FK506 (tacrolimus) inhibits extravasation of lymphoid cells by abrogating VLA-4/VCAM-1 mediated transendothelial migration

Extravasation is a critical process for the physiological lymphocyte traffic as well as the hematogenous spread of malignant hemopoietic cells. Here we report that abrogation of calcineurin activity leads to in vitro transendothelial migration and in vivo infiltration of human lymphoma Nalm-6 cells, which are associated with the abrogation of the VLA-4/VCAM-1 mediated pathway. Rapamycin, which can antagonize FK506 but not CsA to inhibit calcineurin, abrogates FK-506 mediated but not CsA mediated inhibition of in vitro transendothelial migration. FK506 may exert its potent immunosuppressive action partly by inhibiting VLA-4/VCAM-1 mediated transendothelial migration or insinuation of lymphoid cells to tissues.     

Small Molecule Agonist of Very Late Antigen-4 (VLA-4) Integrin Induces Progenitor Cell Adhesion

Activation of the integrin family of cell adhesion receptors on progenitor cells may be a viable approach to enhance the effects of stem cell-based therapies by improving cell retention and engraftment. Here, we describe the synthesis and characterization of the first small molecule agonist identified for the integrin α4β1 (also known as very late antigen-4 or VLA-4). The agonist, THI0019, was generated via two structural modifications to a previously identified α4β1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to α4β1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1α. Molecular modeling predicted that the compound binds at the α/β subunit interface overlapping the ligand-binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin α4β7 as well as α5β1 and αLβ2. When cross-linked to αLβ2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the α/β subunit interface and not at the ligand-binding site in the inserted (“I”) domain of the αL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.     

Homotypic leukocyte aggregation triggered by a monoclonal antibody specific for a novel epitope expressed by the integrin β1 subunit: Conversion of nonresponsive cells by transfecting human integrin α4 subunit cDNA

The monoclonal antibody 33B6 was found to be specific for the β1 integrin subunit. Treatment of leukocytes with this antibody induced a vigorous homotypic aggregation that had similar physiologic conditions as aggregation induced by a monoclonal antibody specific for the α4 subunit. Expression of a β1 subunit on the cell surface was not sufficient for mAb 33B6-mediated aggregation to occur, since cells of the K562 erythroleukemia line failed to respond even though they expressed the β1 subunit and the 33B6 epitope. However, after transfection with cDNA encoding the α4 subunit, K562 cells acquired the ability to aggregate in response to mAb 33B6 binding. By contrast, mAb 33B6 blocked cell binding to the endothelial surface protein vascular cell adhesion molecule-1 and the extracellular matrix protein fibronectin. These results suggest that the β1 epitope defined by mAb 33B6 may play a novel role in regulating leukocyte adhesive interactions.     

LncRNA HOXB-AS1 promotes cell growth in multiple myeloma via FUT4 mRNA stability by ELAVL1

Multiple myeloma (MM) is defined as the second most common hematological tumor in the globe. Long noncoding RNAs (lncRNAs) have been reported to play stimulative or suppressive role in the progression of different carcinomas. The investigation of lncRNAs in MM is still inadequate. LncRNA HOXB cluster antisense RNA 1 (HOXB-AS1) was once revealed to facilitate glioma progression by affecting cellular activities of glioma cells. However, whether HOXB-AS1 participates in the development of MM still remains an enigma. In this study, we unveiled that HOXB-AS1 was highly expressed in MM and loss-of-function assays certified that HOXB-AS1 obstruction suppressed MM cell proliferation, and stimulated cell apoptosis. In addition, HOXB-AS1 could modulate fucosyltransferase 4 (FUT4) and FUT4-mediated Wnt/β-catenin pathway. In subsequence, it was observed from mechanism assays that HOXB-AS1 enhanced the interaction between ELAVL1 and FUT4 so as to stabilize FUT4 messenger RNA. In the end, rescue experiments affirmed that HOXB-AS1 affected the cell growth through FUT4 in MM. In conclusion, the whole modulation mechanism of HOXB-AS1/ELAVL1/FUT4 axis in MM was validated in this study, which suggested that HOXB-AS1 might function as a powerful and promising therapeutic biomarker for the clinical treatment of patients with MM.     

EEN regulates the proliferation and survival of multiple myeloma cells by potentiating IGF-1 secretion

The molecular mechanisms of multiple myeloma are not well defined. EEN is an endocytosis-regulating molecule. Here we report that EEN regulates the proliferation and survival of multiple myeloma cells, by regulating IGF-1 secretion. In the present study, we observed that EEN expression paralleled with cell proliferation, EEN accelerated cell proliferation, facilitated cell cycle transition from G1 to S phase by regulating cyclin-dependent kinases (CDKs) pathway, and delayed cell apoptosis via Bcl2/Bax-mitochondrial pathway. Mechanistically, we found that EEN was indispensable for insulin-like growth factor-1 (IGF-1) secretion and the activation of protein kinase B-mammalian target of rapamycin (Akt-mTOR) pathway. Exogenous IGF-1 overcame the phenotype of EEN depletion, while IGF-1 neutralization overcame that of EEN over-expression. Collectively, these data suggest that EEN may play a pivotal role in excessive cell proliferation and insufficient cell apoptosis of bone marrow plasma cells in multiple myeloma. Therefore, EEN may represent a potential diagnostic marker or therapeutic target for multiple myeloma.     

Expression of a Soluble Functional Form of the Integrin α4β1 in Mammalian Cells

The integrin α₄β₁(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human α₄ and β₁ subunits were fused to the genomic DNA encoding the human γ1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote α₄β₁ heterodimer formation. The soluble α₄β₁ Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent K_d for VCAM-1 binding were similar for both the soluble and native forms of α₄β₁. In addition, the integrin–Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble α₄β₁ should be generally applicable to a range of integrins.     

Sex hormones and immune dysregulation in multiple myeloma

A group of 49 multiple myeloma patients, 20 men and 29 women, were evaluated. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17-oestradiol (E) and testosterone (T) serum concentrations have been detected by radioimmunoassay. Peripheral blood lymphocyte proliferation in response to phytohaemagglutinin (PHA), concanavalin A (ConA), recombinant interleukin-2 (rIL-2) and dextran sulphate (DxS) was investigated. Our findings provide evidence for two different patterns of sex hormone changes and immune dysfunctions presented differently by male and female multiple myeloma patients. In men increased FSH, LH and E concentrations and an augmented E to T ratio were associated with decreased lymphocyte blastogenic response to PHA, ConA and increased proliferation to rIL-2 and DxS. Female patients with multiple myeloma demonstrated normal values of FSH, LH and T, but a diminished E level and decreased E to T ratio correlated with a lymphocyte normal response to PHA and ConA and augmented blastogenesis to IL-2 and DxS. Our data, while admittedly preliminary, suffice to provide an indication of sex hormone changes in multiple myeloma patients, which could be responsible, at least in part, for the immune dysfunction observed in multiple myeloma.     

Overexpression of microRNA-29b induces apoptosis of multiple myeloma cells through down regulating Mcl-1

MicroRNAs (miRNAs) are small, noncoding ribonucleic acids (ncRNAs), which regulate gene expression by targeting mRNAs for translational repression and degradation. Several lines of evidences have indicated that miRNAs act as tumor suppressors and oncogenes. However, the role of miRNAs in pathogenesis of multiple myeloma (MM) remains unclear. In this study, we examined the profile of miRNA expression of primary MM cells, using miRNA microarray and quantitative real-time polymerase chain reaction (qPCR) techniques. These results showed that in the bone marrow specimens analyzed, miRNA-29b was significantly downregulated. Similar results were also observed in human myeloma cell lines (HMCLs). Adenovirus-mediated overexpression of miR-29b induced apoptosis and elevated caspase-3 activation in HMCLs. Using a bioinformatics approach, we found a perfect complementarity between miRNA-29b and the 3′UTR of myeloid-cell-leukemia 1(Mcl-1). It is further confirmed that miRNA-29b downregulated the level of Mcl-1 without effect on the mRNA level using both qRT-PCR assays and Western blot analyses. Moreover, we observed that enforced miR-29b expression by using a retarget miRNA-29b expression vector (Ad5F11p-miR-29b) could induce apoptosis and elevate caspase-3 activation in HMCLs. Our results also indicated that miRNA-29b-induced apoptosis acted antagonistically with IL-6 in HMCLs. These findings suggest that miRNA-29b may play an important role in MM as a tumor suppressor.     

CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.     

二苯乙烯苷对H202诱导血管内皮细胞ICAM-1及VCAM-1表达的影响

目的：通过研究二苯乙烯苷（TSG）对H20：诱导的人脐静脉内皮细胞ICAM-1、VCAM-1表达的影响,探明二苯乙烯苷抗氧化保护内皮细胞的作用机制。方法：体外培养人脐静脉内皮细胞,实验分为空白对照组、H20：组、辛伐他汀组、TSG组,运用逆转录聚合酶链式反应和酶联免疫吸附试验分别检测ICAM-1及VCAM-1mRNA与其蛋白的表达。结果：200μmol·L。的H202作用内皮细胞24h后。ICAM．1和VCAM-1的mRNA和蛋白表达水平均明显上调,与空白对照组比较,差异有显著性（P〈0．01）。而在200μmol·L。的H202作用前用1μmol·L^-1二苯乙烯苷预处理体外培养人脐静脉内皮细胞4h,结果显示二苯乙烯苷能抑制H2O2诱导的内皮细胞ICAM-1、VCAM-1的mRNA和VCAM-1的蛋白水平表达,与H2O2组比较差异有显著性（P〈0．01）;而ICAM-1的蛋白表达水平与H202组比较差异有统计学意义（P〈0．05）;辛伐他汀组ICAM-1和VCAM-1的mRNA及其蛋白水平表达降低,与H20：组比较差异均有显著性（P〈0．01）。实验结果表明二苯乙烯苷可抑制H2O2诱导的内皮细胞粘附分子ICAM-1、VCAM-1表达。结论：二苯乙烯苷可通过降低细胞粘附分子ICAM-1和VCAM-1的表达保护氧化应激引起的人脐静脉内皮细胞损伤。     

Discovery of a potent, orally bioavailable pyrimidine VLA-4 antagonist effective in a sheep asthma model

A series of N-(pyrimidin-4-yl)-phenylalanine VLA-4 antagonists is described. Optimization of substituents at the 2 and 5 positions of the pyrimidine ring gave 14, a very potent VLA-4 inhibitor which is orally active in a sheep asthma model.     

多发性骨髓瘤患者医院感染临床分析

目的 探讨多发性骨髓瘤患者医院感染的临床状况和危险因素。方法 对1994年1月～2001年12月收治的62例多发性骨髓瘤患者医院感染发生情况进行调查分析。结果 多发性骨髓瘤患者发生医院感染33例。感染发生率为53．2％,好发部位为呼吸道、口腔、胃肠道和泌尿道等。结论 多发性骨髓瘤患者医院感染的发生与骨髓瘤细胞含量、疾病Ⅲ期、骨损害、白细胞减少、化疗及住院日等因素有关。应采取相应措施,避免医院感染发生。     

免疫固定电泳在诊断多发性骨髓瘤中的作用

目的 探讨分析免疫固定电泳在诊断多发性骨髓瘤（MML）中的作用。方法 以2014年10月至2016年10月丽水市人民医院收治的110例需进行免疫固定电泳检测的患者为研究对象,对其进行血清M蛋白和浓缩尿中M蛋白的检测,筛选出两种检测均显阳性的患者,并将检测结果与临床资料相比较,对比血清M蛋白和浓缩尿中M蛋白的检测阳性与多发性骨髓瘤之间的关系。结果 110例需进行免疫固定电泳检测的患者中,经临床资料分析,有34例患者为多发性骨髓瘤。这34例患者血清M蛋白的检测显阳性,临床诊断符合率为100.00%;25例患者浓缩尿中M蛋白的检测显阳性,临床诊断符合率为73.53%。血清检验的临床符合率较尿液检验更好,差异具有统计学意义（P＜0.05）;34例血清M蛋白的检测显阳性的患者中,IgG型21例（61.76%）,IgA型8例（23.53%）,IgM型3例（8.82%）,γ轻链型2例（5.88%）。结论 MML患者采用血清及尿液免疫固定电泳检测,血清M蛋白检测阳性率较浓缩尿中M蛋白的检测阳性率要高,血清及尿液进行的免疫固定电泳检测对MML的诊断、检测及预后判断具有重要的提示作用,临床工作者可以根据电泳图谱对MML进行分型,并制定个体化的治疗方案。     

Glycosylation of polyclonal and paraprotein IgG in multiple myeloma

It has previously been shown that in multiple myeloma (MM) each IgG paraprotein exhibits a unique oligosaccharide profile. It has been assumed that this results from a clone specific glycosylation machinery. However, the abnormal physiological environment of the bone marrow in this disease may also affect normal plasma cells producing polyclonal IgG. We present data to show that this is so and that, in two cases, the oligosaccharide profile of the polyclonal IgG reflected that of the paraprotein from the same patient rather than that of normal polyclonal IgG. This revised version was published online in November 2006 with corrections to the Cover Date.     

多发性骨髓瘤预后相关自噬基因筛选及建模

为探讨自噬相关基因(ARGs)在MM发生发展中的作用机制并建立相关的预后模型。基于MMRF与HADb数据库,通过R语言确定多发性骨髓瘤中自噬相关基因的差异表达,GO和KEGG分析自噬相关基因与多发性骨髓瘤发生发展的关系,使用COX回归算法建立多基因预后模型,Kaplan-Meier方法绘制生存曲线,ROC曲线评价预后模型的可靠性。最终从764例多发性骨髓瘤患者骨髓样本及4例正常骨髓样本中共发现104个基因的表达在多发性骨髓瘤样本中具有显著差异,其中上调基因46个,下调基因58个。GO富集主要集中在巨自噬、自噬调节、细胞对外部刺激的反应等本体学注释。KEGG富集主要集中在自噬、细胞凋亡、NOD样受体信号通路、PI3K-Akt信号通路。单因素COX分析发现33个自噬相关基因与多发性骨髓瘤患者整体生存明显相关。多因素COX回归筛选出13个预后相关自噬相关基因(NKX2-3、NCKAP1、BIRC5、PEX3、HGS、RUBCN、PARP1、ARSA、DNAJB9、HSP90AB1、EEF2、FKBP1B和CD46)建立多发性骨髓瘤自噬相关基因预后模型。Kaplan-Meier生存曲线分析显示...     

Geospatial analysis of population-based incidence of multiple myeloma in the United States

BackgroundWe investigated the spatial patterns of multiple myeloma (MM) incidence in the United States (US) between 2013 and 2017 to improve understanding of potential environmental risk factors for MM.MethodsWe analyzed the average county-level age-adjusted incidence rates (“ASR”) of MM between 2013 and 2017 in 50 states and the District of Columbia using the U.S. Cancer Statistics Public Use Databases. We firstly divided the ASR into quintiles and described spatial patterns using a choropleth map. To identify global and local clusters of the ASR, we performed the Spatial Autocorrelation (Global Moran’s I) analysis and the Anselin’s Local Indicator of Spatial Autocorrelation (LISA) analysis. We compared the means of selected demographic and socioeconomic factors between the clusters and counties of the whole US using Welch one-sided t-test.ResultsWe identified distinct spatial dichotomy of the ASR across counties. High ASR were observed in counties in the Southeast of the US as well as the Capital District (metropolitan areas surrounding Albany) and New York City in the state of New York, while low ASR were observed in counties in the Southwest and West of the US. The ASR showed a significant positive spatial autocorrelation. We identified two major high-high local clusters of the ASR in Georgia and Southern Carolina and five major low-low local clusters of the ASR in Alabama, Arizona, New Hampshire, Ohio, Oregon, and Tennessee. The racial population distribution may partly explain the spatial distribution of MM incidence in the US.ConclusionFindings from this study showed distinct spatial distribution of MM in the US and two high-high and five low-low local clusters. The non-random distribution of MM suggests that environmental exposures in certain regions may be important for the risk of MM.     

Involvement of tumor necrosis factor α and very late activation antigen 4/vascular cell adhesion molecule 1 interaction in surgical-stress-enhanced experimental metastasis

 We examined the influence of surgical stress on hematogenous metastasis of malignant tumor cells. The study was performed by focusing on the involvement of inflammatory cytokines in the serum, raised acutely after surgery, and endothelial adhesion molecules in the metastatic process. Surgical stress, given to C57BL/6 mice before B16-BL6 melanoma inoculation, significantly enhanced the pulmonary metastasis. This enhancement was seen when the surgery lasted for more than 2 h. After the 2-h surgery, the enhancement of pulmonary metastasis was seen most remarkably when B16-BL6 was inoculated 24 h after surgery. The serum level of tumor necrosis factor α (TNFα) in the mice that underwent the 2-h surgery peaked 12 h after the surgery. In contrast, serum interferon γ was not detectable. Administration of an anti-TNFα mAb before the surgery inhibited the enhanced metastasis by inhibiting the increased expression of vascular cell adhesion molecule 1 (VCAM-1) on lung vascular endothelium after the surgery. Pretreatment of B16-BL6 cells with an anti-very late activation antigen 4 (anti-VLA-4) mAb completely inhibited the enhanced metastasis after surgery. Administration of an anti-VCAM-1 mAb before surgery also inhibited the enhancement. These results indicate that serum TNFα , raised by surgical stress, is critically involved in the enhanced pulmonary metastasis of mouse melanoma by inducing VCAM-1 expression on lung vascular endothelium. Received: 22 January 1996 / Accepted: 1 April 1996