Chronic lymphocytic leukemia (CLL) cells genetically modified to express B7-1, ICAM-1, and LFA-3 confer APC capacity to T cells from CLL patients

In chronic lymphocytic leukemia (CLL), malignant B cells and nonmalignant T cells exhibit dysfunction. We previously demonstrated that infection of CLL cells with modified vaccinia Ankara (MVA) expressing the costimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM) increased expression of these costimulatory molecules on the surface of CLL cells and thus augmented their antigen-presenting capability. Here, we evaluate the effect of MVA-TRICOM-modified CLL cells on T cells. Following incubation with irradiated MVA-TRICOM-modified CLL cells, allogeneic and autologous CD4⁺ and CD8⁺ T cells expressed significantly higher levels of B7-1, ICAM-1, and LFA-3. We show that this increase was the result of physical acquisition from the antigen-presenting cells (APCs), and that purified T cells that acquired costimulatory molecules from MVA-TRICOM-modified CLL cells were able to stimulate the proliferation of untreated T cells. These results demonstrate for the first time that T cells from CLL patients can acquire multiple costimulatory molecules from autologous CLL cells and can then act as APCs themselves. Given the immunodeficiencies characteristic of CLL, enhancing the antigen-presenting function of CLL cells and T cells simultaneously could be a distinct advantage in the effort to elicit antitumor immune responses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rhein,a Natural Anthraquinone Derivative,Attenuates the Activation of Pancreatic Stellate Cells and Ameliorates Pancreatic Fibrosis in Mice with Experimental Chronic Pancreatitis

Pancreatic fibrosis, a prominent histopathological feature of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma, is essentially a dynamic process that leads to irreversible scarring of parenchymal tissues of the pancreas. Though the exact mechanisms of its initiation and development are poorly understood, recent studies suggested that the activation of pancreatic stellate cells (PSCs) plays a critical role in eliciting such active course of fibrogenesis. Anthraquinone compounds possess anti-inflammatory bioactivities whereas its natural derivative rhein has been shown to effectively reduce tissue edema and free-radical production in rat models of inflammatory conditions. Apart from its anti-inflammatory properties, rhein actually exerts strong anti-fibrotic effects in our current in-vivo and in-vitro experiments. In the mouse model of cerulein-induced CP, prolonged administration of rhein at 50 mg/kg/day significantly decreased immunoreactivities of the principal fibrotic activators alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta (TGF-β) on pancreatic sections implicating the activation of PSCs, which is the central tread to fibrogenesis, was attenuated. Consequently, the overwhelmed deposition of extracellular matrix proteins fibronectin 1 (FN1) and type I collagen (COL I-α1) in exocrine parenchyma was found accordingly reduced. In addition, the expression levels of sonic hedgehog (SHH), which plays important roles in molecular modulation of various fibrotic processes, and its immediate effector GLI1 in pancreatic tissues were positively correlated to the degree of cerulein-induced fibrosis. Such up-regulation of SHH signaling was restrained in rhein-treated CP mice. In cultured PSCs, we demonstrated that the expression levels of TGF-β-stimulated fibrogenic markers including α-SMA, FN1 and COL I-α1 as well as SHH were all notably suppressed by the application of rhein at 10 μM. The present study firstly reported that rhein attenuates PSC activation and suppresses SHH/GLI1 signaling in pancreatic fibrosis. With strong anti-fibrotic effects provided, rhein can be a potential remedy for fibrotic and/or PSC-related pathologies in the pancreas.     

体外诱导成年大鼠骨髓间充质干细胞分化为 5-羟色胺敏感性神经元

体外诱导成年大鼠骨髓间充质干细胞分化为具有神经元表型和部分功能的细胞。在对Woodbury化学诱导法作改良的基础上,加用全反式视黄酸对骨髓间充质干细胞作预诱导。诱导3h后,细胞开始表现神经元的形态特征,细胞折光性增强,形成收缩的双极或多极胞体和细长突起。细胞可以维持神经元样存活72h以上。诱导5h后,对免疫染色的细胞用DAPI进行复染,(92.4±6.9)%的细胞表达神经元特异性烯醇化酶。诱导24h后,(93.9±5.2)%的细胞表达成熟神经元的标志物神经丝M H。在给予5-羟色胺刺激时可以产生与神经元相似的胞内钙离子峰,且免疫组化证实5-羟色胺1A受体在干细胞上表达微弱,但在分化后的神经元中表达较强。实验不仅从形态、细胞标志物而且从功能上证实诱导后的细胞为5-羟色胺敏感性神经元。     

Ventilation inhomogeneity in oleic acid-induced pulmonary edema

Tsang, John Y. C., Michael J. Emery, and Michael P. Hlastala. Ventilation inhomogeneity in oleic acid-inducedpulmonary edema. J. Appl. Physiol.82(4): 1040-1045, 1997.Oleic acid causes permeability pulmonaryedema in the lung, resulting in impairment of gas-exchange andventilation-perfusion heterogeneity and mismatch. Previous studies haveshown that by using the multiple-breath helium washout (MBHW)technique, ventilation inhomogeneity (VI) can be quantitativelypartitioned into two components, i.e., convective-dependent inhomogeneity (cdi) and diffusive-convective-dependent inhomogeneity (dcdi). Changes in VI, as represented by the normalized slope of thephase III alveolar plateau, were studied for 120 min in fiveanesthetized mongrel dogs that were ventilated under paralysis by aconstant-flow linear motor ventilator. These animals received oleicacid (0.1 mg/kg) infusion into the right atrium att = 0. MBHWs were done induplicate for 18 breaths every 40 min afterward. Three other dogs thatreceived only normal saline served as controls. The data show that,after oleic acid infusion, dcdi, which represents VI in peripheralairways, is responsible for the increasing total VI as lung wateraccumulates progressively over time. The cdi, which represents VIbetween larger conductive airways, remains relatively constantthroughout. This observation can be explained by increases in theheterogeneity of tissue compliance in the periphery, distal airwayclosure, or by decreases in ventilation through collateral channels.

     

Id1 interacts and stabilizes the Epstein-Barr virus latent membrane protein 1 (LMP1) in nasopharyngeal epithelial cells

The EBV-encoded latent membrane protein 1 (LMP1) functions as a constitutive active form of tumor necrosis factor receptor (TNFR) and activates multiple downstream signaling pathways similar to CD40 signaling in a ligand-independent manner. LMP1 expression in EBV-infected cells has been postulated to play an important role in pathogenesis of nasopharyngeal carcinoma. However, variable levels of LMP1 expression were detected in nasopharyngeal carcinoma. At present, the regulation of LMP1 levels in nasopharyngeal carcinoma is poorly understood. Here we show that LMP1 mRNAs are transcribed in an EBV-positive nasopharyngeal carcinoma (NPC) cell line (C666-1) and other EBV-negative nasopharyngeal carcinoma cells stably re-infected with EBV. The protein levels of LMP1 could readily be detected after incubation with proteasome inhibitor, MG132 suggesting that LMP1 protein is rapidly degraded via proteasome-mediated proteolysis. Interestingly, we observed that Id1 overexpression could stabilize LMP1 protein in EBV-infected cells. In contrary, Id1 knockdown significantly reduced LMP1 levels in cells. Co-immunoprecipitation studies revealed that Id1 interacts with LMP1 by binding to the CTAR1 domain of LMP1. N-terminal region of Id1 is required for the interaction with LMP1. Furthermore, binding of Id1 to LMP1 suppressed polyubiquitination of LMP1 and may be involved in stabilization of LMP1 in EBV-infected nasopharyngeal epithelial cells.     

Role of voltage‐gated potassium channels in the fate determination of embryonic stem cells

Embryonic stem cells (ESCs) possess two unique characteristics: self‐renewal and pluripotency. In this study, roles of voltage‐gated potassium channels (K_v) in maintaining mouse (m) ESC characteristics were investigated. Tetraethylammonium (TEA⁺), a K_v blocker, attenuated cell proliferation in a concentration‐dependent manner. Possible reasons for this attenuation, including cytotoxicity, cell cycle arrest and differentiation, were examined. Blocking K_v did not change the viability of mESCs. Interestingly, K_v inhibition increased the proportion of cells in G₀/G₁ phase and decreased that in S phase. This change in cell cycle distribution can be attributed to cell cycle arrest or differentiation. Loss of pluripotency as determined at both molecular and functional levels was detected in mESCs with K_v blockade, indicating that K_v inhibition in undifferentiated mESCs directs cells to differentiate instead of to self‐renew and progress through the cell cycle. Membrane potential measurement revealed that K_v blockade led to depolarization, consistent with the role of K_v as the key determinant of membrane potential. The present results suggest that membrane potential changes may act as a “switch” for ESCs to decide whether to proliferate or to differentiate: hyperpolarization at G₁ phase would favor ESCs to enter S phase while depolarization would favor ESCs to differentiate. Consistent with this notion, S‐phase‐synchronized mESCs were found to be more hyperpolarized than G₀/G₁‐phase‐synchronized mESCs. Moreover, when mESCs differentiated, the differentiation derivatives depolarized at the initial stage of differentiation. This investigation is the first study to provide evidence that K_v and membrane potential affect the fate determination of ESCs. J. Cell. Physiol. 224:165–177, 2010 © 2010 Wiley‐Liss, Inc.     

Calibration of prestained protein molecular weight standards for use in the "Western" or enzyme-linked immunoelectrotransfer blot techniques

Prestained protein molecular weight standards allow easy, direct visual location of electrophoretically transblotted lanes on nitrocellulose. They also provide a simple and accurate means for calibrating the molecular weights of resolved bands. Commercial prestained protein molecular weight standards, however, appear to have significantly different molecular weights from the original unstained proteins. We describe a calibration of these prestained molecular weight standards.     

Immunoassay for viable Cryptosporidium parvum oocysts in turbid environmental water samples

Cryptosporidium parvum oocysts in drinking water have been implicated in outbreaks of diarrheal disease. Current methods for monitoring environmental exposures to C. parvum only account for total number of oocysts without regard for the viability of the parasite. Measurement of oocyst viability, as indicated by an oocyst's ability to excyst, is useful because over time oocysts lose the ability to excyst and become noninfective. Thus, correlating the number of viable oocysts in drinking water with incidence and risk for disease should be more reliable than using the total number of oocysts. We have developed a quantitative assay capable of detecting low numbers of excystable, sporozoite-releasing C. parvum oocysts in turbid water samples. Monoclonal (CP7) and polyclonal antibodies have been developed against a sporozoite antigen released only during excystation or when the oocyst is mechanically disrupted. CP7 is specific for C. parvum and does not react with C. baileyi, C. muris, C. serpentis, Giardia spp., Eimeria spp., or E. nieschulzi. In this assay, oocysts in the test sample are first excysted and then centrifuged. The soluble sporozoite antigen is captured by CP7 attached to a magnetic bead. The captured antigen is then detected by ruthenium-labeled polyclonal antibodies via electrochemiluminescence. The CP7 viability assay can detect as few as 50 viable oocysts in a 1-ml assay sample with a turbidity as high as 200 Nephelometric turbidity units. This sensitive, turbidity-tolerant assay for oocyst viability may permit a better assessment of the disease risk associated with the presence of environmental oocysts.