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Coral-Ann M. Almeida Rebecca Laird Imran Ahmed Joanne Turley Andrew Lucas Abha Chopra Mina John 《Journal of immunological methods》2009,344(1):1-2513
The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-γ ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r > 0.83, p < 0.05). The precision results from the automated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time. 相似文献
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M. Venturini L. Del Mastro Franco Testore Marco Danova Ornella Garrone Claudio Lanfranco Fabio Latini Mario R. Sertoli Rita Lionetto Paola Queirolo Andrea Ardizzoni Riccardo Rosso 《Cancer chemotherapy and pharmacology》1996,38(6):487-494
To verify whether the association of granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) would
allow both the acceleration and the dose escalation of the cyclophosphamide/epidoxorubicin/5-fluorouracil (CEF) regimen as
first-line therapy in advanced breast cancer patients, we conducted a dose-finding study. Cohorts of three consecutive patients
received cyclophosphamide (Ctx, dose range 800 –1400 mg/m2), epidoxorubicin (Epidx, dose range 70–100 mg/m2), and 5-fluorouracil (5-Fu, 600 mg/m2, fixed dose) given as an intravenous bolus on day 1 every 14 days; GM-CSF at 5 μg/kg given as a subcutaneous injection from
day 4 to day 11; and EPO at 150 IU/kg given as a subcutaneous injection three times a week. In no single patient was any dose
escalation allowed. A total of 14 patients entered the study. At the 4th dose level (Ctx 1400 mg/m2, Epidx 100 mg/m2, 5-Fu 600 mg/m2), two patients had dose-limiting mucositis and one patient developed dose-limiting neutropenia. Therefore, the 3rd cohort
received the maximum tolerated dose, i.e. Ctx at 1200 mg/m2, Epidx at 90 mg/m2, and 5-Fu at 600 mg/m2, given every 18.5 (±2.5) days. Toxicity was moderate and manageable in an outpatient setting. Only 1 admission at the 4th
dose level was required. Throughout the 4 dose levels there was no toxicity-related death; grade IV leukopenia ranged from
24% to 75% of cycles and grade IV thrombocytopenia ranged from 6% to 8%. No grade IV anemia was recorded. Increasing the doses
of Ctx and Epidx while maintaining a fixed dose of 5-Fu with the support of both EPO and GM-CSF allows safe acceleration and
dose escalation of CEF chemotherapy. Further controlled studies will evaluate the activity and efficacy of this strategy.
Received: 8 October 1995/Accepted: 1 March 1996 相似文献
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Iwata H Nakamura S Toi M Shin E Masuda N Ohno S Takatsuka Y Hisamatsu K Yamazaki K Kusama M Kaise H Sato Y Kuroi K Akiyama F Tsuda H Kurosumi M;Japan Breast Cancer Research Group 《Breast cancer (Tokyo, Japan)》2005,12(2):99-103
PURPOSE: A single-arm phase II multicenter trial of the combination of cyclophosphamide (C), epirubicin (E), and 5-fluorouracil (F) followed by docetaxel as neoadjuvant chemotherapy is being conducted by the Japan Breast Cancer Research Group. This report describes an interim analysis of the clinical response and safety of 79 patients who finished preoperative chemotherapy and surgery. PATIENTS AND METHODS: Patients with operable breast cancer received C at 500 mg/m2, E at 100 mg/m2, and F at 500 mg/m2 every 21 days for 4 cycles followed by docetaxel at 75 mg/m2 every 21 days for 4 cycles. RESULTS: Of the 79 patients evaluable for analysis the median age was 46 years (28-59), and 61 patients (77.2%) had T2 tumors. A total of 312 of 316 (98.7%) cycles of CEF and 296 of 312 (94.9%) cycles of docetaxel were administered. Average total cumulative dose was 92% and 95% for CEF and docetaxel, respectively. The rate and grade of edema, neuropathy, arthralgia and myalgia were higher with docetaxel than with CEF. The overall clinical response rate was 70.9%. Breast conserving surgery was performed in 31 of 42 patients (73.8%) with a base-line tumor size of more than 3 cm. CONCLUSIONS: Interim data suggest that CEF followed by docetaxel is an active and tolerable neoadjuvant chemotherapy regimen. A final analysis is planned for 2005. 相似文献
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目的构建含报告基因增强绿色荧光蛋白(EGFP)的分泌型真核表达载体pSecTag-EGFP,用以研究pSecTag/HygroB转染鸡胚胎成纤维细胞的转染效率,为目的基因的成功表达奠定基础。方法用PCR方法克隆得到EGFP基因,通过酶切、连接、转化构建pSecTag-EGFP分泌型真核表达载体,设计优化实验,通过脂质体介导的方法转染鸡胚胎成纤维细胞,在荧光显微镜下直接观察该基因的表达。用B radford法检测不同组合的细胞培养液中的EGFP蛋白的相对含量。结果脂质体介导的方法能有效转染鸡胚胎成纤维细胞,实验中EGFP蛋白相对表达量随质粒量的增加而明显升高(P<0.01)。其中脂质体2μl、质粒1.0μg时目的蛋白相对表达量最高。结论pSecTag-EGFP质粒构建成功,并成功优化了转染条件。 相似文献
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Luca Pierelli Alessandro Perillo Gabriella Ferrandina Giovanna Salerno Sergio Rutella rea Fattorossi Alessandra Battaglia Aurelia Rughetti Marianna Nuti Enrico Cortesi Giuseppe Leone Salvatore Mancuso Giovanni Scambia 《Transfusion》2001,41(12):1577-1585
BACKGROUND: Peripheral blood progenitor cell (PBPC) transplantation (PBPCT) combined with post-PBPCT administration of myelopoietic growth factors is a valid therapeutic intervention to rapidly restore hematopoiesis after the delivery of intensive, myeloablative cancer chemotherapy. On the other hand, the best growth factor regimen to potentiate PBPC-mediated immunohematopoietic recovery has yet to be determined. STUDY DESIGN AND METHODS: In a randomized evaluation, the effects produced by post-PBPCT G-CSF and GM-CSF on myeloid/lymphoid recovery and transplant outcome in women with chemosensitive cancer were compared. Thirty-seven ovarian cancer patients and 34 breast cancer patients ranging in age from 24 to 60 years were treated with carboplatin, etoposide, and melphalan (CEM) high-dose chemotherapy and then randomly assigned to receive G-CSF (5 microg/kg subcutaneously) or GM-CSF (5 microg/kg subcutaneously) until Day 13 after PBPCT. Patients were compared in regard to hematopoietic recovery, posttransplant clinical management, and immune recovery. Finally, clinical outcome was estimated as time to progression and overall survival. RESULTS: Hematopoietic recovery and posttransplant clinical management were comparable in both the G-CSF and GM-CSF series. Conversely, significantly higher T-cell counts were observed in G-CSF-treated patients during the early and late posttransplant follow-up. Patients who received G-CSF showed a significantly longer median time to progression. A parallel analysis revealed that patients in whom a higher CD3+ count was recovered had a significantly longer overall survival and time to progression. CONCLUSION: The enhancement of post-PBPCT T-cell recovery observed in G-CSF-treated patients encourages the use of G-CSF to ameliorate immune recovery, which seems to play a role in post-PBPCT control of disease in cancer patients. GM-CSF might be administered to prolong immunosuppression after autologous PBPCT for autoimmune diseases or allogeneic PBPCT. 相似文献
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The uptake of 5′-deoxy-5′-S-isobutyl thioadenosine (iBuS)5′ Ado into chick embryo fibroblasts is due to a mixed transport system: (1) a high affinity system with apparent Km values of 0.022 mM and 0.055 mM for normal and Rous Sarcoma Virus transformed cells respectively; (2) a low affinity system with apparent Km values of 0.800mM and 0.720mM for the normal and transformed cells. The drug enters the cells rapidly, and is found in the acid-soluble fraction. Only analogues of (iBuS)5′Ado modified in the 5′-S-side chain are found to be inhibitors of the high affinity system. This indicates that an intact adenosine is required. 2′-Deoxyglucose and factors affecting the cell membrane reduce the (iBuS)5′Ado uptake. 相似文献
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We show here that the immunogenicity of the Modified Vaccinia Ankara MVA vaccine strain can be improved by deletion of the A35 gene, without diminishing the ability of the virus to replicate. Deletion of the A35 gene resulted in increased virus-specific immunoglobulin production, class switching to IgG isotypes, and virus-specific IFNγ-secreting splenocytes. The MVA35 deletion virus provided excellent protective efficacy against virulent virus challenge. These results suggest that A35 deletion mutant strains will have superior vaccine performance for poxvirus vaccines as well as platform vaccines for other infectious diseases and cancer. 相似文献
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Three new fluoroarabinosylpyrimidine nucleosides (FIAC, FIAU and FMAU) were tested for in vitro activity against oncogenic and nononcogenic strains of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT). Marek's disease is a herpesvirus-induced lymphoma in chickens. Nononcogenic strains of MDV and HVT can protect against this disease. All viruses were inhibited by 1 μM of these drugs in chick kidney cell (CKC) cultures, but only FMAU and FIAU were active in chicken embryo fibroblast (CEF) and spleen cell cultures. It was determined that whereas CKC produced the enzyme 2′-deoxycytidine-deaminase which is needed to deaminate FIAC to FIAU, CEF were devoid of this enzyme activity. In addition, the deaminase inhibitor 3,4,5,6-tetrahydrouridine prevented the antiviral activity of FIAC in CKC. FMAU was not active against two Marek's disease-derived lymphoblastoid tumor cell lines. 相似文献
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Valentina Strecker Britta Muster Alexander Bürkle Marina Jendrach 《Mechanisms of ageing and development》2010,131(1):48-59
Elevated reactive oxygen species (ROS) levels have been observed in mammals during aging, implying an important role of ROS in the aging process. Most bird species are known to live longer and to contain lower ROS levels than mammals of the same body weight. The influence of ROS on the aging process of birds has been investigated using pigeon embryonic fibroblasts (PEF) and chicken embryonic fibroblasts (CEF). ROS levels in young avian cells were much lower than in human cells. When cultivated till replicative senescence, PEF proliferated about one-third longer compared to CEF. However, both senescent avian cell populations showed no increased ROS levels or accumulation of ROS-induced damage on the mtDNA or protein level. The investigation for quality control (QC) mechanisms revealed that the autophagosomal/lysosomal pathway was not downregulated in old avian cells and stable overexpression of the autophagy protein ATG5 improved mitochondrial fitness, enhanced the resistance against oxidative stress and prolonged the life span of CEF. Oxidative stress-mediated apoptosis induced a dose-dependent cell proliferation in CEF as well as in PEF. Taken together, our data indicate that autophagy and compensatory proliferation act as QC mechanisms, while ROS did not influence the aging process in avian cells. 相似文献