BACKGROUND & AIMS: :Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.

背景与目标： ：人类胚胎和间充质干细胞疗法可能为许多患者带来重大益处。然而，最近，据报道，在小鼠饲养细胞上培养的人类胚胎干细胞系被异种碳水化合物N-甘氨酰神经氨酸（Neu5Gc）污染，被认为可能不适合人类治疗。为了确定Neu5Gc污染问题对干细胞疗法发展的影响程度，我们调查了它是否也在人类饲养细胞和间充质干细胞中培养的细胞中也发生了，污染的来源是什么，以及污染是否可逆。我们发现，在人类饲养细胞上培养的胚胎干细胞中存在N-羟乙酸神经氨酸，这与商业血清替代培养基成分中Neu5Gc的存在有关。在胎牛血清存在下培养的间充质干细胞中也发生了类似的污染。结果表明，分别通过质谱分析和单克隆抗体染色检测，Neu5Gc同时存在于糖蛋白和脂质连接的聚糖中。值得注意的是，两种细胞类型的后代中的污染在很大程度上都是可逆的，这表明去污染的细胞可能源自现有的干细胞系。尽管在间质干细胞暴露于胎牛血清的临床试验中尚未报告重大并发症，但免疫原性污染可能会反映在移植细胞的生存力和功效中，因此会影响已发表的结果。干细胞安全培养条件的定义对于细胞疗法的未来发展至关重要。

BACKGROUND & AIMS: The mammalian transcription factor LSF (CP2/LBP-1c) binds cellular promoters modulated by cell growth signals. We demonstrate here that LSF-DNA-binding activity is strikingly regulated by induction of cell growth in human peripheral T lymphocytes. Within 15 min of mitogenic stimulation of these cells, the level of LSF-DNA-binding activity increased by a factor of five. The level of LSF protein in the nucleus remained constant throughout this interval. However, a rapid decrease in the electrophoretic mobility of LSF, attributable to phosphorylation, correlated with the increase in DNA-binding activity. pp44 (ERK1) phosphorylated LSF in vitro on the same residue that was phosphorylated in vivo, specifically at amino acid position 291, as indicated by mutant analysis. As direct verification of the causal relationship between phosphorylation and DNA-binding activity, treatment in vitro of LSF with phosphatase both increased the electrophoretic mobility of the protein and decreased LSF-DNA-binding activity. This modulation of LSF-DNA-binding activity as T cells progress from a resting to a replicating state reveals that LSF activity is regulated during cell growth and suggests that LSF regulates growth-responsive promoters.

背景与目标： 哺乳动物转录因子LSF（CP2 / LBP-1c）结合受细胞生长信号调节的细胞启动子。我们在这里证明，LSF-DNA结合活性是由人类外周血T淋巴细胞的细胞生长诱导来显着调节的。在这些细胞的促有丝分裂刺激的15分钟内，LSF-DNA结合活性的水平提高了五倍。在整个间隔期间，核中LSF蛋白的水平保持恒定。但是，可归因于磷酸化的LSF电泳迁移率的快速下降与DNA结合活性的增加有关。突变分析表明，pp44（ERK1）在体外磷酸化的同一残基上，特别是在氨基酸位置291处磷酸化LSF。作为磷酸化与DNA结合活性之间因果关系的直接验证，体外用磷酸酶处理LSF既增加了蛋白质的电泳迁移率，又降低了LSF-DNA结合活性。当T细胞从静止状态发展到复制状态时，对LSF-DNA结合活性的这种调节揭示了LSF活性在细胞生长过程中受到调节，并暗示LSF调节生长响应性启动子。

BACKGROUND & AIMS: Human parvovirus B19 (B19) IgG was studied retrospectively in 66 allogeneic bone marrow transplantation (BMT) patients using an enzyme-linked immunosorbent assay. Recipient and donor sera had been stored pre-BMT together with sequential sera thereafter. Approximately half of donors and recipients had anti-B19 IgG pre-BMT and thus the relative contributions of donor and recipient immunity to antibody production after transplantation could be assessed. For each patient, a serum taken 2 to 3 years after BMT was also tested and the results show that persistence of B19 antibody depends on prior recipient (P = .0003) but not on donor immunity (P = .8). The findings were similar in both sibling and (VUD) BMT volunteer unrelated donor patients. Analysis of sequential post-BMT sera from 41 of the patients, for whom appropriately timed samples were available, showed primary B19 infection in 3 seronegative individuals, whereas 5 others who were seropositive before BMT underwent recurrent infection. Sequential results from the remaining 33 patients without recent B19 infection showed no evidence for donor antibody transfer and confirmed that antibody persistence depends on prior recipient immunity. B19 IgG levels decreased variably with time and some patients eventually became seronegative. It is concluded that this long-term persistence of B19 antibody post-BMT is most probably due to the existence of long-lived recipient plasma cells.

背景与目标： 使用酶联免疫吸附试验对66名同种异体骨髓移植（BMT）患者进行了人类细小病毒B19（B19）IgG回顾性研究。收件人和供体血清已在BMT之前与随后的顺序血清一起存储。大约一半的供体和受体在BMT前具有抗B19 IgG，因此可以评估移植后供体和受体免疫对抗体产生的相对贡献。对于每位患者，还测试了BMT后2至3年的血清，结果表明B19抗体的持久性取决于先前的接受者（P = .0003），而不取决于供体的免疫力（P = .8）。在同级和（VUD）BMT自愿无关供者患者中，发现相似。对41位患者的BMT后连续血清进行分析，发现有适当定时的样本，在3例血清阴性患者中原发性B19感染，而在BMT之前5例血清阳性的患者则进行了复发性感染。其余33例近期未感染B19的患者的顺序结果显示，尚无转移供体抗体的证据，并证实抗体的持久性取决于先前的受体免疫力。 B19 IgG水平随时间而下降，有些患者最终呈血清阴性。结论是BMT后B19抗体的这种长期持久性很可能是由于存在长寿命的受体浆细胞引起的。

BACKGROUND & AIMS: Thrombin activation requires assembly of a prothrombinase complex of activated coagulation factors on an anionic phospholipid surface, classically provided by activated platelets. We have previously shown that anionic phosphatidylserine is exposed by rat vascular smooth muscle cells (VSMCs) undergoing apoptosis after serum withdrawal. In this study, using a chromogenic assay, we have shown thrombin generation by apoptotic VSMCs expressing c-myc (VSMC-myc) with an area under the thrombin-generation curve (AUC) of 305 +/- 17 nmol x min/L and a peak thrombin (PT) of 154 +/- 9 nmol/L. The thrombin-generating potential of the apoptotic VSMC-myc cells was greater than that of unactivated platelets (P = .003 for AUC; P = .0002 for PT) and similar to calcium-ionophore activated platelets (AUC of 332 +/- 15 nmol x min/L, P = .3; PT of 172 +/- 8 nmol/L, P = .2). Thrombin activation was also seen with apoptotic human VSMCs (AUC of 211 +/- 8 nmol x min/L; PT of 103 +/- 4 nmol/L) and was inhibited by annexin V (P < .0001 for AUC and PT). VSMC-myc cells maintained in serum generated less thrombin than after serum withdrawal (P = .0002 for AUC and PT). VSMCs derived from human coronary atherosclerotic plaques that apoptose even in serum also generated thrombin (AUC of 260 +/- 2 nmol x min/L; PT of 128 +/- 4 nmol/L). We conclude that apoptotic VSMCs possess a significant thrombin-generating capacity secondary to phosphatidylserine exposure. Apoptotic cells within atherosclerotic plaques may allow local thrombin activation, thereby contributing to disease progression.

背景与目标： 凝血酶的活化需要活化的凝血因子的凝血酶原酶复合物在阴离子磷脂表面上的组装，这通常是由活化的血小板提供的。先前我们已经表明，在撤离血清后，发生凋亡的大鼠血管平滑肌细胞（VSMC）会暴露出阴离子磷脂酰丝氨酸。在这项研究中，我们使用发色试验显示了表达c-myc（VSMC-myc）的凋亡VSMC的凝血酶生成，其凝血酶生成曲线（AUC）下方的面积为305 /-17 nmol x min / L，凝血酶峰值（PT）为154 9 nmol / L。凋亡的VSMC-myc细胞的凝血酶生成潜能大于未激活的血小板（对于AUC，P = .003；对于PT，P = .0002），类似于钙离子载体激活的血小板（AUC为332 /-15 nmol） xmin / L，P ＝ 0.3； PT为172 / 8-nmol / L，P ＝ 0.2）。在凋亡的人VSMC中也观察到凝血酶活化（AUC为211 /-8 nmol x min / L； PT为103 /-4 nmol / L），并被膜联蛋白V抑制（对于AUC和PT，P <.0001）。血清中维持的VSMC-myc细胞产生的凝血酶少于血清中止后产生的凝血酶（对于AUC和PT，P = .0002）。源自甚至在血清中也会凋亡的人冠状动脉粥样硬化斑块的VSMC也会产生凝血酶（AUC为260 /-2 nmol x min / L； PT为128 /-4 nmol / L）。我们得出结论，凋亡性VSMC具有继磷脂酰丝氨酸暴露后的显着凝血酶生成能力。动脉粥样硬化斑块中的凋亡细胞可能允许局部凝血酶活化，从而促进疾病进展。

BACKGROUND & AIMS: :Etanercept is a recombinant human soluble tumor necrosis factor (TNF-alpha) receptor fusion protein that inhibits TNF-alpha, a major mediator in the pathogenesis of graft-versus-host disease (GVHD). The purpose of our study was to evaluate the safety and efficacy of etanercept therapy in 21 patients with steroid-refractory acute GVHD (aGVHD) (n = 13) and chronic GVHD (cGVHD) (n = 8). Etanercept 25 mg was given subcutaneously twice weekly for 4 weeks followed by 25 mg weekly for 4 weeks. At the time of initiation of etanercept, 14 patients had skin, 13 had gastro-intestinal, 5 had liver, 5 had pulmonary, and 4 had oral involvement. Twelve patients (57%) completed 12 doses of therapy. Overall, 11 of 21 patients (52%) responded to the treatment with etanercept, including 6 patients (46%) with aGVHD [n = 4 complete response (CR), n = 2 partial response (PR)] and 5 patients (62%) with cGVHD (n = 1 CR, n = 4 PR). Clinical responses were most commonly seen in patients with refractory gut aGVHD with 55% of the patients having a CR and 9% having a PR. CMV reactivation occurred in 48% of patients, bacterial infections in 14% of patients, and fungal infections in 19% of patients. Fourteen patients (67%) were alive after a median follow-up of 429 days (range 71-1007 days) since initiation of etanercept. Seven patients died, 3 of infections, 2 of refractory aGVHD, and 2 of disease progression. In conclusion, our preliminary data indicate that etanercept is well tolerated and can induce a high response rate in patients with steroid-refractory aGVHD and cGVHD, particularly in the setting of GI involvement.

背景与目标： ：Etanercept是一种重组人类可溶性肿瘤坏死因子（TNF-alpha）受体融合蛋白，可抑制TNF-alpha（一种在移植物抗宿主病（GVHD）发病机理中的主要介体）。我们的研究目的是评估依那西普治疗21例激素抵抗性急性GVHD（aGVHD）（n = 13）和慢性GVHD（cGVHD）（n = 8）患者的安全性和有效性。每周两次皮下给予Etanercept 25 mg，持续4周，然后每周25 mg，持续4周。在开始使用依那西普时，有14例皮肤，13例胃肠，5例肝，5例肺和4例经口受累。 12名患者（57％）完成了12剂治疗。总体上，21例患者中有11例（52％）对依那西普治疗有反应，其中6例（46％）患有aGVHD [n = 4完全缓解（CR），n = 2部分缓解（PR）]和5例（62 ％）和cGVHD（n = 1 CR，n = 4 PR）。临床反应最常见于顽固性肠aGVHD患者，其中55％的患者为CR，9％的患者为PR。 CMV重新激活发生在48％的患者中，细菌感染发生在14％的患者中，而真菌感染发生在19％的患者中。自依那西普开始接受中位随访429天（71-1007天）后，有14名患者（67％）还活着。 7例患者死亡，3例感染，2例难治性aGVHD死亡，2例疾病进展。总之，我们的初步数据表明，依那西普耐受性好，在类固醇难治性aGVHD和cGVHD患者中可引起较高的应答率，特别是在胃肠道受累的情况下。

BACKGROUND & AIMS: :A photoplethysmographic (PPG) technique to assess blood flow in bone tissue has been developed and tested. The signal detected by the PPG consists of a constant-level (DC) component-which is related to the relative vascularization of the tissue-and a pulsatile (AC) component-which is synchronous with the pumping action of the heart. The PPG probe was applied on the skin over the patella. The probe uses near-infrared (804 nm) and green (560 nm) light sources and the AC component of the PPG signals of the two wavelengths was used to monitor pulsatile blood flow in the patellar bone and the overlying skin, respectively. Twenty healthy subjects were studied and arterial occlusion resulted in elimination of PPG signals at both wavelengths, whereas occlusion of skin blood flow by local surface pressure eliminated only the PPG signal at 560 nm. In a parallel study on a physical model with a rigid tube we showed that the AC component of the PPG signal originates from pulsations of blood flow in a rigid structure and not necessarily from volume pulsations. We conclude that pulsatile blood flow in the patellar bone can be assessed with the present PPG technique.

背景与目标： ：已经开发并测试了用于评​​估骨组织中血流的光电容积描记（PPG）技术。 PPG检测到的信号由与组织的相对血管形成有关的恒定水平（DC）分量和与心脏的泵浦动作同步的脉动（AC）分量组成。将PPG探针施加到over骨上的皮肤上。该探头使用近红外（804 nm）和绿色（560 nm）光源，并且使用两种波长的PPG信号的AC分量分别监视monitor骨和周围皮肤中的脉动血流。对二十名健康受试者进行了研究，动脉阻塞导致两种波长的PPG信号均被消除，而局部表面压力阻塞皮肤血流仅消除了560 nm处的PPG信号。在具有刚性管的物理模型的并行研究中，我们显示了PPG信号的AC分量源自刚性结构中的血流脉动，而不一定源自体积脉动。我们得出的结论是，可以使用当前的PPG技术评估the骨中的搏动性血流。

BACKGROUND & AIMS: :There is an abundance of clinical applications using human umbilical cord blood (HUCB) as a source for stem cell populations. Other than haematopoietic progenitors, there are mesenchymal, endothelial stem cells and neuronal precursors, in varying quantities, that are found in human umbilical cord blood. These may be useful in diseases such as immune deficiency and autoimmune disorders. Considering issues of safety, availability, transplant methodology, rejection and side effects, it is contended that a therapeutic stem cell transplant, utilizing stem cells from HUCB, provides a reliable repository of early precursor cells that can be useful in a great number of diverse conditions. Drawbacks of relatively smaller quantities of mononucleated cells in one unit of cord blood can be mitigated by in-vitro expansion procedures, improved in-vivo signalling, and augmentation of the cellular milieu, while simultaneously choosing the appropriate transplantation site and technique for introduction of the stem cell graft.

背景与目标： ：有大量的临床应用使用人类脐带血（HUCB）作为干细胞群体的来源。除造血祖细胞外，人类脐带血中还存在不同数量的间充质，内皮干细胞和神经元前体。这些可能对诸如免疫缺陷和自身免疫性疾病等疾病有用。考虑到安全性，可用性，移植方法，排斥反应和副作用等问题，可以认为，利用HUCB干细胞进行的治疗性干细胞移植可提供可靠的早期前体细胞库，该库可用于多种条件。可以通过体外扩增程序，改进的体内信号传导和细胞环境增强来缓解一单位脐带血中相对较少数量的单核细胞的缺点，同时选择合适的移植部位和技术引入干细胞移植。

BACKGROUND & AIMS: The analysis of brain extracellular fluid can provide essential information about both the physiology and the pathology of the human nervous system. The introduction of microdialysis into the clinical sciences has provided a new opportunity to study this environment. Using microdialysis, endogenous substances can be obtained and drugs can be delivered in very close proximity to the receptors and ion channels on neuronal membranes. In this sense, microdialysis can be regarded as a novel technique since it can continuously measure interstitial brain activity in living tissue while causing minimal adverse effects. Although it has been well established as an experimental technique for neurochemistry, the true utility of microdialysis as a clinical tool is still being defined. The potential clinical applications of microdialysis to characterize the human brain extracellular environment in patients with pathologic conditions has grown rapidly. The number of publications in which microdialysis has been performed in clinical studies has been increasing during recent years and this article gives a summary of those reports where microdialysis was applied in the study of human brain disorders.

背景与目标： 对大脑细胞外液的分析可以提供有关人类神经系统生理和病理的基本信息。将微透析技术引入临床科学为研究这种环境提供了新的机会。使用微透析，可以获得内源性物质，并且药物可以非常靠近神经元膜上的受体和离子通道递送。从这个意义上讲，微透析可以被视为一种新颖的技术，因为它可以连续测量活组织中的间质性大脑活动，同时将不良影响降到最低。尽管已经将其很好地确立为神经化学的实验技术，但微透析作为临床工具的真正用途仍在定义中。微透析在表征病理状况患者中表征人脑细胞外环境的潜在临床应用迅速增长。近年来，在临床研究中进行微透析的出版物数量不断增加，本文总结了在人类脑部疾病研究中应用微透析的那些报道。

BACKGROUND & AIMS: PURPOSE:Peptide growth factors are known accelerators of corneal wound healing, probably mediated through increased proliferation of the cells; however, information about their effect on keratocyte motility is lacking. The influence of peptide growth factors on keratocyte migratory activity was investigated, using the following growth factors: platelet derived growth factor (PDGF-BB), epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I) and transforming growth factor-beta-1 (TGF-beta 1).

METHODS:Keratocytes were seeded on gels of type 1 collagen, growth factor added, and the cells left to migrate for 72 hours. Subsequently, the number of keratocytes at the different levels in the collagen gel was evaluated by optically sectioning the gel at 20 microns, intervals, with an inverted phase contrast microscope.

RESULTS:PDGF, EGF and bFGF at 10 ng/ml, all increased the number of keratocytes at the different levels of the gel as compared to a non-stimulated control (p < 0.05 or p < 0.01, students t-test). TGF-beta proved to be a strong inhibitor of keratocyte migration, decreasing the number of keratocytes observed at every level in the gel (p < 0.05 and p < 0.01, students t-test), whereas no effect of IGF-I and aFGF was found. During the 72 hours of migration, no contraction of the collagen gels was observed. Autoradiography of histological sections of the gels showed that during the 72-hour period only TGF-beta and 10% fetal bovine serum induced an increase in keratocyte proliferation.

CONCLUSION:PDGF, EGF and bFGF increase keratocyte migration, independent of proliferation in a collagen gel invasion assay and might promote corneal wound healing, not only by increasing cell proliferation, but also through increased motility.

背景与目标： 目的：肽生长因子是已知的角膜伤口愈合促进剂，可能是通过细胞增殖的增加来介导的。然而，缺乏有关它们对角膜细胞运动性影响的信息。使用以下生长因子研究了肽生长因子对角膜细胞迁移活性的影响：血小板衍生生长因子（PDGF-BB），表皮生长因子（EGF），酸性成纤维细胞生长因子（aFGF），碱性成纤维细胞生长因子（bFGF） ），胰岛素样生长因子I（IGF-I）和转化生长因子β-1（TGF-beta 1）。

方法：将角质形成细胞接种在1型胶原蛋白，添加了生长因子，细胞迁移了72小时。随后，通过倒置相差显微镜以20微米的间隔对凝胶进行光学切片，评估胶原蛋白凝胶中不同水平的角质形成细胞的数量。

结果：PDGF ，与未刺激的对照组相比，在10 ng / ml的EGF和bFGF均增加了凝胶水平不同时的角化细胞数量（p <0.05或p <0.01，学生t检验）。 TGF-β被证明是一种强烈的角膜细胞迁移抑制剂，可减少凝胶中各个水平上观察到的角膜细胞数量（p <0.05和p <0.01，学生t检验），而IGF-I和aFGF没有作用成立。在迁移的72小时内，未观察到胶原凝胶的收缩。凝胶组织学切片的放射自显影显示，在72小时内，只有TGF-β和10％的胎牛血清诱导了角膜细胞增殖的增加。

结论：PDGF，EGF bFGF和bFGF可以增加角膜细胞的迁移，而不受胶原凝胶入侵试验中的增殖的影响，不仅可以通过增加细胞增殖，而且可以通过增加运动性来促进角膜伤口愈合。