RESUMEN
Malignant tumor cells often express matrix metalloproteinases (MMPs) at a high level to enable their dissemination and metastasis. Sendai virus (SeV), a nonsegmented negative strand RNA virus, spreads in the target tissues in vivo via cleavage activation of the viral fusion glycoprotein by a tissue-specific, trypsin-like enzyme. By deleting the viral matrix protein, we previously generated a recombinant SeV that does not bud to mature virions, but is highly fusogenic and spreads extensively from cell to cell in a trypsin-dependent manner. Here, we changed the tryptic cleavage site of the fusion glycoprotein of this virus to a site susceptible to MMPs. The resulting recombinant virus was no longer activated by trypsin but spread efficiently in cultured cells supplemented with MMP2 or MMP9 and in human tumor cell lines expressing these MMPs. Furthermore, the virus spread extensively in tumor cells xenotrasplanted to nude mice without disseminating to the surrounding normal cells, leading to the inhibition of the tumor growth in the mice. These results demonstrate the selective targeting and killing of human tumor cells by recombinant SeV technology and greatly advance the reemerging concept of oncolytic virotherapy, which currently appears to rely largely upon a natural preference of certain viruses for cancer cells.
Asunto(s)
Terapia Genética/métodos , Metaloproteinasas de la Matriz/metabolismo , Neoplasias/terapia , Proteínas Recombinantes de Fusión/administración & dosificación , Virus Sendai/genética , Animales , Línea Celular Tumoral , Ingeniería Genética , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Ratones , Ratones Desnudos , Neoplasias/virología , Proteínas Recombinantes de Fusión/metabolismo , Transfección/métodosRESUMEN
Sendai virus (SeV) vector-mediated gene delivery of glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) prevented the delayed neuronal death induced by transient global ischemia in gerbils, even when the vector was administered several hours after ischemia. Intraventricular administration of SeV vector directed high-level expression of the vector-encoded neurotrophic factor genes, which are potent candidates for the treatment of neurodegenerative diseases. After occlusion of the bilateral carotid arteries of gerbils, SeV vector carrying GDNF (SeV/GDNF), NGF (SeV/NGF), brain-derived neurotrophic factor (SeV/BDNF), insulin-like growth factor-1 (SeV/IGF-1) or vascular endothelial growth factor (SeV/VEGF) was injected into the lateral ventricle. Administration of SeV/GDNF, SeV/NGF or SeV/BDNF 30 min after the ischemic insult effectively prevented the delayed neuronal death of the hippocampal CA1 pyramidal neurons. Furthermore, the administration of SeV/GDNF or SeV/NGF as late as 4 or 6 h after the ischemic insult also prevented the death of these neurons. These results indicate that SeV vector-mediated gene transfer of neurotrophic factors has high therapeutic potency for preventing the delayed neuronal death induced by transient global ischemia, and provides an approach for gene therapy of stroke.
Asunto(s)
Isquemia Encefálica/terapia , Terapia Genética/métodos , Factor de Crecimiento Nervioso/genética , Neuronas/patología , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Muerte Celular/genética , Técnicas de Transferencia de Gen , Gerbillinae , Factor Neurotrófico Derivado de la Línea Celular Glial , Hipocampo/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Virus Sendai/genéticaRESUMEN
We scrutinized the applicability and efficacy of Sendai virus (SeV) vectors expressing either LacZ or human insulin-like growth factor-I (hIGF-I) in gene transfer into skeletal muscle. Seven days after the intramuscular injection of LacZ/SeV X-gal labeled myofibers were demonstrated in rat anterior tibialis muscle with/without bupivacaine treatment and the transgene expression persisted up to 1 month after injection. Recombinant hIGF-I was detected as a major protein species in culture supernatants of a neonatal rat myoblast cell line L6 and thus induced the cells to undergo myogenetic differentiation. The introduction of hIGF-I/SeV into the muscle showed a significant increase in regenerating and split myofibers which were indicative of hypertrophy, and also an increase in the total number of myofibers, in comparison to that seen in the LacZ/SeV-treated control muscle. These results demonstrate that SeV achieves high-level transgene expression in skeletal muscle, and that hIGF-I gene transfer using SeV vector may therefore have great potential in the treatment of neuromuscular disorders.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Factor I del Crecimiento Similar a la Insulina/genética , Músculo Esquelético/fisiología , Enfermedades Neuromusculares/terapia , Regeneración , Animales , Células Cultivadas , Expresión Génica , Miembro Posterior , Humanos , Inyecciones Intramusculares , Factor I del Crecimiento Similar a la Insulina/análisis , Operón Lac , Masculino , Modelos Animales , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/análisis , Respirovirus/genética , Transfección/métodos , TransgenesRESUMEN
We examined neuropathologically and immunohistochemically the respiratory centers in the brainstem of two patients with Joubert syndrome (JS), three patients with congenital central hypoventilation syndrome (CCHS) and a patient with apneustic breathing (prolonged inspiratory pause) due to unknown etiology. Immunoreactivity (IR) of tryptophan hydroxylase (TPH) was decreased in the dorsal raphe nuclei of two patients with JS compared with age-matched controls, as well as in two patients with Dandy-Walker malformation. The two JS patients showed vermian defect and elongated cerebellar peduncles, and peculiar vascularities in the midline of the whole brainstem were also noted in one of these patients. These findings, as a whole, confirm that the midline structures of brainstem are disordered both structurally and functionally in JS, conceivably resulting in respiratory patterns and psychomotor deficits. IR of serotonin 1A receptor showed no significant changes in the medulla oblongata of these patients, however. In the parabrachial complex, IR of substance P was increased in two patients with CCHS, and one with apneustic breathing. IR of tyrosine hydroxylase was also increased in the latter. The brainstem of these patients showed reactive astrogliosis. These findings suggest preceding hypoxic episodes as well as an increased activity in the parabrachial complex which plays an important role in conducting the driving force to the medullary respiratory neurons from ascending sensory pathways.
Asunto(s)
Tronco Encefálico/patología , Hipoxia/patología , Neurotransmisores/metabolismo , Centro Respiratorio/patología , Apnea Central del Sueño/patología , Muerte Súbita del Lactante/patología , Niño , Preescolar , Síndrome de Dandy-Walker/patología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Vías Nerviosas/patología , Receptores de Serotonina/metabolismo , Receptores de Serotonina 5-HT1 , Valores de Referencia , Triptófano Hidroxilasa/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
A 12-year-old girl with a main complaint of sever pain on the both knees was admitted to our hospital in October, 1995. She gave a three year history of recurrent arthralgia and purpuric rashes, and persistent microhematuria and proteinuria. She developed vesicles and purpuric rashes on the hands and auricles, morning stiffness, fever, uveitis and pericarditis. Laboratory findings showed an elevated level of erythrocyte sedimentation rate and iron-deficiency anemia. Serum perinuclear pattern ANCA with antimyeloperioxidase specificity (MPO-ANCA) was positive. A renal biopsy specimen disclosed a focal and segmental necrotizing glomerulonephritis with crescents. Our case fulfills the both diagnostic criteria for polyarteritis nodosa and juvenile rheumatoid arthritis. This is a rare case of MPO-ANCA associated vasculitis in children.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/análisis , Artritis Juvenil/complicaciones , Nefritis/inmunología , Peroxidasa/inmunología , Niño , Femenino , HumanosRESUMEN
A 15-year-old boy developed minimal change nephrotic syndrome (MCNS) during remission of Hodgkin's disease. Natural killer (NK) cell activity was practically absent at the onset of MCNS, with a value of 3% compared with the normal value of 44.1% +/- 7.8% (mean +/- SD). Treatment with prednisolone resulted in transient remission of MCNS and partial improvement of NK cell activity. Extensive investigations for Hodgkin's disease were performed at 1- to 3-month intervals; a relapse finally became apparent 25 months after the diagnosis of MCNS. Successful treatment of Hodgkin's disease resulted in complete disappearance of proteinuria and normalisation of NK cell activity. Frequently relapsing MCNS with NK cells deficiency during remission of Hodgkin's disease appears to imply its subclinical relapse.
Asunto(s)
Enfermedad de Hodgkin/complicaciones , Células Asesinas Naturales/patología , Síndrome Nefrótico/etiología , Niño , Enfermedad de Hodgkin/patología , Enfermedad de Hodgkin/terapia , Humanos , Masculino , Síndrome Nefrótico/patología , Síndrome Nefrótico/terapia , RecurrenciaRESUMEN
BACKGROUND: A 12-year-old girl with intractable retroperitoneal paraganglioma experienced increased appetite, acne, obesity, "moon face," and enlargement of the clitoris during the course of the tumor. Plasma cortisol, serum testosterone, and dehydroepiandrosterone sulfate (DHEA-S) levels were increased to 34.1 micrograms/dl, 2.0 ng/ml, and 6.628 ng/ml, respectively. Adrenocorticotrophic hormone (ACTH) levels were not increased, and results of dexamethasone suppression tests were negative. Her condition was diagnosed as Cushing syndrome with virilism. Plasma cortisol levels were increased to a level of 107.1 micrograms/dl before death. METHODS: Tumor samples were obtained at the time of autopsy. The concentrations of cortisol, androgens, ACTH, and catecholamines were assayed in the tumor extracts. The indirect immunoperoxidase procedure was performed on fixed tissues for cortisol, DHEA-S, testosterone, and ACTH. RESULTS: Extracts of the tumor masses contained steroid hormones: the amount of immunoreactive cortisol was 1.64 micrograms/g wet weight; the amount of immunoreactive testosterone was 25.60 ng/g wet weight; immunoreactive DHEA-S, 579.00 ng/g wet weight; and immunoreactive ACTH, 891.00 pg/g wet weight in the metastatic mass of the lung. Immunohistochemically, immunoreactive cortisol, testosterone, and DHEA-S were detectable in the tumor cells. The adrenal gland was atrophic. CONCLUSIONS: The patient is the first reported with malignant paraganglioma with the capacity to produce cortisol, androgens, and ACTH.
Asunto(s)
Hormona Adrenocorticotrópica/biosíntesis , Andrógenos/biosíntesis , Síndrome de Cushing/diagnóstico , Hidrocortisona/biosíntesis , Paraganglioma/diagnóstico , Paraganglioma/metabolismo , Neoplasias Retroperitoneales/diagnóstico , Neoplasias Retroperitoneales/metabolismo , Virilismo/diagnóstico , Hormona Adrenocorticotrópica/análisis , Andrógenos/análisis , Catecolaminas/análisis , Catecolaminas/biosíntesis , Niño , Deshidroepiandrosterona/análogos & derivados , Deshidroepiandrosterona/análisis , Deshidroepiandrosterona/biosíntesis , Sulfato de Deshidroepiandrosterona , Diagnóstico Diferencial , Femenino , Humanos , Hidrocortisona/análisis , Paraganglioma/secundario , Testosterona/análisis , Testosterona/biosíntesisRESUMEN
The relationship of glomerular anionic sites to proteinuria was examined ultrastructurally in human nephrotic syndrome. The anionic sites were analysed morphometrically in patients with minimal-change nephrotic syndrome (MCNS, 11 patients) and in other glomerulonephritides complicated with nephrotic syndrome (4 patients) by the high-iron diamine-thiocarbohydrazide-silver proteinate method. The anionic sites in MCNS patients in remission (7 patients) were normal. In contrast, the anionic sites in nephrotic patients with MCNS (4 patients) and the other glomerulonephritides were decreased in number. Moreover, smaller and irregularly distributed anionic sites or the greater loss of them from the paramesangial region were observed in the nephrotic patients. The loss of glomerular anionic sites may induce structural alteration of the glomerular basement membrane and mesangial matrix. The loss and structural abnormalities of glomerular anionic sites in nephrotic patients may be one of the mechanisms responsible for massive proteinuria.
Asunto(s)
Aniones/análisis , Glomérulos Renales/química , Glomérulos Renales/ultraestructura , Nefrosis Lipoidea/patología , Adolescente , Adulto , Membrana Basal/química , Membrana Basal/ultraestructura , Niño , Preescolar , Femenino , Glomerulonefritis/patología , Humanos , Lactante , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Proteinuria/patología , Coloración y EtiquetadoRESUMEN
Histopathological analysis was performed in the first renal biopsy specimens of patients over and under 10 yrs of IgA nephritis. They were divided clinically into two groups, the one with remission and the other with prolonged disease state respectively. Increased mesangial sclerosis, frequent occurrence of segmental glomerular lesions and tubulointerstitial change were significantly evident in the group with prolonged disease state. It is suggested that similar glomerular events are progressing in IgA nephritis which is carried over to adult age.
Asunto(s)
Glomerulonefritis por IGA/patología , Glomérulos Renales/patología , Adolescente , Factores de Edad , Biopsia , Niño , Femenino , Humanos , Glomérulos Renales/ultraestructura , Masculino , Microscopía ElectrónicaRESUMEN
Changes in glomerular anionic sites were analyzed in 31 patients with various types of glomerulonephritis using the high-iron diamine-thiocarbohydrazide-silver proteinate method. On the basis of the degree of proteinuria at the time of biopsy, the patients were divided into 3 groups as follows: Group 1, less than 10 mg/kg/24 h; Group 2, 10-50 mg/kg/24 h; Group 3, more than 50 mg/kg/24 h. The number of glomerular anionic sites per 300 nm length of the lamina rare externa was 16.20 +/- 3.37 in Group 1, 12.19 +/- 3.42 in Group 2 and 9.97 +/- 2.51 in Group 3. Moreover, smaller and irregularly distributed anionic sites, and a greater loss of anionic sites in the paramesangial region were observed in Groups 2 and 3. On the other hand, there was no significant correlation between the mesangial sclerosis index and the number of anionic sites (r = -0.40) in patients with IgA nephropathy (9 cases) and nephritis of Henoch-Schönlein purpura (6 cases). These results suggest that the proteinuria seen in various types of glomerulonephritis is related to the loss of glomerular anionic sites, i.e., dysfunction of the charge-selective barrier.
Asunto(s)
Aniones , Glomerulonefritis/patología , Glomérulos Renales/patología , Adolescente , Adulto , Membrana Basal/patología , Sitios de Unión/fisiología , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Manejo de EspecímenesRESUMEN
Changes in glomerular anionic sites were examined morphometrically in nephrotic rats receiving daily subcutaneous injections of puromycin aminonucleoside (PAN). The high-iron diamine-thiocarbohydrazide-silver proteinate method showed that anionic sites composed of heparan sulfate proteoglycans formed a continuous band within the lamina rara externa of the glomerular basement membrane (GBM) in control rats. Only one day after the first injection of PAN, anionic site loss was already detectable, preceding the morphological changes in the epithelial cells. The number and size of the anionic sites decreased greatly between days 7 and 10, when urinary protein excretion began to appear. The number of anionic sites in the paramesangial regions (epithelial side of the mesangium) was slightly more reduced than that in the capillary walls. These results suggested that PAN directly injured the glomerular anionic sites and increased the permeability of the glomerulus to macromolecules. However, there was no complete correlation between the number of lost anionic sites and the level of urinary protein excretion. Thus protein excretion into the urinary space may be related not only to the loss of subepithelial anionic sites but also to dysfunction of the protein absorption mechanism in epithelial cells due to excessive permeation of macromolecules through the GBM.