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1.
We describe here a protocol for the detection of epithelial cells in effusions combined with quantification of apoptosis by flow cytometry (FCM). The procedure described consists of the following stages: culturing and induction of apoptosis by staurosporine in control ovarian carcinoma cell lines (SKOV-3 and OVCAR-8); preparation of effusion specimens and cell lines for staining; staining of cancer cells in effusions and cell lines for cell surface markers (Ber-EP4, EpCAM and CD45) and intracellular/nuclear markers of apoptosis (cleaved caspase-3 and caspase-8, and incorporated deoxyuridine triphosphates); and FCM analysis of stained cell lines and effusions. This protocol identifies a specific cell population in cytologically heterogeneous clinical specimens and applies two methods to measure different aspects of apoptosis in the cell population of interest. The cleaved caspase and deoxyuridine triphosphate incorporation FCM assays are run in parallel and require (including sample preparation, staining, instrument adjustment and data acquisition) 8 h. The culturing of cell lines requires 2-3 days and induction of apoptosis requires 16 h. 相似文献
2.
Chrisanthar R Knappskog S Løkkevik E Anker G Ostenstad B Lundgren S Risberg T Mjaaland I Skjønsberg G Aas T Schlichting E Fjösne HE Nysted A Lillehaug JR Lønning PE 《PloS one》2011,6(4):e19249
Background
TP53 mutations have been associated with resistance to anthracyclines but not to taxanes in breast cancer patients. The MDM2 promoter single nucleotide polymorphism (SNP) T309G increases MDM2 activity and may reduce wild-type p53 protein activity. Here, we explored the predictive and prognostic value of TP53 and CHEK2 mutation status together with MDM2 SNP309 genotype in stage III breast cancer patients receiving paclitaxel or epirubicin monotherapy.Experimental Design
Each patient was randomly assigned to treatment with epirubicin 90 mg/m2 (n = 109) or paclitaxel 200 mg/m2 (n = 114) every 3rd week as monotherapy for 4–6 cycles. Patients obtaining a suboptimal response on first-line treatment requiring further chemotherapy received the opposite regimen. Time from last patient inclusion to follow-up censoring was 69 months. Each patient had snap-frozen tumor tissue specimens collected prior to commencing chemotherapy.Principal Findings
While TP53 and CHEK2 mutations predicted resistance to epirubicin, MDM2 status did not. Neither TP53/CHEK2 mutations nor MDM2 status was associated with paclitaxel response. Remarkably, TP53 mutations (p = 0.007) but also MDM2 309TG/GG genotype status (p = 0.012) were associated with a poor disease-specific survival among patients having paclitaxel but not patients having epirubicin first-line. The effect of MDM2 status was observed among individuals harbouring wild-type TP53 (p = 0.039) but not among individuals with TP53 mutated tumors (p>0.5).Conclusion
TP53 and CHEK2 mutations were associated with lack of response to epirubicin monotherapy. In contrast, TP53 mutations and MDM2 309G allele status conferred poor disease-specific survival among patients treated with primary paclitaxel but not epirubicin monotherapy. 相似文献3.
Secondary damage following primary spinal cord injury extends pathology beyond the site of initial trauma, and effective management is imperative for maximizing anatomical and functional recovery. Bisperoxovanadium compounds have proven neuroprotective effects in several central nervous system injury/disease models, however, no mechanism has been linked to such neuroprotection from bisperoxovanadium treatment following spinal trauma. The goal of this study was to assess acute bisperoxovanadium treatment effects on neuroprotection and functional recovery following cervical unilateral contusive spinal cord injury, and investigate a potential mechanism of the compound's action. Two experimental groups of rats were established to 1) assess twice-daily 7 day treatment of the compound, potassium bisperoxo (picolinato) vanadium, on long-term recovery of skilled forelimb activity using a novel food manipulation test, and neuroprotection 6 weeks following injury and 2) elucidate an acute mechanistic link for the action of the drug post-injury. Immunofluorescence and Western blotting were performed to assess cellular signaling 1 day following SCI, and histochemistry and forelimb functional analysis were utilized to assess neuroprotection and recovery 6 weeks after injury. Bisperoxovanadium promoted significant neuroprotection through reduced motorneuron death, increased tissue sparing, and minimized cavity formation in rats. Enhanced forelimb functional ability during a treat-eating assessment was also observed. Additionally, bisperoxovanadium significantly enhanced downstream Akt and mammalian target of rapamycin signaling and reduced autophagic activity, suggesting inhibition of the phosphatase and tensin homologue deleted on chromosome ten as a potential mechanism of bisperoxovanadium action following traumatic spinal cord injury. Overall, this study demonstrates the efficacy of a clinically applicable pharmacological therapy for rapid initiation of neuroprotection post-spinal cord injury, and sheds light on the signaling involved in its action. 相似文献
4.
Regional cerebral blood flow (rCBF), a parameter of neuronal activity in the brain, was measured by the 133Xe inhalation method in 43 patients undergoing stereotactic thalamotomy. A postoperative flow reduction of about 2% in the operated hemisphere was found, persisting in further measurements performed after a year. There was no consistent change in the pattern of regional flow distribution. The results indicate a diminished level of activity in the hemisphere subjected to thalamotomy, but the change could not be linked to any specific area or function. 相似文献
5.
Comparison of oxidative metabolism in vitro in endothelial cells from different species and vessels 总被引:1,自引:0,他引:1
Oxygen consumption was compared in confluent cultures of endothelial cells from human umbilical cord veins, rat pulmonary arteries, and bovine aortas. A microrespirometric method utilizing oxygenated hemoglobin as oxygen supply and indicator of respiration was used. Respiratory rate was equal in human and murine cells (2.0 X 10(-6) and 2.2 X 10(-6) microliters O2/cell/hour respectively), compared on a cell-to-cell basis, while respiratory rate of the bovine endothelium was significantly lower (0.4 X 10(-6) microliters O2/cell/hour) (P less than .001). 相似文献
6.
Jon Amund Kyte Sissel Trachsel Bente Risberg Per thor Straten Kari Lislerud Gustav Gaudernack 《Cancer immunology, immunotherapy : CII》2009,58(10):1609-1626
Cancer vaccine trials frequently report on immunological responses, without any clinical benefit. This paradox may reflect
the challenge of discriminating between effective and pointless immune responses and sparse knowledge on their long-term development.
Here, we have analyzed T cell responses in long-term survivors after peptide vaccination. There were three main study aims:
(1) to characterize the immune response in patients with a possible clinical benefit. (2) To analyze the long-term development
of responses and effects of booster vaccination. (3) To investigate whether the Th1/Th2-delineation applies to cancer vaccine
responses. T cell clones were generated from all nine patients studied. We find that surviving patients harbor durable tumor-specific
responses against vaccine antigens from telomerase, RAS or TGFβ receptor II. Analyses of consecutive samples suggest that
booster vaccination is required to induce robust T cell memory. The responses exhibit several features of possible clinical
advantage, including combined T-helper and cytotoxic functionality, recognition of naturally processed antigens and diverse
HLA-restriction and fine-specificity. CD4−CD8− T cell clones display unconventional cytotoxicity and specifically kill tumor cells expressing mutated TGFβ receptor II.
Cytokine profiling on the long-term survivors demonstrates high IFNγ/IL10-ratios, favoring immunity over tolerance, and secretion
of multiple chemokines likely to mobilize the innate and adaptive immune system. Interestingly, these pro-inflammatory cytokine
profiles do not follow a Th1/Th2-delineation. Most IFNγhigh/IL4low/IL10low cultures include high concentrations of hallmark Th2-cytokines IL-5 and IL-13. This does not reflect a mixture of Th1- and
Th2-clones, but applies to 19/20 T cell clones confirmed to be monoclonal through TCR clonotype mapping. The present study
identifies several factors that may promote clinical efficacy and suggests that cytokine profiling should not rely on the
Th1/Th2-paradigm, but assess the overall inflammatory milieu and the balance between key cytokines.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
7.
Bruna Jardim Pereira Lima Gabriel Rodrigues Leal de Oliveira Thainá Cavalleri Sousa Ariana Musa de Aquino Karianne Delalibera Hinokuma Maria Luiza Silva Ricardo Wellerson Rodrigo Scarano Anthony César de Souza Castilho Francis Lopes Pacagnelli Francisco Eduardo Martinez Leonardo de Oliveira Mendes 《Animal Reproduction》2023,20(3)
8.
Organ blood flow and cardiac contractility in anaesthetized cats at 5 bar (500 kPa) ambient pressure
Jan Risberg Ingvald Tyssebotn 《European journal of applied physiology and occupational physiology》1992,64(5):389-394
Previous in vivo and in vitro experiments have demonstrated increased cardiac contractility and increased total myocardial blood flow (Qmyocardial) when rats were exposed to normoxic 5-bar (500 kPa) ambient pressure. In the present study, regional blood flow was measured using the microsphere method on nine anaesthetized cats at surface and normoxic 5-bar (500 kPa) ambient pressure. Left ventricular pressure (LVP) and cardiac contractility, measured as peak left ventricular +dP/dt and -dP/dt were measured in six of the cats. Arterial pressure, heart rate and cardiac output remained unchanged after compression, but total Qmyocardial increased by 29% (P less than 0.01) and cerebral blood flow increased by 66% (P less than 0.05). At the same time +dP/dt and -dP/dt was increased by 83% and 102%, respectively (P less than 0.01), while LVP was enhanced by 14% (P less than 0.05). Except for a moderate decrease in partial pressure of oxygen, acid base status in arterial blood remained unchanged. The results indicate that the effects of increased ambient pressure on the heart are general physiological phenomena, which are not only limited to the laboratory rat. 相似文献
9.
10.
Karianne Johansen Mette Krogh Alf Terje Andresen Asbjrg S. Christophersen Gustav Lehne Knut E. Rasmussen 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,669(2)
A fully automated method for determination of the free and total concentration of drugs with a varying degree of protein binding is described. The antiepileptic drugs phenytoin, carbamazepine and phenobarbitone were chosen to demonstrate the utility of this technique. The method was based on the ASTED system and combined on-line equilibrium dialysis at 37°C with concentration of the dialysate on a trace enrichment column and HPLC determination with UV detection. The dialysis cell was a modification of the ASTED dialysis cell and 22% of the free concentration of the drugs were recovered in the recipient channel of the dialyser after 10 min of dialysis at 37°C. The free concentration, the total concentration as well as the drugs protein binding could be determined. The method was shown to be well suited for routine monitoring of the free and the total concentrations of the drugs in plasma from epileptic patients. 相似文献