RESUMEN
INTRODUCTION: Tenascin-X (TNX) is an extracellular matrix (ECM) glycoprotein, the absence of which in humans leads to a recessive form of Ehlers-Danlos syndrome (EDS), a group of inherited connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. A mouse model of TNX-deficient type EDS has been used to characterize the dermatological, orthopedic, and obstetrical features. The growing insight in the clinical overlap between myopathies and inherited connective tissue disorders asks for a study of the muscular characteristics of inherited connective tissue diseases. Therefore, this study aims to define the muscular phenotype of TNX knockout (KO) mice. MATERIALS AND METHODS: We performed a comprehensive study on the muscular phenotype of these TNX KO mice, consisting of standardized clinical assessment, muscle histology, and gene expression profiling of muscle tissue. Furthermore, peripheral nerve composition was studied by histology and electron microscopy. RESULTS: The main findings are the presence of mild muscle weakness, mild myopathic features on histology, and functional upregulation of genes encoding proteins involved in ECM degradation and synthesis. Additionally, sciatic nerve samples showed mildly reduced collagen fibril density of endoneurium. DISCUSSION: The muscular phenotype of TNX KO mice consists of mild muscle weakness with histological signs of myopathy and of increased turnover of the ECM in muscle. Furthermore, mildly reduced diameter of myelinated fibers and reduction of collagen fibril density of endoneurium may correspond with polyneuropathy in TNX-deficient EDS patients. This comprehensive assessment can serve as a starting point for further investigations on neuromuscular function in TNX KO mice.
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Músculos/patología , Tenascina/deficiencia , Animales , Modelos Animales de Enfermedad , Síndrome de Ehlers-Danlos/patología , Femenino , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Noqueados , Actividad Motora , Músculo Esquelético/fisiopatología , Músculos/fisiopatología , Nervio Ciático/patologíaRESUMEN
INTRODUCTION: In aromatic L-amino acid decarboxylase (AADC) deficiency, a neurotransmitter biosynthesis defect, paradoxical normal or increased levels of urinary dopamine have been reported. Genotype/phenotype correlations or alternative metabolic pathways may explain this remarkable finding, but were never studied systematically. METHODS: We studied the mutational spectrum and urinary dopamine levels in 20 patients with AADC-deficiency. Experimental procedures were designed to test for alternative metabolic pathways of dopamine production, which included alternative substrates (tyramine and 3-methoxytyrosine) and alternative enzymes (tyrosinase and CYP2D6). RESULTS/DISCUSSION: In 85% of the patients the finding of normal or increased urinary levels of dopamine was confirmed, but a relation with AADC genotype could not be identified. Renal microsomes containing CYP2D were able to convert tyramine into dopamine (3.0 nmol/min/g protein) but because of low plasma levels of tyramine this is an unlikely explanation for urinary dopamine excretion in AADC-deficiency. No evidence was found for the production of dopamine from 3-methoxytyrosine. Tyrosinase was not expressed in human kidney. CONCLUSION: Normal or increased levels of urinary dopamine are found in the majority of AADC-deficient patients. This finding can neither be explained by genotype/phenotype correlations nor by alternative metabolic pathways, although small amounts of dopamine may be formed via tyramine hydroxylation by renal CYP2D6. CYP2D6-mediated conversion of tyramine into dopamine might be an interesting target for the development of new therapeutic strategies in AADC-deficiency.
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Descarboxilasas de Aminoácido-L-Aromático/deficiencia , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Dopamina/orina , Adolescente , Adulto , Animales , Descarboxilasas de Aminoácido-L-Aromático/genética , Niño , Preescolar , Citocromo P-450 CYP2D6/metabolismo , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Humanos , Lactante , Corteza Renal/enzimología , Masculino , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Ratas , Tiramina/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Adulto JovenRESUMEN
The tumour microenvironment (TME) forms a major obstacle in effective cancer treatment and for clinical success of immunotherapy. Conventional co-cultures have shed light onto multiple aspects of cancer immunobiology, but they are limited by the lack of physiological complexity. We develop a human organotypic skin melanoma culture (OMC) that allows real-time study of host-malignant cell interactions within a multicellular tissue architecture. By co-culturing decellularized dermis with keratinocytes, fibroblasts and immune cells in the presence of melanoma cells, we generate a reconstructed TME that closely resembles tumour growth as observed in human lesions and supports cell survival and function. We demonstrate that the OMC is suitable and outperforms conventional 2D co-cultures for the study of TME-imprinting mechanisms. Within the OMC, we observe the tumour-driven conversion of cDC2s into CD14+ DCs, characterized by an immunosuppressive phenotype. The OMC provides a valuable approach to study how a TME affects the immune system.
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Plasticidad de la Célula/fisiología , Células Dendríticas/metabolismo , Melanoma/metabolismo , Microambiente Tumoral/fisiología , Comunicación Celular , Supervivencia Celular , Técnicas de Cocultivo , Fibroblastos/patología , Humanos , Queratinocitos/patología , Melanoma/inmunología , Melanoma/patología , Piel/patología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Microambiente Tumoral/inmunología , Melanoma Cutáneo MalignoRESUMEN
To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin-free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.
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Desmina/metabolismo , Filamentos Intermedios/metabolismo , Secuencia de Aminoácidos , Animales , Biopolímeros , Línea Celular , Deleción Cromosómica , Cricetinae , Desmina/química , Desmina/genética , Técnica del Anticuerpo Fluorescente , Humanos , Filamentos Intermedios/ultraestructura , Queratinas/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Membrana Nuclear/metabolismo , Relación Estructura-Actividad , Transfección , Vimentina/fisiologíaRESUMEN
How and why tumors metastasize is still a matter of debate. The assumption is that mutations render tumor cells with a metastatic phenotype, enabling entrance in and transport through lymph or blood vessels. Distant outgrowth is thought to occur only in a suitable microenvironment (the seed and soil hypothesis). However, the anatomical location of most metastases in cancer patients suggests entrapment of tumor cells in the first microcapillary bed that is encountered. We here investigated how vascular endothelial growth factor-A (VEGF-A) attributes to the metastatic process. We describe here that VEGF-A enhances spontaneous metastasis by inducing intravasation of heterogeneous tumor cell clusters, surrounded by vessel wall elements, via an invasion-independent mechanism. These tumor clusters generate metastatic tissue embolisms in pulmonary arteries. Treatment of tumor-bearing mice with the antiangiogenic compound ZD6474 prevented the development of this metastatic phenotype. This work shows that tumors with high constitutive VEGF-A expression metastasize via the formation of tumor emboli and provides an alternative rationale for anti-VEGF-A therapy, namely to inhibit metastasis formation.
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Neoplasias Encefálicas/secundario , Neoplasias Pulmonares/secundario , Melanoma/secundario , Células Neoplásicas Circulantes/metabolismo , Embolia Pulmonar/patología , Neoplasias Cutáneas/patología , Factor A de Crecimiento Endotelial Vascular/fisiología , Inhibidores de la Angiogénesis/farmacología , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/prevención & control , Regulación Neoplásica de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevención & control , Metástasis Linfática/patología , Masculino , Melanoma/metabolismo , Melanoma/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Neoplásicas Circulantes/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Piperidinas/farmacología , Quinazolinas/farmacología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/prevención & control , Transfección , Células Tumorales CultivadasRESUMEN
BACKGROUND/PURPOSE: The aim of the study was to evaluate whether a collagen biomatrix is useful for delayed intrauterine coverage of a surgically created spina bifida in a fetal lamb. METHODS: In 20 fetal lambs, surgery was performed at 72 or 79 days' gestation. In 15 lambs a spina bifida was created surgically. In 8 lambs it was covered with a collagen biomatrix 2 weeks later and in 7 lambs it was left uncovered. Five lambs served as sham operated controls. Neurological examination was performed at 1 week of age and afterwards the lambs were sacrificed for further histological evaluation. RESULTS: None of the 5 surviving lambs with the defect covered showed loss of spinal function and the architecture of the spinal cord was preserved in 4 of the 5 lambs. In the uncovered group, 1 of the 4 surviving lambs had loss of spinal function, 5 lambs were available for histological evaluation and 4 of them showed disturbance of the architecture of the spinal cord. CONCLUSIONS: Collagen biomatrices can be used for intrauterine coverage of an experimental spina bifida and can preserve the architecture of the spinal cord. Neurological outcome is not different between fetuses with their spinal cord covered and fetuses with uncovered cords.
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Colágeno Tipo I/administración & dosificación , Modelos Animales de Enfermedad , Atención Prenatal/métodos , Disrafia Espinal/cirugía , Animales , Femenino , Embarazo , Oveja Doméstica , Disrafia Espinal/patología , Factores de TiempoRESUMEN
Specific binding of [125I]-(-)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (Kd = 5.3 +/- 0.9 pmol/l and RT = 78 +/- 7 fmol/g tissue). The beta 1-selective antagonists atenolol and LK 203-030 inhibited specific [125I]-(-)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous beta 2-adrenoceptor population. In one subject using LK 203-030 a small beta 1-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pKH- and pKL-values for the high and low affinity sites were 8.0 +/- 0.2 and 5.9 +/- 0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD2-value of 6.63 +/- 0.19.
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Contracción Muscular , Relajación Muscular , Músculo Liso/metabolismo , Receptores Adrenérgicos beta/metabolismo , Tráquea/metabolismo , Atenolol/metabolismo , Unión Competitiva , Humanos , Técnicas In Vitro , Pindolol/análogos & derivados , Pindolol/metabolismo , Propranolol/metabolismo , Receptores Adrenérgicos beta/fisiología , Sarcolema/metabolismo , Estereoisomerismo , Tráquea/fisiologíaRESUMEN
Collapsing focal segmental glomerulosclerosis (cFSGS) is characterized by hyperplasia of glomerular epithelial cells. In a mouse model of FSGS and in a patient with recurrent idiopathic FSGS, we identified the proliferating cells as parietal epithelial cells (PECs). In the present study, we have evaluated the origin of the proliferating cells in cFSGS associated with human immunodeficiency virus (HIV) and pamidronate. We performed a detailed study of glomerular lesions in biopsies of two patients with HIV-associated cFSGS and a nephrectomy specimen of a patient with pamidronate-associated cFSGS. Glomeruli were studied by serial sectioning using light and electron microscopy and immunohistochemistry to determine the epithelial cell phenotype. We used Synaptopodin, vascular endothelial growth factor, and CD10 as podocyte markers, CK8 and PAX2 as PEC markers and Ki-67 as marker of cell proliferation. The newly deposited extracellular matrix was characterized using antiheparan sulfate single-chain antibodies. The proliferating cells were negative for the podocyte markers, but stained positive for the PEC markers and the cell proliferation marker Ki-67. The proliferating PAX-2 and CK8 positive cells that covered the capillary tuft were always in continuity with PAX-2/CK8 positive cells lining Bowman's capsule. The matrix deposited by these proliferating cells stained identically to Bowman's capsule. Our study demonstrates that PECs proliferate in HIV and pamidronate-associated cFSGS. Our data do not support the concept of the proliferating, dedifferentiated podocyte.
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Nefropatía Asociada a SIDA/patología , Células Epiteliales/patología , Glomeruloesclerosis Focal y Segmentaria/patología , Glomérulos Renales/patología , Antineoplásicos/efectos adversos , Biomarcadores , Biopsia , División Celular , Difosfonatos/efectos adversos , Glomeruloesclerosis Focal y Segmentaria/inducido químicamente , Humanos , Pamidronato , Podocitos/patologíaRESUMEN
This paper describes a new automated system to prepare slides of cytological material from suspension. The system collects material on a filter tape by filtration and transfers it to glass slides by means of pressure-fixation. Using cervical cells as a model, results show that a well-defined cell number is evenly deposited over a standardized area, while a small number of cells is retained on the tape and a negligible number lost in the filtrate. Contamination is very small. Application of the system to other cytological material (fine needle aspirations, monolayer and cell suspension cultures, agar cultures, and isolated nuclei) is shown. In general, more than one slide can be made from one sample. Several histological staining procedures as well as immunofluorescence labeling protocols can be applied to the preparations obtained in this way. This system thus introduces a method that will standardize specimen preparation, is quick, saves operator time, and can be used for both diagnostic and research applications.
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Autoanálisis/instrumentación , Técnicas Histológicas/instrumentación , Autoanálisis/métodos , Biopsia con Aguja , Carcinoma/patología , Recuento de Células/instrumentación , Recuento de Células/métodos , Línea Celular , Cuello del Útero/patología , Epitelio/patología , Femenino , Humanos , Microscopía Fluorescente/métodos , Neoplasias de la Vejiga Urinaria/patología , Neoplasias Uterinas/patologíaRESUMEN
Degradation of the extracellular matrix and other tissue barriers by proteases like plasminogen activators (PAs) is a prerequisite for neoplastic growth and metastasis. Recently, we reported that highly metastatic behavior of human melanoma cells in nude mice correlates with urokinase-type PA (u-PA) expression and activity and with PA inhibitor type 1 and 2 (PAI-1, PAI-2) expression. Here we report on the occurrence of components of the PA system in the various stages of human melanoma tumor progression in situ. We studied the protein distribution on freshly frozen lesions of common nevocellular nevi (n = 25), dysplastic (= atypical) nevi (n = 16), early primary melanomas (n = 8), advanced primary melanomas (n = 11), and melanoma metastases (n = 17). Tissue-type PA was present in endothelial cells in all lesions, whereas in metastases it could be detected in tumor cells in a minority of the lesions. u-PA, its receptor, PAI-1, and PAI-2 could not be detected in benign and in early stages but appeared frequently in advanced primary melanoma and melanoma metastasis lesions. u-PA was detected in stromal cells and in tumor cells at the invasive front, the u-PA receptor and PAI-2 in tumor cells, and PAI-1 in the extracellular matrix surrounding tumor cells. Localization of the corresponding messenger RNAs and enzyme activities revealed a similar distribution. We conclude that plasminogen activation is a late event in melanoma tumor progression.