma1 chains and differ from each other on their alpha chain, which is alpha1 and alpha2 for laminin-1 and -2, respectively. The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial-mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin alpha1 chain. Synthesis of laminin alpha1 chain however returns upon re-establishment of epithelial-mesenchymal contact. Cell-cell contact is critical, since laminin alpha1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial-mesenchymal cocultures in which heterotypic cell-cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin alpha1 chain upon heterotypic cell- cell contact. In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin alpha1, alpha2, and beta/gamma chains. Lung explants exposed to monoclonal antibodies to laminin alpha1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle alpha actin and desmin. Taken together, our studies suggest that laminin alpha1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway smooth muscle development.
Asunto(s)
Bronquios/fisiología , Células Epiteliales/fisiología , Laminina/biosíntesis , Pulmón/embriología , Mesodermo/fisiología , Músculo Liso/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Bronquios/citología , Bronquios/embriología , Comunicación Celular , Células Cultivadas , Técnicas de Cocultivo , Embrión de Mamíferos , Células Epiteliales/citología , Sustancias Macromoleculares , Mesodermo/citología , Ratones , Músculo Liso/citología , Músculo Liso/embriología , Técnicas de Cultivo de ÓrganosRESUMEN
Bronchial smooth muscle (SM) mesenchymal cell precursors change their shape from round to spread/elongated while undergoing differentiation. Here we show that this change in cell shape induces the expression of laminin (LM) alpha2 chain not present in round mesenchymal cells. LM alpha2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM beta1 and gamma1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM alpha2. Dy/dy mice express very low levels of LM alpha2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM alpha-actin, desmin, and SM-myosin than controls. These deficiencies were completely corrected by exogenous LM-2. On histological examination, dy/dy mouse airways and gastrointestinal tract had shorter SM cells, and lungs from dy/dy mice contained less SM-specific protein. The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity. This study therefore demonstrated a novel role for the LM alpha2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.
Asunto(s)
Regulación de la Expresión Génica , Laminina/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Músculo Liso/citología , Músculo Liso/embriología , Actinas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Desmina/metabolismo , Sistema Digestivo/citología , Sistema Digestivo/metabolismo , Sistema Digestivo/patología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Laminina/genética , Laminina/inmunología , Laminina/farmacología , Mesodermo/efectos de los fármacos , Ratones , Ratones Endogámicos , Ratones Mutantes , Modelos Biológicos , Músculo Liso/efectos de los fármacos , Músculo Liso/patología , Distrofia Muscular Animal/congénito , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Miosinas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sistema Respiratorio/citología , Sistema Respiratorio/embriología , Sistema Respiratorio/metabolismo , Sistema Respiratorio/patologíaRESUMEN
Smooth muscle (SM) develops only in organs and sites that sustain mechanical tensions. Therefore, we determined the role of stretch in mouse and human bronchial myogenesis. Sustained stretch induced expression of SM proteins in undifferentiated mesenchymal cells and accelerated the differentiation of cells undergoing myogenesis. Moreover, bronchial myogenesis was entirely controlled in lung organ cultures by the airway intraluminal pressure. Serum response factor (SRF) is a transcription factor critical for the induction of muscle-specific gene expression. Recently, a SRF-truncated isoform produced by alternative splicing of exon 5 has been identified (SRFDelta5). Here we show that undifferentiated mesenchymal cells synthesize both SRF and SRFDelta5 but that SRFDelta5 synthesis is suppressed during bronchial myogenesis in favor of increased SRF production. Stretch induces the same change in SRF alternative splicing, and its myogenic effect is abrogated by overexpressing SRFDelta5. Furthermore, human hypoplastic lungs related to conditions that hinder cell stretching continue to synthesize SRFDelta5 and show a marked decrease in bronchial and interstitial SM cells and their ECM product, tropoelastin. Taken together, our findings indicate that stretch plays a critical role in SM myogenesis and suggest that its decrease precludes normal bronchial muscle development.
Asunto(s)
Empalme Alternativo/genética , Bronquios/metabolismo , Proteínas de Unión al ADN/genética , Pulmón/metabolismo , Músculo Liso/metabolismo , Proteínas Nucleares/genética , Estrés Mecánico , Animales , Bronquios/citología , Diferenciación Celular , Dextranos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Feto , Regulación de la Expresión Génica , Humanos , Immunoblotting , Inmunohistoquímica , Pulmón/anomalías , Pulmón/embriología , Masculino , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Ratones , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso/citología , Técnicas de Cultivo de Órganos , Isoformas de Proteínas/genética , ARN/genética , ARN/metabolismo , Factor de Respuesta SéricaRESUMEN
Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive activity. To date, despite the mounting evidence implicating the potential diagnostic/prognostic and therapeutic value of maspin in breast and prostate carcinoma, the lack of a suitable animal model hampers the in vivo investigation on the role of maspin at different stages of tumor progression. In this study, we used MMTV/TGF-alpha transgenic mouse model to study the expression profile of maspin in mammary tumor progression. Histopathological examinations of MMTV/TGF-alpha transgenic mice revealed TGF-alpha expression leading to hyperproliferation, hyperplasia, and occasional carcinoma in mammary gland. Interestingly, when MMTV/TGF-alpha transgenic mice were breed to homozygocity, they also developed characteristic skin papillomas. Immunohistochemistry analysis of maspin expression in the breast tissues of TGF-alpha transgenic mice showed a direct correlation between down-regulation of maspin expression and tumor progression. The loss of maspin expression was concomitant with the critical transition from carcinoma in situ to invasive carcinoma. Subsequent in-situ hybridization analyses suggest that the down-regulation of maspin expression is primarily a transcriptional event. This data is consistent with the tumor suppressive role of maspin. Furthermore, our data suggests that MMTV/TGF-alpha transgenic mouse model is advantageous for in vivo evaluation of both the expression and the biological function of maspin during the slow multi-stage carcinogenesis of mammary gland.
Asunto(s)
Carcinoma in Situ/metabolismo , Regulación hacia Abajo , Neoplasias Mamarias Experimentales/metabolismo , Virus del Tumor Mamario del Ratón/genética , Biosíntesis de Proteínas , Serpinas/biosíntesis , Factor de Crecimiento Transformador alfa/genética , Animales , Carcinoma in Situ/genética , Carcinoma in Situ/patología , Progresión de la Enfermedad , Femenino , Genes Supresores de Tumor , Hiperplasia/genética , Hiperplasia/metabolismo , Inmunohistoquímica , Hibridación in Situ , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Invasividad Neoplásica , Proteínas/genética , Proteínas/inmunología , ARN Neoplásico/biosíntesis , Serpinas/genética , Serpinas/inmunología , Transcripción GenéticaRESUMEN
Mouse embryonic mesenchymal cells undergo spontaneous smooth muscle (SM) differentiation upon spreading/elongation in culture (Relan et al., J. Cell Biol. 147 (1999) 1341; Yang et al., Development 125 (1998) 2621; Yang et al., Development 126 (1999) 3027). Using these cells we generated a subtracted cDNA library to identify potential suppressors of SM myogenesis. One of the differentially expressed genes was heterogeneous nuclear ribonucleoprotein-H (hnRNP-H), which is involved in pre-mRNA alternative splicing. hnRNP-H was highly expressed in mesenchymal cells prior to the onset of SM differentiation, but its expression rapidly decreased in mesenchymal cells undergoing SM myogenesis. In vivo, the drop in hnRNP-H expression was restricted to visceral SM cells. Antisense oligodeoxynucleotide and antisense RNA were used to inhibit hnRNP-H synthesis in SM-differentiating mesenchymal cells and in embryonic lung explants. A decrease in hnRNP-H levels resulted in upregulation of SM-specific gene expression and increased bronchial SM development in lung explants. hnRNP-H overexpression in cell cultures had the opposite effect. These studies, therefore, indicate a novel role for hnRNP-H in the control of visceral myogenesis.
Asunto(s)
Músculo Liso/embriología , Ribonucleoproteínas/fisiología , Empalme Alternativo , Animales , Northern Blotting , Western Blotting , Bronquios/embriología , Diferenciación Celular , Células Epiteliales/metabolismo , Biblioteca de Genes , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H , Ribonucleoproteínas Nucleares Heterogéneas , Inmunohistoquímica , Pulmón/embriología , Pulmón/patología , Mesodermo/metabolismo , Ratones , Ratones Endogámicos ICR , Oligonucleótidos Antisentido/metabolismo , Técnicas de Cultivo de Órganos , ARN Mensajero/metabolismo , Factores de Tiempo , Distribución Tisular , TransfecciónRESUMEN
We used an antisense oligonucleotide (ODN) to inhibit laminin (LM) beta1 chain synthesis in mouse embryonic lung explants and cell cultures. The ODN spanned 17 bases located 13 bases downstream the initiation codon and contained phosphorothioate and C-5 propynyl pyrimidine modifications. Penetration of the ODN into the lung explants was confirmed by fluorescein isothiocyanate (FITC) tagging. 50 microM of antisense ODN decreased LM beta1 chain synthesis by 82+/-6.9% with no significant changes in the synthesis of other LM chains. The same antisense probe but without C-5 propynyl pyrimidine modification, another 17-mer ODN complementary to the LM beta1 initiation codon, and a 17-mer ODN complementary to the LM alpha1 initiation codon had no antisense activity. Lung explants exposed to the active LM beta1 antisense ODN showed decreased LM-1 and collagen type IV deposition at the epithelial-mesenchymal interface and an arrest in bronchial smooth muscle (SM) development. Histological examination and cell motility assays suggested that this arrest was due to impaired spreading and migration of SM cell precursors over the defective basement membrane (BM). Our studies indicate that beta1-chain containing LMs play a role in bronchial myogenesis.
Asunto(s)
Laminina/genética , Pulmón/efectos de los fármacos , Pulmón/embriología , Músculo Liso/patología , Oligonucleótidos Antisentido/farmacología , Animales , Bronquios/efectos de los fármacos , Bronquios/embriología , Bronquios/patología , Movimiento Celular/efectos de los fármacos , Técnicas de Cocultivo , Células Epiteliales/efectos de los fármacos , Fluoresceína-5-Isotiocianato , Inmunohistoquímica , Laminina/biosíntesis , Laminina/efectos de los fármacos , Mesodermo/efectos de los fármacos , Ratones , Ratones Endogámicos , Músculo Liso/efectos de los fármacos , Músculo Liso/embriología , Oligonucleótidos Antisentido/química , Pirimidinas/química , Tionucleótidos/química , Tionucleótidos/farmacologíaRESUMEN
Laminins (LMs), the main constituents of basement membranes (BMs), are heterotrimeric glycoproteins composed of alpha, beta, and gamma chains held together by disulfide bonds. In the presence of Ca2+, some laminins, such as laminin-1 self-assemble into a polymer through the interaction of their three NH2 termini. Here we exposed lung organotypic cultures to a proteolytic fragment of laminin-1 that blocks laminin polymerization. This fragment, referred as E4, comprises the outer globular region of laminin beta1 chain. Inhibition of laminin polymerization in lung organotypic cultures resulted in impaired basement membrane assembly and failure of epithelial cells to polarize. In addition, we found that in control organotypic cultures, the bronchial smooth muscle cells were arranged in concentric layers around the newly formed epithelium. However, in E4-treated cultures, the smooth muscle cells were in disarray. Exposure of organotypic cultures to laminin-1 proteolytic fragment P1', that comprises part of alpha1, beta1, and gamma1 chains, but does not overlap with fragment E4, had no effect in basement membrane assembly. Exposure to fragment E4 also caused an increased release of laminin-1 into the culture medium, suggesting a failure to retain laminin at the epithelial-mesenchymal interface. These studies provide the first direct evidence linking epithelial cell polarization to laminin polymerization at the epithelial-mesenchymal interface and assign a key role to the outer globular region of laminin beta1 chain.
Asunto(s)
Polaridad Celular , Células Epiteliales/citología , Laminina/antagonistas & inhibidores , Laminina/química , Pulmón/embriología , Fragmentos de Péptidos/antagonistas & inhibidores , Animales , Técnicas de Cocultivo , Inmunohistoquímica , Polímeros/metabolismo , Desnaturalización Proteica , Células Tumorales CultivadasRESUMEN
Previous studies have demonstrated that a number of membrane-active agents are capable of binding to the surface of polymorphonuclear leukocytes (PMN) resulting in an augmentation of superoxide anion and hydrogen peroxide (H2O2) production in response to soluble stimuli. It is now demonstrated that these same membrane-active agents can bind to the surface of endothelial cells and enhance their susceptibility to killing by H2O2. Membrane-active agents which are capable of synergizing with H2O2 include cationic proteins, cationic poly-amino acids, lysophosphatides and enzymes which are capable of degrading membrane phospholipids (e.g., phospholipase C, phospholipase A2 and streptolysin S). In each case, treatment of the target cells with the membrane-active agent and H2O2 produces greater damage than the sum of the damage produced by either agent separately. Since inflammatory lesions, particularly sites of bacterial infection, may contain a rich mixture of cationic substances, phospholipases and phospholipid breakdown products, these substances may contribute to the tissue damage observed at sites of inflammation by enhancing endothelial cell sensitivity to PMN-generated H2O2 as well as by augmenting the generation of H2O2 by PMNs.
Asunto(s)
Citotoxicidad Inmunológica/fisiología , Endotelio Vascular/fisiología , Peróxido de Hidrógeno/metabolismo , Animales , Catalasa/metabolismo , Cromo/metabolismo , Endotelio Vascular/metabolismo , Radicales Libres , Glucosa Oxidasa/metabolismo , Histonas/metabolismo , Técnicas In Vitro , Lisofosfatidilcolinas/metabolismo , Microscopía de Contraste de Fase , Neutrófilos/metabolismo , Fosfolipasas/metabolismo , Polianetolsulfonato/metabolismo , Ratas , Estreptolisinas/metabolismoRESUMEN
In a patient with a large atrial myxoma, the phonocardiographic timing of the tumor plop has been correlated with the two-dimensional echocardiographic motion pattern of the cardiac mass. The tumor plop occurred at the time when the mass stopped its diastolic forward motion into the ventricle and made a strong impact on the interventricular septum and right ventricular posterior wall. Occurrence of tumor plop may require a large mass or long enough tumor stalk to allow the impact of the mass on the ventricular wall.
Asunto(s)
Neoplasias Cardíacas/diagnóstico , Mixoma/diagnóstico , Adulto , Ecocardiografía , Femenino , Atrios Cardíacos , Neoplasias Cardíacas/fisiopatología , Tabiques Cardíacos , Humanos , Contracción Miocárdica , Mixoma/fisiopatología , Fonocardiografía , SonidoRESUMEN
Donor lymphocyte infusion mediates most effective graft- versus-leukemia (GVL) effects following induction of host-versus-graft tolerance by transplantation of donor stem cells. This study was designed to maximize GVL effects across both major (MHC) and minor (mHgs) histocompatibility barriers in recipients inoculated with murine B-cell leukemia (BCL1), using specifically immune donor lymphocytes. GVL effects were induced with donor spleen cells from mice immunized across MHC or mHgs barriers with BCL/1 cells or normal BALB/c spleen cells. Our data suggest that spleen cells from donor mice immunized against murine B-cell leukemia of BALB/c origin, or to a lesser extent against normal host alloantigens, induce better therapeutic GVL effects with less great-versus-host disease (GVHD) across both mHgs and MHC. The cytokine profile of effector cells inducing predominantly GVL effects with reduced GVHD across MHC and mHg barriers consisted preferentially of upregulated IFN-gamma, IL-2, IL-10 and IL-12 in donors, implying a Th-1 to Th-2 cytokine shift. We hypothesize that immunotherapy with immune donor lymphocytes sensitized in vivo or in vitro with allogeneic tumor cells or normal host cells together with allogeneic BMT may provide an effective approach for amplifying GVL effects, while reducing procedure-related morbidity and mortality due to uncontrolled GVHD.
Asunto(s)
Efecto Injerto vs Leucemia , Inmunización/métodos , Inmunoterapia Adoptiva/métodos , Leucemia de Células B/terapia , Transfusión de Linfocitos/métodos , Animales , Citocinas/biosíntesis , Enfermedad Injerto contra Huésped , Leucemia de Células B/complicaciones , Leucemia de Células B/mortalidad , Ratones , Ratones Endogámicos , Bazo/citología , Bazo/inmunología , Bazo/trasplante , Inmunología del Trasplante , Trasplante HomólogoRESUMEN
Gangliogliomas are rare tumors, primarily seen in patients under the age of 30 years. They occur least commonly in the spinal cord. This report presents a 2 1/2-year-old boy who harbored an intramedullary conus tumor with the light microscopic appearance of a ganglioglioma. The neurobiological behavior of this tumor is yet to be determined.
Asunto(s)
Neuroblastoma/patología , Neoplasias de la Médula Espinal/patología , Preescolar , Humanos , MasculinoRESUMEN
Seven cardiac myxomas were studied by immunoperoxidase and immunofluorescence in formalin fixed and paraffin embedded tissues. Specific antisera to factor VIII related antigens, vimentin, myosin of smooth muscle, actin, desmin, alpha-1-antitrypsin, muramidase, fibrin and prekeratin antigens were used. All myxoma cells reacted positively with antibodies to vimentin and showed no staining reaction with antibodies to alpha-1-antitrypsin, muramidase, myosin, or prekeratin. Factor VIII related antigen was found only in endothelial cells and not in myxoma cells proper. Fibrin was found in patchy areas within the stroma. Antisera to actin and desmin failed to react with formalin fixed tissue. Our results suggest that the main cellular component of cardiac myxoma is a primitive mesenchymal cell without immunohistochemical evidence of more specific differentiation.
Asunto(s)
Neoplasias Cardíacas/análisis , Mixoma/análisis , Actinas/análisis , Adulto , Anciano , Factor VIII/análisis , Femenino , Histocitoquímica , Humanos , Queratinas/análisis , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Vimentina/análisisRESUMEN
We report two cases of benign ovarian cysts that revealed cytomegaly of the epithelial lining cells. One case was a large solitary luteinized follicle cyst of pregnancy and puerperium. The other case was an endometrial cyst. Changes in the cells included marked pleomorphism and hyperchromasia of the nuclei, large amounts of cytoplasm, and lack of mitotic figures. We ascribed these cellular features to hormonal effects and stress the importance of recognizing such changes and differentiating them from malignancy.
Asunto(s)
Cuerpo Lúteo/patología , Células Lúteas/patología , Quistes Ováricos/patología , Adulto , Núcleo Celular/patología , Citoplasma/patología , Epitelio/patología , Femenino , Humanos , Quistes Ováricos/cirugía , Periodo Posparto , EmbarazoRESUMEN
Laminins are essential components of basement membranes, playing important roles in cell adhesion, proliferation, and differentiation. These heterotrimeric glycoproteins are composed of an alpha, beta, and gamma chains held together by disulfide bonds. The first laminin identified, from the mouse Engelbreth-Holm-Swarm (EHS) tumor, is now referred to as laminin-1. Laminin-1 is expressed in the mouse developing lung by epithelial and mesenchymal cells and plays a role in branching morphogenesis. Since laminins are multidomain proteins, different laminin sites are engaged in promoting lung organogenesis by serving different functions at different stages of development. This study shows that the cross region of the molecule selectively promotes epithelial cell proliferation. The outer globular region of alpha 1 and beta 1 chains mediates laminin polymerization and thereby basement membrane formation and epithelial cell polarization. The inner globular region of laminin beta 1 chain binds to heparan sulfate proteoglycan and both stimulate lumen formation. While the combined effect of these laminin active sites results in normal lung tissue structure and branching morphogenesis, different developmental abnormalities of the lung may result from alterations in each of them.
Asunto(s)
Laminina/metabolismo , Pulmón/embriología , Secuencia de Aminoácidos , Animales , Membrana Basal/metabolismo , Desarrollo Embrionario y Fetal , Células Epiteliales , Epitelio/embriología , Matriz Extracelular/metabolismo , Pulmón/citología , Pulmón/metabolismo , Ratones , Datos de Secuencia Molecular , MorfogénesisRESUMEN
Retinoic acid (RA) stimulated proliferation of both epithelial and mesenchymal cells in cocultures isolated from developing mouse lungs. There was a corresponding increase in epithelial branching activity in organ culture of embryonic lungs exposed to similar doses of RA. Stimulation was maximal with concentrations of 1 microM and progressively decreased with either lower or higher concentrations. However, when lung cell monocultures of isolated epithelial and mesenchymal cells were exposed to RA, the mitogenic effect was observed only in the mesenchymal population. This suggests that RA may not have a direct mitogenic effect on epithelial cells but rather functions indirectly through the mesenchyme. The cellular response to RA was correlated with an increase in the expression of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) also stimulated terminal branch formation in the developing lung. Unlike RA, EGF stimulated proliferation in both epithelial cells and mesenchymal cells in monoculture. In comparison, transforming growth factor-alpha, which also binds to the EGFR, elicited no response. We conclude that RA stimulates cell proliferation and branching activity in the developing mouse lung by a mechanism involving epithelial-mesenchymal interactions. The effect is, in part, produced by stimulation of EGFR expression, with the resulting amplification of the cellular response to EGF or other EGFR ligands. In this process the mesenchyme provides a paracrine support to the epithelium, otherwise unresponsive to RA. Further studies identified the mesenchyme as a major source of EGF in the embryonic lung, suggesting that mesenchymal EGF may represent a paracrine factor involved in the epithelial response to RA.
Asunto(s)
Receptores ErbB/biosíntesis , Pulmón/embriología , Mesodermo/fisiología , Tretinoina/farmacología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales , Epitelio/fisiología , Femenino , Pulmón/efectos de los fármacos , Mesodermo/citología , Ratones , EmbarazoRESUMEN
The pattern of deposition and the role of laminin, a major glycoprotein constituent of basement membranes, were investigated during lung morphogenesis in the fetal mouse. Lung primordia were removed from Day 13 embryos, right lower lobes were further dissected and placed in filter membrane assemblies. Explants were then cultured at the liquid-air interface for 3 days in the presence of anti-laminin, anti-thrombospondin (another extracellular matrix constituent), preimmune serum, laminin-neutralized anti-laminin, or medium alone. Cultures were monitored by (direct) phase-contrast microscopy, light microscopy, and immunofluorescence. We found that anti-laminin antibodies altered normal lung morphogenesis in a dose-dependent manner. The anti-laminin-treated explants presented a marked inhibition of branching morphogenesis and a distortion of the bronchial tree. A lower rate of growth was also observed in the explants exposed to this antibody. High concentrations of anti-thrombospondin antibodies, normal rabbit serum, or laminin-neutralized anti-laminin antibodies had no effect on lung morphogenesis. These results were not modified by culturing the explants in submersion culture or on Vitrogen 100-coated surfaces.
Asunto(s)
Laminina/fisiología , Pulmón/embriología , Animales , Anticuerpos , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Immunoblotting , Laminina/inmunología , Laminina/farmacología , Pulmón/citología , Pulmón/efectos de los fármacos , Ratones , Ratones Endogámicos , Peso Molecular , Morfogénesis/efectos de los fármacos , Técnicas de Cultivo de Órganos , EmbarazoRESUMEN
Glycerol induced acute renal failure (ARF) is known to attenuate subsequent mercuric chloride nephrotoxicity. This protection was evaluated in rats. Glycerol induced varying degrees of renal insufficiency. After 14 days, when serum creatinine (SCr) creatinine clearance (CCr) and fractional excretion of sodium (FENa) had returned to baseline, injection of mercuric chloride caused significantly milder renal insufficiency in recovered rats than in controls (SCr 356 +/- 46 vs. 475 +/- 19 mumol/L; CCr 0.12 +/- 0.02 vs. 0.02 +/- 0.02 mL/min, p < .05; and mortality 0 vs. 45%, respectively, p < .01). A striking finding was that the degree of renal insufficiency induced by mercuric chloride correlated inversely with the degree of renal insufficiency previously induced by glycerol (r = -0.496, p < .05 for SCr and CCr), but there was no correlation with other measures of previous renal function such as urine volume, sodium excretion, or FENa. Glycerol induced ARF also attenuated the renal toxicity of mercuric chloride injected 4 days after glycerol, before full recovery of renal function. The decrements in renal function after the two insults were also inversely related (r = -0.76, p < .01). A third renal insult with a second mercuric chloride injection after three weeks was still attenuated. However, after the third insult, there was no longer an inverse or any statistical relationship with previous measurements of renal function. Histopathology revealed a good correlation between peak Scr after glycerol, and percentage of tubules undergoing re-generation 14 days later (r = 0.97, p < .01). There was an inverse correlation between Scr after mercuric chloride (administered 14 days after glycerol) and percentage of tubular regeneration seen two days later (r = -0.79, p < .05). The correlations of SCr and CCr with regeneration was greater than the correlations with tubular necrosis, suggesting that the regenerative process is involved in the protection from repeated renal insults. In conclusion, glycerol-induced ARF attenuates subsequent mercuric chloride renal insult. The attenuation correlates directly with the initial glycerol-induced damage, so that the more severe the initial renal insufficiency, the milder the renal insufficiency following subsequent mercuric chloride. This protection can be seen as early as 4 days and also 14 days after previous renal insult. The degree of renal tubular regeneration correlates well with the protection seen, and probably plays a role in acquired renal resistance to repeated insults.
Asunto(s)
Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/fisiopatología , Glicerol , Riñón/fisiopatología , Cloruro de Mercurio , Animales , Pruebas de Función Renal , Túbulos Renales/fisiología , Masculino , Ratas , Regeneración , Factores de TiempoRESUMEN
The histological findings in the liver in four fatal cases of infantile cytomegalovirus (CMV) infection are presented, three occurred in premature infants, and all died at the ages of 2-4 months. Most previously reported cases showed various degrees of hepatitis with giant cell transformation. In three of our four cases, however, the main feature was cholestasis, portal fibrosis and bile duct proliferation, not unlike the findings seen in extrahepatic biliary obstruction. In one case, massive hepatic necrosis was found, a finding not previously reported in this disease. The diversity of liver findings in infantile CMV infection is stressed.
Asunto(s)
Colestasis/patología , Infecciones por Citomegalovirus/patología , Hígado/patología , Conductos Biliares/patología , Enfermedades en Gemelos , Femenino , Fibrosis , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro , MasculinoRESUMEN
The behavior of embryonic murine lung cells on a basement membrane extract (Matrigel) was investigated. Single cell suspensions generated by trypsinization of lungs removed from day 12 embryos were plated on Matrigel and cultured for up to one week. The basement membrane extract was used as a gel, and as a wet or dried film. In all of these instances, organotypic arrangement of the embryonic lung cells was observed. This process consisted of cell aggregation, sorting, polarization and formation of a tridimensional organization resembling embryonic lung. The maximal degree of organotypic development was obtained by using a thick gel; minimal reorganization was observed using a dried film. A rabbit polyclonal serum to laminin inhibited organotypic pattern formation while normal rabbit serum did not. Culture of lung cells on laminin gels promoted epithelial cyst formation but poor mesenchymal organization. By studying the behavior of epithelial and/or mesenchymal enriched cell populations on Matrigel, it was concluded that organotypic pattern formation on Matrigel required the presence of both cell populations. Cultivation of dissociated lung cells on a gel consisting of a mixture of collagens type I and III (Vitrogen-100) produced only cell aggregation. Cultivation of lung cells on a thin film of Vitrogen-100 or on uncoated tissue culture plastic produced monolayers of mesenchymal cells alone. Cultivation of lung cells in suspension also failed to induce organotypic arrangement even at maximal cell densities. The present study strongly supports a role for the basement membrane in the organotypic rearrangement of embryonic lung cells and subsequent in vitro cyst formation and budding of the reestablished epithelium. This, in turn, reinforces the concept of the basement membrane as a major regulator of organogenesis.
Asunto(s)
Membrana Basal/fisiología , Laminina/fisiología , Pulmón/embriología , Animales , Epitelio/fisiología , Pulmón/fisiología , Pulmón/ultraestructura , Mesodermo/fisiología , Ratones , Ratones Endogámicos , Microscopía Electrónica , Microscopía de Contraste de Fase , Técnicas de Cultivo de ÓrganosRESUMEN
The recent establishment of a role for laminin in mouse lung organogenesis (Schuger et al. 1990a,b, 1991) prompted us to study its expression in the developing lung. Laminin A and B chains were detected in the murine lung from the first hours of development onward. In situ hybridization of mRNA as well as SDS-PAGE studies of lung cells in monoculture indicated that both epithelium and mesenchyme produce complete laminin molecules. Quantitative analysis of the in situ hybridization studies showed a gradual increase in laminin expression during development which was further supported by immunohistochemistry and ELISA. The overall pattern of expression suggested that the effects of laminin in morphogenesis were not restricted to a particular stage of development. Furthermore, the increase in expression during late development supported a role for the molecule in the fetal lung, which was not previously established. We next determined whether the increase in laminin production modulated the behavior of fetal lung cells as compared with their embryonic counterparts. We previously showed that organotypic pattern formation does not occur in cultures of mixed embryonic lung cells unless exogenous laminin is added (Schuger et al., 1990b). Organotypic pattern formation is the result of cell sorting into epithelial and mesenchymal compartments and further rearrangement in a pattern resembling the tissue of origin. In the present study, we demonstrated that organotypic pattern formation occurs spontaneously in cultures of mixed fetal lung cells, which express high laminin levels. Pattern formation was abolished by antibodies to laminin. These studies suggest a correlation between laminin expression and the ability of lung cells in culture to reproduce normal tissue patterns. We conclude that laminin is critical for epithelial-mesenchymal recognition and further morphogenic interaction during both the embryonic and fetal stages of lung development.