Tarte K, Olsen S J, Yang Lu Z, Legouffe E, Rossi J F, Chang Y, Klein B
Institute for Molecular Genetics, CNRS, Montpellier, France.
Blood. 1998 Mar 15;91(6):1852-7.
Bone marrow dendritic cells (DC) from patients with multiple myeloma (MM) were recently reported to be infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Because immunotherapy strategies using DC are very promising in this disease, we looked for KSHV DNA in clinical-grade DC generated in vitro from MM patients. Adherent apheresis cells from MM patients were maintained for 7 days in clinical-grade X-VIVO 15 culture medium supplemented with granulocyte-macrophage colony-stimulating factor, interleukin-4, or interleukin-13. Tumor necrosis factor alpha was added for the last 2 days. We obtained a cell population with a DC phenotype able to endocytose fluorescein isothiocyanate (FITC)-dextran and efficiently activate resting allogenic T lymphocytes. To detect KSHV DNA, we used polymerase chain reaction (PCR) followed by Southern blotting of PCR product with a sensitivity detecting a few copies of viral DNA. All the PCR were repeated in a blinded fashion three times, on 1 mug and 0.2 mug of genomic DNA, in two different laboratories. Clinical-grade DC from 10 (91%) of 11 patients were not infected with KSHV. The apheresis cells and the purified CD34(+) cells from the same patients were also negative. A very weak PCR band was detected with DC from one patient, but the initial apheresis cells were negative. The detection of KSHV infection in 1 (9%) of 11 MM patients probably represents background seroprevalence. It seems likely that functional and clinical-grade DC from MM patients can safely be used in clinical trials.
最近有报道称，多发性骨髓瘤（MM）患者的骨髓树突状细胞（DC）感染了卡波西肉瘤相关疱疹病毒（KSHV）。由于使用DC的免疫治疗策略在这种疾病中非常有前景，我们在体外从MM患者产生的临床级DC中寻找KSHV DNA。将MM患者的贴壁单采细胞在补充有粒细胞巨噬细胞集落刺激因子、白细胞介素-4或白细胞介素-13的临床级X-VIVO 15培养基中培养7天。在最后2天添加肿瘤坏死因子α。我们获得了具有DC表型的细胞群体，该群体能够内吞异硫氰酸荧光素（FITC）-葡聚糖并有效激活静息的同种异体T淋巴细胞。为了检测KSHV DNA，我们使用聚合酶链反应（PCR），随后对PCR产物进行Southern印迹分析，其灵敏度可检测到几份病毒DNA拷贝。所有PCR均在两个不同实验室中以盲法对1μg和0.2μg基因组DNA重复进行三次。11例患者中有10例（91%）的临床级DC未感染KSHV。同一患者的单采细胞和纯化的CD34(+)细胞也为阴性。在一名患者的DC中检测到一条非常弱的PCR条带，但最初的单采细胞为阴性。11例MM患者中有1例（9%）检测到KSHV感染，这可能代表背景血清阳性率。MM患者的功能性和临床级DC似乎可以安全地用于临床试验。
