动物外周血单个核细胞分离方法探讨

目的 探索用人淋巴细胞分离液分离多种属动物外用血单个核细胞（PBMCs）的可行性及评价其分离效果,建立一种适于野外大规模多种属动物PBMCs的分离方法.方法 采用人淋巴细胞分离液分离来源于新疆的伊犁马、新疆驴、不同品种犬、新疆双峰驼、天山马鹿和来源于重庆的荣昌猪、中国荷斯坦奶牛、简阳大耳黑山羊外周血PBMCs.随机抽样检测分离的动物PBMCs细胞总数、细胞纯度和细胞活率.结果 用人淋巴细胞分离液成功分离上述8种动物PBMCs,每毫升动物外周血分离的PBMCs细胞总数为0.52×106～2.03×106个,纯度为67%～93%,细胞活率为92.5%-98.0%.结论 用人淋巴细胞分离液分离多种动物PB-MCs的方法宜在动物的病毒分子流行学调查中进一步推广.     

Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology. Received 1 August 2002; received after revision 17 September 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation

A challenging task for the adaptive immune system of vertebrates is to identify and eliminate intracellular antigens. Therefore a highly specialized antigen presentation machinery has evolved to display fragments of newly synthesized proteins to effector cells of the immune system at the cell surface. After proteasomal degradation of unwanted proteins or defective ribosome products, resulting peptides are translocated into the endoplasmic reticulum by the transporter associated with antigen processing and loaded onto major histocompatibility complex (MHC) class I molecules. Peptide-MHC I complexes are transported via the secretory pathway to the cell surface where they are then inspected by cytotoxic T lymphocytes, which can trigger an immune response. This review summarizes the current view of the intracellular machinery of antigen processing and of viral immune escape mechanisms to circumvent destruction by the host. Received 4 October 2005; received after revision 19 November 2005; accepted 24 November 2005     

Deoxyribozymes: useful DNA catalysts in vitro and in vivo

Deoxyribozymes (DNA enzymes; DNAzymes) are catalytic DNA sequences. Using the technique of in vitro selection, individual deoxyribozymes have been identified that catalyze RNA cleavage, RNA ligation, and a growing range of other chemical reactions. DNA enzymes have been used in vitro for applications such as biochemical RNA manipulation and analytical assays for metal ions, small organic compounds, oligonucleotides, and proteins. Deoxyribozymes have also been utilized as in vivo therapeutic agents to destroy specific mRNA targets. Although many conceptual and practical challenges remain to be addressed, deoxyribozymes have substantial promise to contribute meaningfully for applications both in vitro and in vivo.     

Interleukin-17 stimulates inducible nitric oxide synthase-dependent toxicity in mouse beta cells

The influence of the proinflammatory cytokine interleukin (IL)-17 on inducible nitric oxide (NO) synthase (iNOS)-mediated NO release was investigated in the mouse insulinoma cell line MIN6 and mouse pancreatic islets. IL-17 markedly augmented iNOS mRNA/protein expression and subsequent NO production induced in MIN6 cells or pancreatic islets by different combinations of interferon-γ, tumor necrosis factor-α, and IL-1β. The induction of iNOS by IL-17 was preceded by phosphorylation of p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK activation completely abolished IL-17-stimulated NO release. IL-17 enhanced the NO-dependent toxicity of proinflammatory cytokines toward MIN6 cells, while IL-17-specific neutralizing antibody partially reduced the NO production and rescued insulinoma cells and pancreatic islets from NO-dependent damage induced by activated T cells. Finally, a significant increase in blood IL-17 levels was observed in a multiple low-dose streptozotocin model of diabetes, suggesting that T cell-derived IL-17 might be involved in NO-dependent damage of beta cells in this disease. Received 14 June 2005; received after revision 17 September 2005; accepted 21 September 2005     

The neurotrophic factors in non-neuronal tissues

Although neurotrophic factors are defined as molecules that maintain neuronal cells, they possess a range of functions outside the nervous system. For example, glial cell line-derived neurotrophic factor is essential for ureteric branching in kidney morphogenesis and for regulating the fate of stem cells during spermatogenesis. Leukemia inhibitory factor, a member of the interleukin-6 (IL-6) ciliary neurotrophic factor family, inhibits differentiation of embryonic stem cells, induces tubulogenesis in the embryonic kidney, and regulates sperm differentiation. Other IL-6 family members are important in cardiac differentiation and they have pleiotropic functions in the hematopoietic and immune systems. Although neurotrophin receptors have been found on a number of non-neuronal tissues, they represent mostly truncated receptor isoforms that are incapable of signal transduction and may have scavenger or dominant negative functions. However, several examples can be presented of essential non-neuronal functions played by neurotrophins in e.g., cardiac, hair follicle, and vascular differentiation, and the maintenance of immune cells.     

The translocation motif of hepatitis B virus improves protein vaccination

Cell-penetrating peptides (CPPs) have been shown to improve antigen loading of dendritic cell vaccines. Here we asked whether fusion of a CPP to a protein improves its immunogenicity when this fusion protein is directly applied as vaccine. We used the cell-penetrating translocation motif (TLM) derived from the hepatitis B virus, because no size limitation of cargos has been observed. Increased immunogenicity was observed when TLM was fused to ovalbumin (TLM-ova). TLM-ova was found to be superior to ova in inducing proliferation and cytotoxicity of ova-specific CD8+ T cells in vitro and in vivo. Using ovalbumin-expressing thymoma cells (EG7-ova), an improved anti-tumor immune response was observed for TLM-ova vaccination versus vaccination with ova. Moreover, TLM-ova vaccination induced a higher titer of anti-ovalbumin IgG2a antibodies compared to ova. These data demonstrate that CPP-protein vaccines can improve cellular as well as humoral immune responses. Received 16 November 2005; received after revision 12 December 2005; accepted 10 January 2006 †These authors contributed equally to this work     

Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment

The fatality of cancer predominantly results from the dissemination of primary tumor cells to distant sites and the subsequent formation of metastases. During tumor progression, some of the primary tumor cells as well as the tumor microenvironment undergo characteristic molecular changes, which are essential for the metastatic dissemination of tumor cells. In this review, we will discuss recent insights into pro-metastatic events occurring in tumor cells themselves and in the tumor stroma. Tumor cell-intrinsic alterations include the loss of cell polarity and alterations in cell-cell and cell-matrix adhesion as well as deregulated receptor kinase signaling, which together support detachment, migration and invasion of tumor cells. On the other hand, the tumor stroma, including endothelial cells, fibroblasts and cells of the immune system, is engaged in an active molecular crosstalk within the tumor microenvironment. Subsequent activation of blood vessel and lymph vessel angiogenesis together with inflammatory and immune-suppressive responses further promotes cancer cell migration and invasion, as well as initiation of the metastatic process. Received 4 July 2005; received after revision 3 November 2005; accepted 14 November 2005     

Aloe emodin decreases the ERK-dependent anticancer activity of cisplatin

The present study describes the ability of an anthraquinone derivative aloe emodin (AE) to reduce the cytotoxic activity of the platinum(II)-based anticancer agent cisplatin toward murine L929 fibrosarcoma and C6 glioma cell lines. The protective effect of AE was demonstrated by MTT and crystal violet assays for cell viability, and involved supression of cisplatin-induced apoptosis and necrosis, as assessed by lactate dehydrogenase release and flow cytometric analysis of DNA fragmentation or phosphatidylserine exposure. Cell-based ELISA and Western blot analysis revealed that AE abolished cisplatin-triggered activation of extracellular signal-regulated kinase (ERK) in tumor cells, while activation of c-Jun N-terminal kinase was not significantly altered. A selective blockade of ERK activation with PD98059 mimicked the protective effect of AE treatment in both tumor cell lines. Moreover, AE failed to protect tumor cells against the ERK-independent toxicity of the Pt(IV)-based complex tetrachloro(O,O-dibutyl-ethylenediamine-N,N′-di-3-propanoate)platinum(IV). Taken together, these data indicate that herbal anthraquinone AE can downregulate the anticancer activity of cisplatin by blocking the activation of ERK in tumor cells.Received 30 January 2005; received after revision 21 March 2005; accepted 31 March 2005     

High frequency of human cytomegalovirus (HCMV)-specific CD8+-T cells detected in a healthy CMV-seropositive donor

Human cytomegalovirus (HCMV) persists after infection but is controlled by cellular immune responses, particularly by CD8⁺ T cells. If infected individuals are immunosuppressed, HCMV can be reactivated. Upon testing the blood of healthy donors with human lymphocyte antigen tetramers, we found one individual with about 50 % of his CD8⁺ T cells being specific for the immunodominant pp65 epitope NLVPMVATV. Over a period of 2 years the high level of HCMV-specific T cells was maintained, and no HCMV DNA could be detected. At one timepoint, however, HCMV-specific DNA was detected, while 65 % of CD8⁺ T cells were specific for HCMV. When virus was detectable, a lower percentage of HCMV-specific CD8⁺ T cells showed interferon γ (IFN-γ) production after peptide stimulation in vitro. These data suggest that HCMV reactivation may also occur in immunocompetent persons, accompanied by the presence of HCMV-specific CD8⁺ T cells which are not producing IFNγ, and therefore potentially anergic or in vivo exhausted. Received 6 March 2002; received after revision 15 April 2002; accepted 17 April 2002     

Receptor communication within the lymphocyte plasma membrane: a role for the thrombospondin family of matricellular proteins

Lymphocytes, the principal cells of the immune system, carry out immune surveillance throughout the body by their unique capacity to constantly reposition themselves between a free-floating vascular state and a tissue state characterized by migration and frequent adhesive interactions with endothelial cells and components of the extracellular matrix. Therefore, mechanisms co-ordinating adhesion and migration with signals delivered through antigen recognition probably play a pivotal role for the regulation of lymphocyte behaviour and function. Endogenous thrombospondin-1 (TSP-1) seems to be the hub in such a mechanism for autocrine regulation of T cell adhesion and migration. TSP-1 functions as a mediator of cis interaction of vital receptors within the T lymphocyte plasma membrane, including integrins, low density lipoprotein receptor-related protein, calreticulin and integrin-associated protein. Received 1 June 2006; received after revision 28 June 2006; accepted 11 October 2006     

Surface exposure of phosphatidylserine in pathological cells

The asymmetric phospholipid distribution in plasma membranes is normally maintained by energy-dependent lipid transporters that translocate different phospholipids from one monolayer to the other against their respective concentration gradients. When cells are activated, or enter apoptosis, lipid asymmetry can be perturbed by other lipid transporters (scramblases) that shuttle phospholipids non-specifically between the two monolayers. This exposes phosphatidylserine (PS) at the cells outer surface. Since PS promotes blood coagulation, defective scramblase activity upon platelet stimulation causes a bleeding disorder (Scott syndrome). PS exposure also plays a pivotal role in the recognition and removal of apoptotic cells via a PS-recognizing receptor on phagocytic cells. Furthermore, expression of PS at the cell surface can occur in a wide variety of disorders. This review aims at highlighting how PS expression in different cells may complicate a variety of pathological conditions, including those that promote thromboembolic complications or produce aberrations in apoptotic cell removal.Received 26 November 2004; received after revision 3 January 2005; accepted 10 January 2005 Available online 09 March 2005     

Identification of factor XIII-A as a marker of alternative macrophage activation

Factor XIII subunit A of blood coagulation (FXIII-A) is known to be synthesized but not secreted by the monocyte/macrophage cell line. On the basis of its intracellular localization and substrate profile, FXIII-A is thought to be involved in certain intracellular processes. Our present study was designed to monitor the changes in FXIII-A gene expression and protein production in long-term culture of human monocytes during their differentiation into macrophages in the presence of activating agents (interleukin-4, interferon-γ, Mycobacterium bovis BCG) inducing classical and alternative activation pathways. By using quantitative RT-PCR and fluorescent image analysis at the single-cell level we demonstrated that the expression of FXIII-A both at the mRNA as well as at the protein level is inversely regulated during the two activation programmes. Here we conclude that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophages indicates their classically activated state.Received 2 June 2005; received after revision 12 July 2005; accepted 22 July 2005     

The innate immunity of the central nervous system in chronic pain: The role of Toll-like receptors

Toll-like receptors (TLRs) are a family of pattern recognition receptors that mediate innate immune responses to stimuli from pathogens or endogenous signals. Under various pathological conditions, the central nervous system (CNS) mounts a well-organized innate immune response, in which glial cells, in particular microglia, are activated. Further, the innate immune system has emerged as a promising target for therapeutic control of development and persistence of chronic pain. Especially, microglial cells respond to peripheral and central infection, injury, and other stressor signals arriving at the CNS and initiate a CNS immune activation that might contribute to chronic pain facilitation. In the orchestration of this limited immune reaction, TLRs on microglia appear to be most relevant in triggering and tailoring microglial activation, which might be a driving force of chronic pain. New therapeutic approaches targeting the CNS innate immune system may achieve the essential pharmacological control of chronic pain. Received 21 November 2006; received after revision 8 January 2007; accepted 7 February 2007     

Pluripotent stem cells reveal the developmental biology of human megakaryocytes and provide a source of platelets for clinical application

Human pluripotent stem cells [PSCs; including human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] can infinitely proliferate in vitro and are easily accessible for gene manipulation. Megakaryocytes (MKs) and platelets can be created from human ESCs and iPSCs in vitro and represent a potential source of blood cells for transfusion and a promising tool for studying the human thrombopoiesis. Moreover, disease-specific iPSCs are a powerful tool for elucidating the pathogenesis of hematological diseases and for drug screening. In that context, we and other groups have developed in vitro MK and platelet differentiation systems from human pluripotent stem cells (PSCs). Combining this co-culture system with a drug-inducible gene expression system enabled us to clarify the novel role played by c-MYC during human thrombopoiesis. In the next decade, technical advances (e.g., high-throughput genomic sequencing) will likely enable the identification of numerous gene mutations associated with abnormal thrombopoiesis. Combined with such technology, an in vitro system for differentiating human PSCs into MKs and platelets could provide a novel platform for studying human gene function associated with thrombopoiesis.     

Decision-making at the surface of the intact or barrier disrupted skin: potential applications for vaccination or therapy

The skin is a highly accessible organ and constitutes an active immunological site. Both these properties make this surface an attractive route for what promises to be a cost-effective, simple, practical and needle-free delivery of vaccines and immunomodulators. Less obvious is the fact that the state of the skin barrier can influence quantitative and qualitative aspects of antigen-specific immune responses. The everyday decision-making at the skin epithelium concerns the choice between the induction of an immune response and the establishment of a state of non-responsiveness (tolerance). This decision is influenced by various factors such as the dose, the route (intact vs barrier-disrupted skin), the cytokine microenvironment and the nature of the antigenic stimulus. By increasing our understanding of how immune responses are regulated in the epidermis we can envisage the development of immunisation protocols aimed at eliciting a protective immune response or inducing tolerance, with direct applications to preventive or therapeutic vaccination, respectively.Received 29 November 2004; received after revision 2 February 2005; accepted 22 February 2005     

Recombinant anti-P protein autoantibodies isolated from a human autoimmune library: reactivity,specificity and epitope recognition

The ribosomal P proteins are specific and important autoantigens in patients affected by systemic lupus erythematosus. In this study, we describe for the first time the selection and characterization of recombinant human monoclonal anti-P protein (auto)-antibody fragments from an autoimmune patient-derived phage display antibody library. The selected recombinant anti-P antibodies specifically recognize the P proteins in immunofluorescence assays on HEp-2 cells and in immunoblotting assays, and they immunoprecipitate the P proteins under native conditions. Using both anti-P-positive patient sera and the selected recombinant anti-P antibodies, the immunodominant epitope was determined and shown to be located at the C-terminal end of the P proteins (amino acids 111-115). Inhibition of in vitro protein translation demonstrated that interaction of the monoclonal patient-derived anti-P antibodies with their native epitope functionally inhibits the activity of the P proteins on the ribosome, confirming the notion that patient autoantibodies are often directed to the functional centre of their autoantigenic target.