Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady- state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.     

A biomaterial model of tumor stromal microenvironment promotes mesenchymal morphology but not epithelial to mesenchymal transition in epithelial cells

The stromal tissue surrounding most carcinomas is comprised of an extracellular matrix densely packed with collagen-I fibers, which are often highly aligned in metastatic disease. Here we developed an in vitro model to test the effect of an aligned fibrous environment on cancer cell morphology and behavior, independent of collagen ligand presentation. We grew cells on a biomimetic surface of aligned electrospun poly-l-lactic acid (PLLA) fibers and then examined the effect of this environment on growth rate, morphology, cytoskeletal organization, biochemical and genetic markers of epithelial to mesenchymal transition (EMT), cell surface adhesion, and cell migration. We grew a phenotypically normal breast epithelial cell line (MCF10A) and an invasive breast cancer cell line (MDA-MB-231) on three different substrates: typical flat culture surface (glass or plastic), flat PLLA (glass coated with PLLA) or electrospun PLLA fibers. Cells of both types adopted a more mesenchymal morphology when grown on PLLA fibers, and this effect was exaggerated in the more metastatic-like MDA-MB-231 cells. However, neither cell type underwent the changes in gene expression indicative of EMT despite the changes in cell shape, nor did they exhibit the decreased adhesive strength or increased migration typical of metastatic cells. These results suggest that changes in cell morphology alone do not promote a more mesenchymal phenotype and consequently that the aligned fibrous environment surrounding epithelial cancers may not promote EMT solely through topographical cues.     

河北省儿童医院住院患儿EB病毒感染流行病学特征

目的了解河北省儿童医院住院患儿EB病毒(EBV)感染的流行病学特征,为儿童EBV感染的诊断和预防提供科学依据。方法收集2017年1—12月河北省儿童医院0~14岁EBV感染住院患儿的全血样本,采用酶联免疫吸附试验(ELISA)检测其EBV衣壳抗原(VCA)IgG及IgM抗体,抗早期抗原(EA)IgG抗体和抗核抗原1(NA1)IgG抗体,以检测结果为研究样本的抗体谱。根据4种EBV抗体的检测结果分为现症感染(抗VCA-IgM抗体阳性,抗NA1-IgG抗体阴性、抗VCA-IgG抗体、抗EA-IgG抗体阳性或阴性)、亚急性感染(抗VCA-IgG抗体阳性,抗VCA-IgM抗体、抗NA1-IgG抗体、抗EA-IgG抗体阳性或阴性)、既往感染(抗NA1-IgG抗体阳性,抗VCA-IgG抗体阳性或阴性,其他抗体均为阴性)和未感染(4种抗体均阴性)。按照患儿年龄、检出月份和性别分析各组的阳性率。结果共纳入符合要求的样本4 451例,其中3 257例(73.17%)抗体谱提示EBV感染,包括现症感染380例(8.54%)、亚急性感染616例(13.84%)、既往感染2 261例(50.80%)。不同年龄组原发阳性检出率差异有统计学意义(P<0.05),其中学龄前(>3岁)组的阳性检出率最高(P<0.05);不同检出月份组阳性检出率差异有统计学意义(P<0.05),7月份阳性检出率高于其他月份(P<0.05);男性患儿与女性患儿EBV感染率差异无统计学意义(P>0.05)。380例现症感染患儿的疾病谱以血液系统疾病[传染性单核细胞增多症、急性粒细胞缺乏症、血小板减少性紫癜、EBV相关嗜血细胞综合征]为主,其中传染性单核细胞增多症为临床常见疾病;其次是呼吸系统疾病(急性支气管炎、疱疹性咽峡炎、急性扁桃体炎);其他疾病谱包括神经系统疾病及血流感染、肾病综合征、川崎病。结论河北省儿童医院住院患儿EBV阳性检出率有年龄和检出月份差异,现症感染以血液系统疾病患儿为主,医院应根据流学病学特征制定相应预防措施。     

Role of cholinergic neuromuscular transmission in the neuroregulation of the autophosphorylatable regulatory subunit of cyclic AMP-dependent protein kinase type II and the acetylcholine receptor content in skeletal muscle

Previously, we found that the in vitro [32P]-autophosphorylation of the regulatory subunit of cyclic AMP-dependent protein kinase type II in rat soleus muscles is subject to a nerve-stump-length-dependent neuroregulation which indicates that this event is dependent upon some neural signal other than the impulse-directed release of acetylcholine. In this investigation, tetrodotoxin and alpha-bungarotoxin were also administered to further differentiate the effect of impulse-directed and spontaneously released acetylcholine upon this event and also upon the appearance of new acetylcholine receptors as measured by the binding of radioiodinated bungarotoxin. A 24 h blockade of cholinergic transmission with either neurotoxin did not change the phosphorylation level of the regulatory subunit, while a significant increase is observed when solei are surgically denervated for this period. The phosphorylation level and also the acetylcholine receptor content were increased only after more prolonged (48-96 h) muscle inactivity was produced with the neurotoxins. However, then their effects may not be solely related to alterations in cholinergic transmission. Taken together, our results do not support a trophic role for spontaneously released acetylcholine with respect to the two neurotrophic events studied.     

Integration of Merged Delayed‐Enhanced Magnetic Resonance Imaging and Multidetector Computed Tomography for the Guidance of Ventricular Tachycardia Ablation: A Pilot Study

MDCT/MRI Fusion for the Guidance of VT Ablation . Background: Delayed enhancement (DE) MRI can assess the fibrotic substrate of scar‐related VT. MDCT has the advantage of inframillimetric spatial resolution and better 3D reconstructions. We sought to evaluate the feasibility and usefulness of integrating merged MDCT/MRI data in 3D‐mapping systems for structure–function assessment and multimodal guidance of VT mapping and ablation. Methods: Nine patients, including 3 ischemic cardiomyopathy (ICM), 3 nonischemic cardiomyopathy (NICM), 2 myocarditis, and 1 redo procedure for idiopathic VT, underwent MRI and MDCT before VT ablation. Merged MRI/MDCT data were integrated in 3D‐mapping systems and registered to high‐density endocardial and epicardial maps. Low‐voltage areas (<1.5 mV) and local abnormal ventricular activities (LAVA) during sinus rhythm were correlated to DE at MRI, and wall‐thinning (WT) at MDCT. Results: Endocardium and epicardium were mapped with 391 ± 388 and 1098 ± 734 points per map, respectively. Registration of MDCT allowed visualization of coronary arteries during epicardial mapping/ablation. In the idiopathic patient, integration of MRI data identified previously ablated regions. In ICM patients, both DE at MRI and WT at MDCT matched areas of low voltage (overlap 94 ± 6% and 79 ± 5%, respectively). In NICM patients, wall‐thinning areas matched areas of low voltage (overlap 63 ± 21%). In patients with myocarditis, subepicardial DE matched areas of epicardial low voltage (overlap 92 ± 12%). A total number of 266 LAVA sites were found in 7/9 patients. All LAVA sites were associated to structural substrate at imaging (90% inside, 100% within 18 mm). Conclusion: The integration of merged MDCT and DEMRI data is feasible and allows combining substrate assessment with high‐spatial resolution to better define structure–function relationship in scar‐related VT. (J Cardiovasc Electrophysiol, Vol. 24, pp. 419‐426, April 2013)     

Impact of the microenvironment on the pathogenesis of mucosa-associated lymphoid tissue lymphomas

Approximately 8% of all non-Hodgkin lymphomas are extranodal marginal zone B cell lymphomas of mucosa-associated lymphoid tissue (MALT), also known as MALT lymphomas. These arise at a wide range of different extranodal sites, with most cases affecting the stomach, the lung, the ocular adnexa and the thyroid. The small intestine is involved in a lower percentage of cases. Lymphoma growth in the early stages is associated with long-lasting chronic inflammation provoked by bacterial infections (e.g., Helicobacter pylori or Chlamydia psittaci infections) or autoimmune conditions (e.g., Sjögren’s syndrome or Hashimoto thyroiditis). While these inflammatory processes trigger lymphoma cell proliferation and/or survival, they also shape the microenvironment. Thus, activated immune cells are actively recruited to the lymphoma, resulting in either direct lymphoma cell stimulation via surface receptor interactions and/or indirect lymphoma cell stimulation via secretion of soluble factors like cytokines. In addition, chronic inflammatory conditions cause the acquisition of genetic alterations resulting in autonomous lymphoma cell growth. Recently, novel agents targeting the microenvironment have been developed and clinically tested in MALT lymphomas as well as other lymphoid malignancies. In this review, we aim to describe the composition of the microenvironment of MALT lymphoma, the interaction of activated immune cells with lymphoma cells and novel therapeutic approaches in MALT lymphomas using immunomodulatory and/or microenvironment-targeting agents.     

流式细胞术的培训实践及技术平台建设探讨

流式细胞分析技术(flow cytometry,FCM),又称流式细胞术,是一种在功能水平上对单细胞或其他生物粒子进行定量分析和分选的技术,具有广泛的科研应用价值.采用理论与实践并举,结合讨论的教学方式,在教师队伍开设针对性的培训课程,对深刻理解并在科研工作中有效利用该技术具有及其重要意义.同时以教学促科研,以科研促技...     

Inhibition of melanoma cell motility by the snake venom disintegrin eristostatin.

Eristostatin, an RGD-containing disintegrin isolated from the venom of Eristicophis macmahoni, inhibits lung or liver colonization of melanoma cells in a mouse model. In this study, transwell migration and in vitro wound closure assays were used to determine the effect of eristostatin on the migration of melanoma cells. Eristostatin significantly impaired the migration of five human melanoma cell lines. Furthermore, it specifically inhibited cell migration on fibronectin in a concentration-dependent manner, but not that on collagen IV or laminin. In contrast, eristostatin was found to have no effect on cell proliferation or angiogenesis. These results indicate that the interaction between eristostatin and melanoma cells may involve fibronectin-binding integrins that mediate cell migration. Mutations to alanine of seven residues within the RGD loop of eristostatin and four residues outside the RGD loop of eristostatin resulted in significantly less potency in both platelet aggregation and wound closure assays. For six of the mutations, however, decreased activity was found only in the latter assay. We conclude that a different mechanism and/or integrin is involved in these two cell activities.