Fluorometric assessment of acetaminophen-induced toxicity in rat hepatocyte spheroids seeded on micro-space cell culture plates

Hepatotoxicity induced by the metabolic activation of drugs is a major concern in drug discovery and development. Three-dimensional (3-D) cultures of hepatocyte spheroids may be superior to monolayer cultures for evaluating drug metabolism and toxicity because hepatocytes in spheroids maintain the expression of various metabolizing enzymes and transporters, such as cytochrome P450 (CYP). In this study, we examined the hepatotoxicity due to metabolic activation of acetaminophen (APAP) using fluorescent indicators of cell viability and intracellular levels of glutathione (GSH) in rat hepatocyte spheroids grown on micro-space cell culture plates. The mRNA expression levels of some drug-metabolizing enzymes were maintained during culture. Additionally, this culture system was compatible with microfluorometric imaging under confocal laser scanning microscopy. APAP induced a decrease in intracellular ATP at 10 mM, which was blocked by the CYP inhibitor 1-aminobenzotriazole (ABT). APAP (10 mM, 24 h) decreased the levels of both intracellular ATP and GSH, and GSH-conjugated APAP (APAP-GSH) were formed. All three effects were blocked by ABT, confirming a contribution of APAP metabolic activation by CYP to spheroid toxicity. Fluorometric imaging of hepatocyte spheroids on micro-space cell culture plates may allow the screening of drug-induced hepatotoxicity during pharmaceutical development.     

Differential survival of cell subpopulations in spheroids after treatment with bleomycin

Summary The cytotoxic effect of bleomycin (BLM) on Chinese hamster V79-cells grown as spheroids is compared to that on monolayer cultures in exponential vs. plateau phase of growth. Cells treated as intact spheroids with BLM (50 g/ml for 1 h) are more sensitive than monolayer cultures. Cell subpopulations derived from different zones of the spheroids survive BLM-treatment to different degrees: When treated as single cells in suspension, the formerly outer cells are less affected than the inner cells. Within intact spheroids, however, progressive cellular resistance with increasing distance from the outer rim is found.     

Chemotherapeutic drugs as indirect oxygen radiosensitizers

We have characterized the oxygen distribution in V-79 spheroids which were grown by the spinner flask method. Using ultramicroelectrodes with tip diameters of 1–5 microns and a perfusion system whereby the spheroids' milieu could be maintained and controlled, we found plateau pO₂ values of less than 10 mm Hg in spheroids of greater than 500 microns in diameter. Data from experiments using respiration inhibitory drugs indicate that the characteristics of the outer layers of cells is the major determinant of the oxygen profile in the interior. Parallel control radiobiological experiments confirmed the-control pO₂ measurements, and formed the basis for the experiments using the potential indirect radiation sensitizers: Chlorambucil and Mustargen.     

Overexpression of heat shock proteins in pallido-nigral axonal spheroids of nonhuman aged primates

The occurrence of spheroids has been described in the globus pallidus (GP) and substantia nigra pars reticulata (SNr) of aged rhesus monkeys. Opinions vary as to the origin of spheroids. Ultrastructural and immunohistochemical analysis suggested that spheroids originate from degenerating axons or astroglia. In the present study, we have investigated the GP and SNr of aged monkeys (Macaca fascicularis and Macaca mulatta). Although immunoreactive for microtubule-associated protein (MAP) 1A, tau, amyloid precursor protein, synaptophysin and phosphorylated neurofilament, spheroids were not immunoreactive for MAP1B and MAP2. We confirmed the axonal nature of pallido-nigral spheroids in aged rhesus monkeys. Pallido-nigral spheroids have been reported to overexpress stress proteins, such as ubiquitin, B-crystallin, and heat shock protein (Hsp) 27. We further evaluated the expression of Hsps in pallido-nigral spheroids. As well as being intensely immunoreactive for ubiquitin, B-crystallin, Hsp27, and Hsp70, spheroids were immunoreactive for Hsp32 (heme oxygenase-1), Hsp40, Hsp60, and Hsp90. On the basis of these findings, we speculate that Hsp32-immunoreactive spheroids might be expressed as an oxidative stress response. Induction of other Hsps might play a role in protection of axons from the aggregation of neurofilament, MAPs and other proteins, and failure to protect degenerating axons might result in their proteolysis by the ubiquitin-proteasome system.     

The combination of bleomycin with suicide or interferon-β gene transfer is able to efficiently eliminate human melanoma tumor initiating cells

We explored the potential of a chemogene therapy combination to eradicate melanoma tumor initiating cells, key producers of recurrence and metastatic spread. Three new human melanoma cell lines, two obtained from lymph nodes and one from spleen metastasis were established and characterized. They were cultured as monolayers and spheroids and, in both spatial configurations they displayed sensitivity to single treatments with bleomycin (BLM) or human interferon-β (hIFNβ) gene or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) lipofection. However, the combination of bleomycin with SG or hIFNβ gene transfer displayed greater antitumor efficacy. The three cell lines exhibited a proliferative behavior consistent with melan A and gp100 melanoma antigens expression, and BRAF V600E mutation. BLM and both genetic treatments increased the fraction of more differentiated and treatment-sensitive cells. Simultaneously, they significantly decreased the sub-population of tumor initiating cells. There was a significant correlation between the cytotoxicity of treatments with BLM and gene transfer and the fraction of cells exhibiting (i) high proliferation index, and (ii) high intracellular levels of reactive oxygen species. Conversely, the fraction of cells surviving to our treatments closely paralleled their (i) colony and (ii) melanosphere forming capacity. A very significant finding was that the combination of BLM with SG or hIFNβ gene almost abrogated the clonogenic capacity of the surviving cells. Altogether, the results presented here suggest that the combined chemo-gene treatments are able to eradicate tumor initiating cells, encouraging further studies aimed to apply this strategy in the clinic.     

Infantile neuroaxonale Dystrophie

Zusammenfassung Bericht über die Gehirnbefunde des jüngsten bisher bekannten Falles von Infantiler neuroaxonaler Dystrophie (InaD). Der im Alter von 6 Monaten interkurrent verstorbene Knabe mit negativer Familienanamnese bot bald nach der Geburt progressive neurologische Ausfälle (Bulbusdivergenz, Muskelhypotonie, Beugestellung der Extremitäten). Die Hirnsektion ergab eine diffuse Kleinhirnatrophie. Histologisch fanden sich in der verödeten Kleinhirnrinde zahlreiche Dendrit- und Axonschollen mit eigenartiger vacuoliger Degeneration der Purkinjezellen. Nur vereinzelt waren Spheroide in der Hirnstammhaube sowie im gut bemarkten Pallidum anzutreffen, das wenig Neutralfettablagerungen bot. Hinterstrangskerne und Telencephalon waren frei. Die Beobachtung entspricht formal den bekannten Fällen von InaD mit längerem Verlauf, ist aber durch den fast elektiven Befall des Kleinhirns gekennzeichnet, was für dessen frühe Beteiligung am Krankheitsprozeß spricht. Die nosologische Stellung der InaD innerhalb der Syndromgruppe der neuro-axonalen Dystrophien wird kurz diskutiert. Im Anhang wird eine weitere verifizierte Beobachtung von InaD mit voll ausgeprägtem Gewebssyndrom erwähnt.

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Infantile neuroaxonal dystrophy

Summary The neuropathological findings of a sporadic case of infantile neuroaxonal dystrophy (InaD) are reported who died at an earlier age than any of the previously recorded ones. The patient was a boy with negative family history who showed progressive neurologic symptoms (deviation of bulbi, muscle hypotonia and quadruplegia in flexion) soon after birth and died at 6 months of age. Brain section revealed severe cerebellar atrophy. The characteristic histological findings were axonal and dendritic swellings in the cerebellar cortex with peculiar vacuolic desintegration of the Purkinje cells. Very few spheroids were seen in the brain stem and in the pallidum which showed normal myelination and few amounts of sudanophilic material. The nuclei of Goll and the telencephalon were free of spheroids. The changes were those of the reported cases of InaD with longer duration of illness. However, there was almost elective affection of the cerebellum indicating the early onset of cerebellar changes in InaD. Its nosological position within the group of neuroaxonal dystrophies is discussed. In the addendum a further verified case of InaD with fully developed morphological syndrome is mentioned.

Mitgeteilt in der Wissenschaftl. Sitzung der Vereinigung Österr. Pathologen, am 18. 4. 1967, in Wien.     

1例遗传性弥漫性白质脑病合并轴索球样变患者行造血干细胞移植的护理

对1例无法行走,认知功能低下,大小便失禁,抑郁状态又易激惹的遗传性弥漫性白质脑病合并轴索球样变患者进行异基因造血干细胞移植,护理要点包括：对陪护家属进行医院感染相关知识和技能的培训及考核;化疗期间提前使用止吐药物干预,饮食上添加增稠剂并联合呼吸肌训练来减少患者呛咳及误吸的发生;进行四肢主动及被动运动,维持患者肌肉功能状态;不断变换思路来应对患者排便失禁;易激惹患者及照护者的心理护理;移植后密切监测感染、出血倾向及移植物抗宿主病等并发症。经过精心的治疗及护理,造血干细胞成功植入,入无菌仓26 d后患者转至普通病房继续治疗。     

Effects of irradiation and cisplatin on human glioma spheroids: inhibition of cell proliferation and cell migration

Purpose: Investigation of cell migration and proliferation of human glioma cell line spheroids (CLS) and evaluation of morphology, apoptosis, and immunohistochemical expression of MIB-1, p53, and p21 of organotypic muticellular spheroids (OMS) following cisplatin (CDDP) and irradiation (RT). Material and methods: Spheroids of the GaMg glioma cell line and OMS prepared from biopsy tissue of six glioblastoma patients were used. Radiochemosensitvity (5 μg/ml CDDP followed by RT) was determined using migration and proliferation assays on CLS. In OMS, histology and immunohistochemical studies of MIB-1, p53, and p21 expression were examined 24 and 48 h following treatment. Results: Combination treatment led to a migration inhibition of 38% (CDDP 13%; RT 27%) and specific growth delay of 2.6 (CDDP 1.3; RT 2.1) in CLS. Cell cycle analysis after combination treatment showed an accumulation of cells in the G2/M phase. In OMS, apoptosis increased, cell proliferation decreased, and p53/p21 expression increased more pronounced following CDDP+RT. No morphological damage was observed. Conclusion: CDDP can lead to enhancement of the RT effect in spheroids of both human glioma cell line spheroids and biopsy spheroids from glioblastoma specimens. The exerted effect is additive rather than synergistic.