Selective in vivo growth of lymphocyte function- associated antigen-1-positive murine myeloma cells. Involvement of function-associated antigen-1-mediated homotypic cell-cell adhesion

OBJECTIVES: Lymphocyte function-associated antigen-1 (LFA-1) expression on multiple myeloma cells and its potential role in myeloma biology have been the subject of conflicting literature reports. In this study we used the 5T experimental mouse model to analyze the involvement of LFA-1 in myeloma cell bone marrow homing, survival, and growth. MATERIALS AND METHODS: The 5T33MM vitro (5T33MMvt) myeloma line was used. LFA-1 and intercellular adhesion molecule-1 (ICAM-1) expression were analyzed by flow cytometry. A small molecule antagonist of LFA-1/ICAM interactions, BIRT 377, was used to block LFA-1 in vitro. Transendothelial migration was assessed by measuring migration through Transwells coated with bone marrow endothelial cells. Immediate in vivo homing was analyzed by tracing 51Cr-labeled cells. Invert microscopic cell counting was used to analyze homotypic cell adhesion. Cell cycle analysis was used to analyze apoptosis. S+G(2)/M phase analysis and 3H-thymidine incorporation were used to assess proliferation. Cells were separated into LFA-1(+) and LFA-1(-) fraction by magnetic activated cell sorting. RESULTS: 5T33MMvt cells had a heterogeneous LFA-1 expression and all cells were positive for the LFA-1 ligand ICAM-1. LFA-1 inhibition with BIRT 377 did not affect transendothelial migration of the 5T33MMvt cells; however, it did result in cell cluster scattering, indicating LFA-1 involvement in homotypic cell-cell adhesion. No effect was observed on apoptosis, but the percentage of cells in S+G(2)/M phase was decreased by 39%. 3H-thymidine incorporation confirmed this effect on 5T33MMvt cell proliferation (38% reduction). When 5T33MMvt cells were injected into animals, all myeloma cells isolated at the end stage of the disease were LFA-1(+) in contrast to the situation before injection. LFA-1(+) and LFA-1(-) MM cells had similar in vivo bone marrow homing capacities. Mice injected with LFA-1(+) 5T33MMvt cells developed myeloma (5/5) within 12 weeks after injection. In contrast, LFA-1(-) recipients did not develop the disease (0/5), even 1 year after tumor inoculation. CONCLUSIONS: Our data suggest that LFA-1-mediated homotypic cell-cell adhesion is involved in myeloma cell proliferation and raises the possibility that this interaction may have a crucial role in in vivo myeloma cell growth. LFA-1 does not appear to play a role in the bone marrow homing of these cells.     

Decreased lymphocyte function-associated antigen-1 molecule expression on peripheral blood lymphocytes from patients with Graves' disease

Humoral and cellular abnormalities have been described in autoimmune thyroid diseases, but a deficient cooperation between humoral and cellular systems has been poorly investigated in such diseases. Lymphocyte function-associated antigen-1 (LFA-1), a glycoprotein expressed on T- and B-lymphocytes, plays an important role in cell-cell interaction mechanisms and is involved in the homing phenomenon. The expression of this molecule was investigated on peripheral blood lymphocytes from patients suffering from various thyroid disorders. Decreased numbers of LFA-1-positive cells were observed in 30 patients with Graves' disease, while normal values were present in 8 patients with Hashimoto's thyroiditis and 37 subjects with nonautoimmune thyroid disorders. Treatment-induced improvement of Graves' disease was associated with an increase in LFA-1-positive cell number.     

Phagocytic plasma cells in a patient with multiple myeloma

A female patient with IgG multiple myeloma and phagocytosing plasma cells is presented. Electron microscopical investigation showed an unusually large number of small mitochondria in the myeloma cells. In 21%, intracytoplasmic incorporation of one or more red cells or occasionally of erythroblasts or cells of the myeloid series were found. Uptake of platelets was seen rarely only. Studies of the in vitro phagocytic activity of myeloma cells did not reveal phagocytosis of opsonized bacteria or of latex particles by the malignant plasma cells.     

Distribution of myeloma plasma cells in peripheral blood and bone marrow correlates with CD56 expression

There is a wide variation in the degree of marrow and blood involvement between patients with multiple myeloma. Both of these parameters are known to be highly significant prognostic factors, and the differences between patients may be due to variable expression of adhesion molecules. To test this we used three-colour flow cytometry to study three adhesion molecules associated with myeloma, namely CD38, CD56 and CD138. The level of expression of these molecules was compared with the distribution of myeloma plasma cells in bone marrow (n=59) and peripheral blood (n=26) in patients at presentation or relapse. The extent of marrow infiltration on the trephine biopsy correlated inversely with CD56 expression (Mann-Whitney U Test, P=0.022); there was no difference in CD38 or in CD138 expression. CD56 expression also correlated inversely with the number of circulating plasma cells (linear regression, R2=0.4268, slope=-0.58, P=0.0003). Peripheral blood plasma cell numbers correlated weakly with bone marrow plasmacytosis, and inversely with CD38 expression. The level of CD56 expression by neoplastic plasma cells was assessed in 37 patients over a median of 11 months (range 6-25). There was no significant change in expression (Wilcoxon Signed Rank, P=0.6271). We conclude that plasma cell CD56 expression is constant over the course of the disease; unlike CD138 expression, it is significantly linked to the degree of both bone marrow and peripheral blood involvement.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

系统性红斑狼疮患者CpG基序甲基化状态与淋巴细胞功能相关抗原-1表达的研究

目的 研究系统性红斑狼疮患者(SLE)CpG基序甲基化状态和淋巴细胞功能相关抗原-1(LFA-1)mRNA的表达水平,并探讨两者的关系.方法 提取26例SLE患者与17名健康人外周血淋巴细胞,分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化状态和反转录-聚合酶链反应(RT-PCR)分析LFA-1 mRNA表达水平.结果 SLE患者的CpG基序甲基化水平(10.0±1.2)低于健康对照组(11.9±1.0,P<0.05),并与SLE疾病活动指数(SLEDAI)旱负相关(r=-0.62,P<0.05);SLE患者IJFA-1 mRNA表达(0.55±0.11)明显高于健康对照组(0.25±0.08,P<0.05),并与SLEDAI呈正相关(r=0.54,P<0.05);SLE患者CpG基序甲基化水平与LFA-1 mRNA表达呈负相关(r=-0.57,P<0.05).结论 SLE患者存在CpG基序低甲基化状态,并与LFA-1高表达相关联,表观遗传学在SLE发病机制中具有重要作用.     

Treatment response to bortezomib in multiple myeloma correlates with plasma hepatocyte growth factor concentration and bone marrow thrombospondin concentration

Multiple myeloma (MM) is associated with increased rate of bone marrow angiogenesis. Increased concentration of hepatocyte growth factor (HGF) is associated with poor prognosis in patients treated with conventional chemotherapy or thalidomide. We have shown previously that decreased level of thrombospondin, an angiogenesis inhibitor, correlates with poor response to high‐dose chemotherapy. The aim of our current study was to evaluate association between therapeutic response to bortezomib and thrombospondin and HGF levels. Peripheral blood plasma concentration of HGF and bone marrow plasma concentration of thrombospondin were measured in patients with MM prior to the initiation of bortezomib therapy. Overall, 58 patients were enrolled, 44/58 (76%) of them with relapsed disease. Treatment outcomes were analyzed for possible associations with pretreatment HGF and thrombospondin levels. Patients who achieved complete response had significantly higher pretreatment HGF levels and lower pretreatment thrombospondin levels than others. More than 70% of patients with low pretreatment HGF and high pretreatment thrombospondin concentrations achieved very good partial response or complete response, in contrast to only 20% of patients with high HGF and low thrombospondin levels. High pretreatment thrombospondin and low pretreatment HGF concentrations are associated with therapeutic response to bortezomib in patients with MM.     

Sialyltransferase activity in plasma cells of multiple myeloma

A marked elevation of sialyltransferase activity (STA) was observed in a solid tumor of plasma cells, which had been removed from a patient with multiple myeloma (MM), as compared to normal lymphatic tissues. STA was also determined in mononuclear bone marrow cells of 10 patients with MM and found to be 12 times higher than that of bone marrow mononuclear cells from 5 patients with non-malignant disorders (with less than 1% plasma cells in the bone marrow aspirate). A significant correlation was found between STA and the number of plasma cells in the bone marrow aspirate.     

The AKT kinase is activated in multiple myeloma tumor cells.

Immunohistochemistry (IHC) was performed on archived bone marrow (BM) with a phosphospecific anti-AKT antibody. IHC on 26 BM biopsies from patients with multiple myeloma (MM) demonstrated phospho-AKT staining of malignant plasma cells in a cell membrane-specific pattern, whereas nonmalignant hematopoietic cells did not stain. Preabsorption of the antibody with phosphorylated AKT peptide, but not nonphosphorylated peptide, abrogated staining. Frequency of plasma cell staining in BMs of patients with stage I or smoldering MM was significantly less than that of stage III MM marrows. Plasma cells in 10 patients with monoclonal gammopathy of undetermined significance were not stained by the antibody. To investigate the significance of AKT activation, 2 cell lines initiated from cultures of primary MM cells were also studied. Both demonstrated constitutive AKT activation. Interruption of AKT activation and activity, achieved by either exposure to wortmannin or by ectopic expression of a dominant negative AKT mutant, resulted in inhibition of MM cell growth in vitro. These results indicate that activation of the AKT kinase is a characteristic of MM cells and suggest that AKT activity is important for MM cell expansion.