RESUMEN
Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic potential. Detailed preclinical information regarding the safety of oncolytic NDV is scarce. In this study, we evaluated the toxicity, biodistribution and shedding of intravenously injected oncolytic NDVs in non-human primates (Macaca fascicularis). Two animals were injected with escalating doses of a non-recombinant vaccine strain, a recombinant lentogenic strain or a recombinant mesogenic strain. To study transmission, naive animals were co-housed with the injected animals. Injection with NDV did not lead to severe illness in the animals or abnormalities in hematologic or biochemistry measurements. Injected animals shed low amounts of virus, but this did not lead to seroconversion of the contact animals. Postmortem evaluation demonstrated no pathological changes or evidence of virus replication. This study demonstrates that NDV generated in embryonated chicken eggs is safe for intravenous administration to non-human primates. In addition, our study confirmed results from a previous report that naïve primate and human sera are able to neutralize egg-generated NDV. We discuss the implications of these results for our study and the use of NDV for virotherapy.
Asunto(s)
Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Virus de la Enfermedad de Newcastle/genética , Viroterapia Oncolítica/métodos , Alantoides/virología , Animales , Antineoplásicos/administración & dosificación , Línea Celular , Embrión de Pollo , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inyecciones Intravenosas , Macaca fascicularis , Masculino , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas Virales/administración & dosificación , Esparcimiento de VirusRESUMEN
The virulence of morbilliviruses for toothed whales (odontocetes) appears to differ according to host species. In 4 species of odontocetes, morbilliviruses are highly virulent, causing large-scale epizootics with high mortality. In 8 other species of odontocetes, including white-beaked dolphins (Lagenorhynchus albirostris), morbilliviruses have been found as an incidental infection. In these species, the virulence of morbilliviruses is not clear. Therefore, the admission of 2 white-beaked dolphins with morbillivirus infection into a rehabilitation center provided a unique opportunity to investigate the virulence of morbillivirus in this species. By phylogenetic analysis, the morbilliviruses in both animals were identified as a dolphin morbillivirus (DMV) most closely related to that detected in a white-beaked dolphin in Germany in 2007. Both animals were examined clinically and pathologically. Case No. 1 had a chronic neural DMV infection, characterized by polioencephalitis in the cerebrum and morbillivirus antigen expression limited to neurons and glial cells. Surprisingly, no nervous signs were observed in this animal during the 6 months before death. Case No. 2 had a subacute systemic DMV infection, characterized by interstitial pneumonia, leucopenia, lymphoid depletion, and DMV antigen expression in mononuclear cells and syncytia in the lung and in mononuclear cells in multiple lymphoid organs. Cause of death was not attributed to DMV infection in either animal. DMV was not detected in 2 contemporaneously stranded white-beaked dolphins. Stranding rate did not increase in the region. These results suggest that DMV is not highly virulent for white-beaked dolphins.
Asunto(s)
Delfines/virología , Enfermedades de los Peces/patología , Infecciones por Morbillivirus/veterinaria , Morbillivirus/patogenicidad , Animales , Secuencia de Bases , Femenino , Enfermedades de los Peces/virología , Alemania , Masculino , Morbillivirus/clasificación , Morbillivirus/genética , Infecciones por Morbillivirus/patología , Infecciones por Morbillivirus/virología , Países Bajos , Filogenia , Análisis de Secuencia de ADN/veterinaria , VirulenciaRESUMEN
In the present study, the distribution of genetic aberrations in a glioblastoma resection specimen of unusually large size (9x8x2 cm) was investigated using comparative genomic hybridization (CGH). CGH was performed on 20 samples taken from the specimen, and the genetic aberrations found were compared with the regional histology. The samples were histopathologically graded according to WHO criteria, and a division in high- and low-grade areas and infiltration rims was made. In high-grade areas, low-grade areas as well as infiltration rims, gains on 10p11.2-pter (14/20), 11q12-q22 (6/20) and losses on 4q13-qter (9/20), 10q22-qter (8/20), 11p14-pter (5/20), 13q12-qter (7/20) were revealed. Gains on 1q21-32 (2/4) and losses on 7p21-pter (3/4) were exclusively found in the high-grade areas. In the low-grade tumor samples and in the infiltration rim, gains on 16p11.2-pter (6/16), 17p11.2-pter (6/16), 17q11.2-qter (5/16), 20q11.2-q13 (3/16) and deletions on 5q31-qter (4/16) were detected. Gains on 7q21-qter (8/11) and 8q11.2-qter (6/11), and loss of chromosome 9 (4/11) and the Y-chromosome (4/11) were found in the high-grade and low-grade samples, not in the infiltration rims. The finding of a set of identical chromosomal aberrations throughout the resection specimen, most of which have been previously reported in gliomas, confirms a mechanism of clonal tumor proliferation operative in gliomas. The previously unreported genetic alterations which were predominantly traced in the tumor rims, might reflect either selection for properties related to infiltrating behavior, or genomic instability of subclones. The findings illustrate the importance of searching for high-grade genetic aberrations in low-grade tumor samples taken from cases in which sampling error is suspected.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Variación Genética , Glioblastoma/genética , Glioblastoma/patología , Neoplasias Encefálicas/diagnóstico , Genoma , Genotipo , Glioblastoma/diagnóstico , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Hibridación de Ácido NucleicoRESUMEN
We investigated the usefulness of membrane grown human term trophoblast cells in transferrin-mediated iron transfer studies. We showed that diferric transferrin is taken up both at the microvillous and at the basal membrane by means of receptor-mediated endocytosis. Uptake from the microvillous side is predominant. This corresponded with a much higher expression of transferrin receptors at the microvillous membrane as compared to the basal one. Iron appeared to accumulate in the cell. Accumulation was higher when transferrin was supplied at the microvillous side. Transfer of iron could not be assessed because uptake of transferrin by the cells was much less than passive diffusion of transferrin through the cell-free filter. The observation of iron accumulation was unexpected for a transfer epithelium. Could it be that part of the iron taken up by the cells is rapidly released whereas the remaining part accumulates? In this case the rate of iron uptake should be higher than the rate of iron accumulation. This question was assessed with non-polarly cultured trophoblast cells. We showed that like in polar cells iron accumulated in ferritin. A new experimental design enabled us to demonstrate that indeed the rate of transferrin-mediated iron is in excess over iron accumulation. We thus provide evidence for a mechanism that enables rapid transfer of iron across the syncytiotrophoblast cell layer.
Asunto(s)
Hierro/metabolismo , Trofoblastos/metabolismo , Transporte Biológico , Polaridad Celular , Células Cultivadas , Femenino , Humanos , Radioisótopos de Yodo , Radioisótopos de Hierro , Cinética , Microscopía Electrónica , Microvellosidades/metabolismo , Embarazo , Receptores de Transferrina/análisis , Transferrina/metabolismo , Trofoblastos/química , Trofoblastos/ultraestructuraRESUMEN
Four low-grade oligodendrogliomas, nine anaplastic oligodendrogliomas and two mixed oligoastrocytomas were investigated for chromosomal aberrations by comparative genomic hybridization on formalin-fixed, paraffin-embedded tissue samples. The most frequent losses observed involved 1p, 9p, 10pq, 14q, 16p, 19q, while the most frequent gains were seen on 7pq, 11pq, 17p, 19pq, and Xp. In one oligodendroglioma, a highly specific amplification of 1q32.1 was seen. The frequent losses of 14q have not been reported previously. In the two cases of mixed oligoastrocytomas multiple gains and losses were found that did not show a clear overlap with the alterations found in the pure oligodendrogliomas.
Asunto(s)
Aberraciones Cromosómicas , Glioma/genética , Oligodendroglioma/genética , Adulto , Femenino , Humanos , Hibridación in Situ , Masculino , Persona de Mediana Edad , Oligodendroglioma/patología , Adhesión en ParafinaRESUMEN
OBJECTIVES: To detect in situ the precise osteopontin (OPN) localization in papillary stones. METHODS: Immunocytochemical labelling procedures are applied to detect OPN localizations in crystalline material of renal papillary stones. The tissue-processing procedure for electron microscopy, which includes OsO4 postfixation, preserves both immunocytochemical OPN reactivity and cellular membrane contrast up to the ultrathin section. Reflection-contrast light microscopical images are correlated with high resolution transmission-electron microscopical observations from consecutive ultrathin epon sections. RESULTS: Preserved crystalline material in interstitial and peripheral papillary stones is recognized as calcium oxalate monohydrate. After section incubation with markers conjugated to an antibody against OPN (alpha OPN) the crystals are converted into ghosts. In the ghosts, alpha OPN markers are present around microcrystals. The size of these microcrystals ranges from several nanometers to micrometers. It is observed (due to the OsO4-preserved membranes) that interstitial cells are separated from the stone surfaces by unidentified extracellular material, also present in the center as a stone matrix. CONCLUSION: The microcrystal-growth inhibitor OPN is detected in situ in interstitial stones induced in the rat's papilla and at the surface of the papilla.
Asunto(s)
Cálculos Renales/química , Médula Renal/ultraestructura , Sialoglicoproteínas/análisis , Animales , Cálculos Renales/ultraestructura , Médula Renal/química , Microscopía Electrónica , Tetróxido de Osmio , Osteopontina , RatasRESUMEN
The mechanism and regulation of iron transport to the brain are largely unknown. The large surface area of the blood-brain barrier capillaries and the presence of transferrin receptors on the luminal plasma membranes of the blood-brain barrier endothelial cells (BBB-ECs) suggest that these cells actively participate in the transport of iron into the brain. In this paper, we describe the ultrastructural morphology of primary and first-passage cultures of BBB-ECs grown on different types of porous membranes. To investigate the mechanism of iron transport into and across the BBB-ECs, porous membrane grown first-passage cells were incubated with 6.6-nm gold-labeled transferrin and studied with electron microscopy. Results are suggestive for a transcytosis of transferrin through the BBB-ECs.
Asunto(s)
Barrera Hematoencefálica/fisiología , Endotelio Vascular/metabolismo , Oro Coloide/farmacocinética , Transferrina/farmacocinética , Animales , Transporte Biológico/fisiología , Células Cultivadas/metabolismo , Células Cultivadas/ultraestructura , Endotelio Vascular/citología , Endotelio Vascular/ultraestructura , Hierro/metabolismo , Microscopía Electrónica , PorcinosRESUMEN
Lectin reactivity in epithelial apical cell coats of normal rat kidneys was compared to that from animals subjected to crystal inducing diets (CID). The aim was to see whether the absence of lectin reactivity in cell coats is related to intratubular calcium oxalate crystal retention. In normal rat kidneys, after a pre-embedding procedure, it was observed that at the ultrastructural level, reactivity was present but that the lectin specificity for the various parts of the nephron might have to be reconsidered. There was heterogeneity between the epithelial cells with respect to the presence of coat material in the tubular cell apices. Tubular epithelial cell apices from CID rats showed no obvious changes in lectin reactivity pattern. Lectin reactivity was present at the periphery of intratubular crystals but undetectable at true crystal attachment sites or reduced at cell apices in the vicinity of recently attached crystals or agglomerates. After a post-embedding reaction procedure, wheat-germ agglutinin (WGA)-lectin reactivity confirmed the presence of coat material in the cleft between cell apex and retained crystal at crystal-attachment sites. The WGA/Au-10 nm reaction products were also seen inside epithelial cells. WGA/Au-10 nm reaction products mark a crystal matrix component inside intratubular and retained crystals. A similar matrix was also marked by an alpha-osteopontin (alpha OPN/Au-10 nm) reaction product.
Asunto(s)
Cálculos Renales/metabolismo , Riñón/química , Lectinas/metabolismo , Sialoglicoproteínas/análisis , Animales , Cristalización , Inmunohistoquímica , Riñón/ultraestructura , Cálculos Renales/ultraestructura , Osteopontina , Polisacáridos/farmacología , Ratas , Sialoglicoproteínas/genética , Adhesión del TejidoRESUMEN
In the present study, we exposed rats to a crystal-inducing diet (CID) consisting of vitamin D3 and 0.5% ethylene glycol (EG), and we investigated histologically the kidney damage induced by the deposition of calcium oxalate (CaOx) crystals. After 28 days, 50% of the animals had renal CaOx crystals, of which 60% also had small papillary stones. Most crystals were present in the cortex. The occurrence of these crystals coincided with morphological and cytochemical changes: glomerular damage, tubular dilatation and necrosis, and an enlargement of the interstitium. The number of epithelial and interstitial cells positive for the proliferating cell nuclear antigen (PCNA) was increased. Tamm-Horsfall protein (THP) was not only demonstrable in the thick ascending limb of the loop of Henle (TAL), but also frequently in glomeruli, in the proximal tubular epithelium, and in the papilla. In the lumen of the tubular system, it was associated with urinary casts. Reflection contrast microscopy (RCM) showed that the crystals were coated with a thin layer of THP. In spite of the high urinary oxalate concentrations, the above described cellular changes were not observed in CID-fed rats without renal crystals. We conclude, therefore, that in the kidney, the retained CaOx crystals rather than the urinary oxalate ions are responsible for the observed morphological and immunocytochemical changes.
Asunto(s)
Oxalato de Calcio/química , Cálculos Renales/patología , Riñón/patología , Animales , Enfermedad Crónica , Inmunohistoquímica , Riñón/química , Cálculos Renales/metabolismo , Masculino , Mucoproteínas/análisis , Antígeno Nuclear de Célula en Proliferación/análisis , Ratas , Ratas Wistar , UromodulinaRESUMEN
Crystal retention is studied in a rat-model system as a possible mechanism for the etiology of human nephrolithiasis. A crystal-inducing diet (CID) of ethylene glycol plus NH4Cl in their drinking-water is offered to healthy rats to generate intratubular crystals. Subsequently, the fate of retained crystals is investigated by allowing the rats a tissue recovery/crystalluria phase for three, five and ten days, respectively, on normal drinking water. The process of exotubulosis is observed in cortex and medulla of aldehyde-fixed kidneys after three days recovery. After five days, crystals are predominantly seen there in the interstitium. After ten days, cortex and medulla are virtually free of crystals. However, in the papillary regions after five and ten days recovery, three types of calcium oxalate monohydrate (COM) crystals are present: (1) free in the calycine space, (2) sub-epithelially located surrounded by interstitial cells within, and (3) covered by macrophage-like cells, outside the original papillary surface. After a CID plus three days recovery, a further thirty-seven days extra oxalate challenge with solely 0.3 vol% ethylene glycol induced intratubular and interstitial oxalate crystals. In the papillary region, large sub-epithelial crystals are seen. However, no crystals are seen in kidneys from rats given solely (0.5 or 0.8 vol.%) ethylene glycol for thirty days. An oxalate re-challenge retards crystal removal.
Asunto(s)
Oxalato de Calcio/orina , Cálculos Renales/etiología , Cálculos Urinarios/etiología , Cloruro de Amonio/administración & dosificación , Animales , Cristalización , Modelos Animales de Enfermedad , Glicol de Etileno , Glicoles de Etileno/administración & dosificación , Hiperoxaluria/etiología , Hiperoxaluria/patología , Cálculos Renales/patología , Corteza Renal/ultraestructura , Médula Renal/ultraestructura , Túbulos Renales/ultraestructura , Masculino , Ratas , Ratas Wistar , Cálculos Urinarios/patologíaRESUMEN
In kidneys of healthy rats submitted to a crystal-inducing diet (CID) with ethylene glycol (EG) and NH4Cl, the fate of retained crystals in the papillar region is studied during a recovery period of one, five or ten days, as model system for human nephrolithiasis. Scanning electron microscopy (SEM) shows, at papillary tips bulging into the calycine space, crystal masses covered either by the epithelium or a thin fibrous veil, or by unidentified mobile cuboidal cells. After CID plus one or five days recovery, small sub-epithelial swellings are seen of large sub-epithelial crystals at or around the papillary tip. After CID plus ten days, massive sub-surface crystal-containing micrometer-sized stones are seen in which the presence of calcium is confirmed by X-ray microanalysis. The papillary tip of rats after a re-challenge with an oxalate load from 0.1 vol% EG for twelve or forty-two days shows minor lesions. But a re-challenge with 0.3 vol% EG for thirty-seven days induces large sub-epithelial papillary millimeter-sized stones. The Von Kossa section staining converts the crystals into a black precipitate, but large peri-tubular or peri-vascular calcium deposits are absent. A new hypothesis about the etiology of an inductive calcium oxalate monohydrate nephrolithiasis is formulated which differs from the one proposed by Randall based on his deductive human kidney studies.
Asunto(s)
Oxalato de Calcio/orina , Cálculos Renales/etiología , Médula Renal/fisiología , Cálculos Urinarios/etiología , Cloruro de Amonio/administración & dosificación , Animales , Cristalización , Modelos Animales de Enfermedad , Microanálisis por Sonda Electrónica , Glicol de Etileno , Glicoles de Etileno/administración & dosificación , Humanos , Hiperoxaluria/etiología , Hiperoxaluria/patología , Cálculos Renales/patología , Cálculos Renales/fisiopatología , Médula Renal/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Ratas , Ratas Wistar , Cálculos Urinarios/patología , Cálculos Urinarios/fisiopatologíaRESUMEN
In a rat-model system, tubular crystal retention as a possible mechanism for the etiology of nephrolithiasis in man, was studied by conventional transmission electron microscopy. The animals were supplied for nine days with a crystal-inducing diet, with ethylene glycol plus NH4Cl in their drinking-water. After this induction period, a two day regime with fresh drinking-water was included, to allow crystals to be removed by spontaneous crystalluria. After aldehyde fixation of the rat kidneys, large crystals were seen inside the tubular lumen. The crystals were attached to cell surfaces and covered by neighboring epithelial cells. Some crystals were overgrown by several epithelial cells and underwent a process of so-called exotubulosis, resulting in free or cell-surrounded crystals in the interstitium, and possibly in crystals in Giant cells. To investigate the fate of the retained crystals, some animals were additionally exposed to a low-oxalate challenge from drinking water containing 0.1 volume per cent of ethylene glycol for 12 or 30 days, respectively. It was assumed that this would interfere with the retained intratubular or interstitial crystals, and allow the crystals to grow into mini-stones. This was not observed. After the oxalate challenge, no crystals were found to be retained in the tubules (free or covered by cells). Interstitial crystals were observed, but it remains to be demonstrated whether such crystals actually grow into mini-stones or that they are removed by the sterile inflammation process observed.