T cell vaccination as an immunotherapy for autoimmune diseases

Immunization with inactivated autoreactive T cells(T cell vaccination) selected from individual's own T cellrepertoire provides a unique in vivo setting for testing immune regulation that is known to involve interactionsof a variety of related surface molecules(1).It induces regulatory immune responses that closely resemble thein vivo situation where the immune system is challenged by clonal activation and expansion of given T cellpopulations in various autoimmune diseases.T cell vaccination provides a powerful means of eliciting naturalreactions of the immune system in response to clonal expansion of T cells,which can be used as a therapeuticapproach to suppress or eliminate specific pathogenic autoreactive T cells in autoimmune conditions.Clinicaltrials using T cell vaccination to deplete autoreactive T cells in human autoimmune conditions have begun toreveal the pathologic relevance of various autoimmune T cell populations in the disease processes,providing aunique opportunity to test the autoimmune theories in a clinical setting.Cellular & Molecular Immunology.2004;1(5):321-327.     

Intercellular trogocytosis plays an important role in modulation of immune responses

Intercellular communication is an important means of molecular information transfer through exchange of membrane proteins from cells to cells. Advent of the latest analytical and imaging tools has allowed us to enhance our understanding of the cellular communication through the intercellular exchange of intact membrane patches, also called trogocytosis, which is a ubiquitous phenomenon. Immune responses against pathogens or any foreign antigens require fine immune regulation, where cellular communications are mediated by either soluble or cell surface molecules. It has been demonstrated that the membrane molecule transfer between immune cells such as dendritic and T cells can be derived through internalization/recycling pathway, dissociation-associated pathway, uptake of exosomes and membrane nanotube formations. Recent evidence implicates the trogocytosis as an important mechanism of the immune system to modulate immune responses. Exchange of membrane molecules/ antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. In this review, we discuss the possible mechanisms of trogocytosis and its physiological relevance to immune system, with special reference to T cells and the stimulatory or suppressive immune responses derived from T cells with acquired dendritic cell membrane molecules. Cellular ＆ Molecular Immunology. 2008;5（4）：261-269.     

Applying Surface Fitting in the Medical Thermal Tomography Technology

Based on the thermal tomography technology, this thesis tries to calculate quantity and distribution of heat source in vivo from body surface temperature. A superposition temperature image of a number of point heat sources is surface fitted to get the Q of heat sources (information of cancer cells) quantitatively. The result can reflect the disease area information because cancer cell’s Q value is much higher than that of normal cell. This application is a new try in the diagnosis of breast cancer, which has an important value on the early detection and diagnosis of disease source.     

Compressive Stress Enhances Invasive Phenotype of Cancer Cells via Piezo1 Activation

In this study,we hypothesized that Piezo 1 channels mediate the compression-enhanced invasive phenotype of cancer cells via a caveolae-dependent mechanism.To test this hypothesis,we examined in vitro cultured human breast cancer cells for their ability to invade and degrade extracellular matrix in the presence or absence of compressive stress,together with corresponding changes in Piezo1 as well as cytoskeletal remodeling and calcium signaling.Here we show that compressive stress enhanced invasion,matrix degradation,and invadopodia formation of breast cancer cells.We further identified Piezo1 as the putative mechanosensitive cellular component that transmits compression to induce calcium influx,which in turn triggers several downstream pathways.Interestingly,for the first time we observed inv-adopodia with matrix degradation ability on the apical side of the cells, similar to those commonly observed at the cell s ventral side.Furthermore,we demonstrate that Piezo1 and caveolae were both involved in mediating the compressive stress-induced cancer cell invasive phenotype as Piezo 1 and caveolae were often colocalized,and reduction of Cav-1 expression or disruption of caveolae with methyl-β-cyclodextrin led to not only reduced Piezo1 expression but also attenuation of the invasive phenotypes promoted by compressive stress.Taken together,we first observed that in breast cancer cells,simulating uncontrolled growth-induced compressive stress enhanced cancer cell invasion,matrix degradation,and invadopodia and stress fiber formation.Our study also confirmed that Piezo1 channels are highly expressed in breast cancer cells compared to normal breast cells,and is consistent with the data that compressive stress regulates cell migration of breast cancer cells but not normal breast cells.Additionally,we identified that Piezol mediated these processes and the invasive phenotypes also depended on the integrity of caveolae.These findings provide the first demonstration that compressive stress enhances matrix degradation by breast cancer cells and Piezo1 is an essential mechanosensor and transducer for such stress in breast cancer.Additionally,our data supports the model where caveolae might be the"mechanical force foci"which concentrates Piezol to facilitate force sensing and transduction in mammalian cells.Our work may have relevance to human tumors in vivo.As solid tumor experiences high compressive stress due to uncontrolled proliferation and confinement by the stiff extracellular matrix environment,this microenvironment facilitates compression-enhanced cell invasion.The identification of Piezo1's crucial role in this process provides the first demonstration of the dependence of Piezo1 channels on the response of breast cancer cells to physiological compressive stress.The functional dependence of Piezo1 on caveolae further highlights the importance of membrane organization and composition on forcegated ion channels.Both of these findings underscore the cardinal role that Piezo1 channels play in regulating cell invasion and may inspire further development targeting Piezol as a potential cancer therapeutic target.     

Selective Function of PKC-θ in T cells

T cell activation is a critical process in initiating adaptive immune response since only through this process the naive antigen specific T cells differentiate into armed effector T cells that mediate the actual immune response. During T cell activation, naive T cells undergo clonal expansion and acquire the capability to kill target cells infected with pathogens or produce cytokines essential for regulating immune response. Inappropriate activation or inactivation of T cells leads to autoimmunity or severe immunodeficiencies. PKC-θ is selectively expressed in T cells and required for mediating T cell activation process. Mice deficient in PKC-θ exhibit defects in T cell activation, survival and activation-induced cell death. PKC-θ selectively translocates to immunological synapse and mediates the signals required for activation of NF-κB, AP1 and NFAT that are essential for T cell activation. Furthermore, PKC-θ^-│- mice displayed multiple defects in the development of T cell-mediated immune responses in vivo. PKC-θ is thus a critical molecule that regulates T cell function at multiple stages in T cell-mediated immune responses in vivo.     

Intratumoral Expression of MIP-1β Induces Antitumor Responses in a Pre-Established Tumor Model through Chemoattracting T Cells and NK Cells

Direct intratumoral introduction of therapeutic or regulatory genes is a developing technology with potential application for cancer gene therapy. Macrophage inflammatory protein-1 beta (MIP-1β) is a chemokine which can chemoattract immune cells such as T cells. In the present study, murine colorectal adenocarcinoma CT26 cells were transfected with a recombinant adenovirus (AdhMIP-1β) carrying the human MIP-1β gene. 24h post-transfection, hMIP-1β levels reached approximately 980 pg/ml in supernatants of 10^6 hMIP-1β-transfected CT26 cells. Moreover, the supernatants exhibited chemotactic activity for CD8^ T cells, CD4^ T cells, NK cells and immature DCs. Intratumoral injection of AdhMIP-1β significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice. Intratumoral hMIP-1β gene transfer also induced powerful tumor-specific CTL responses in vivo. The therapeutic effects of hMIP-1β gene therapy were greatly reduced following in vivo depletion of both CD4^ and CD8~ T cells, but were unaffected by depletion of single T cell subsets. Immune cell depletion experiments also revealed that NK cells played an important role in hMIP-1β-induced antitumor responses. These results suggest that intratumoral expression of hMIP-1β has the potential effect to induce host antitumor immunity and may prove to be a useful form of cancer gene therapy.     

Expression, purification and identification of recombinant mouse interleukin 21 protein in E. coli

Interleukin 21 （IL-21） is a novel type I cytokine that is significantly homologous to IL-2, IL-4 and IL-15. Its receptor complex contains γc chain which is also a component of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, so there may be overlapping or relevancies in their biological functions. IL-21 is capable of co-stimulating mature T cells, B cells, NK cells, and of stimulating CD16 expression on the surface of NK cells to induce ADCC in innate immune response. It can also strengthen the anti-tumor effect of the cellular immunity, especially v/a enhancing the activities of NK and antigen specific CTL cells. Thus, IL-21 is a potential useful therapeutic molecule for immunotherapy of malignancies, by eliciting innate and adaptive anti-tumor immune responses in tumor-bearing hosts. In order to study the biological functions of IL-21, we constructed a mIL-21 prokaryotic expression plasmid and expressed the recombinant mIL-21 protein in E. coli in present study. The recombinant plasmid pET28a/mIL-21 with a carboxyl terminal His-tag was subcloned from the pcDNA3.1/mIL-21 and expressed in E. coli. The induced protein was detected by SDS-PAGE, and identified by Western-blot assay with anti-mIL-21 antibody. The recombinant protein was purified v/a Ni^＋ affinity chromatography, and renatured with GSH/GSSG system. Our mouse T cell proliferation experiment showed that the recombinant mIL-21 protein could enhance the mouse T cell proliferation either by itself alone or in the presence of Con A.     

Functional identification of the stable transfection C5aR cell line Molt-4

The complement C5 anaphylatoxin receptor is a member of the seven transmembrane-spanning G protein-coupled receptor superfamily that signals through Gcxi and Gtz16. C5aR is mostly expressed on neutrophils, macrophages and endothelial cells. C5a and C5aR interaction plays an important role in numerous biological effects such as in vivo cytokine storm which results in inflammatory damage. Considering the limitation of collection of human peripheral blood neutrophils and their short half life, the stably transfected cell line for studying the biological effects of C5aR is needed. In this study, we transfected C5aR gene into Molt-4 cell line and examined the function of ectopic C5aR. Our results showed stable expression of the C5aR in Molt-4 cell line and their interaction with human C5a induced ERKI/2 phosphorylation, Ca＋＋ influx. This stable transfected cell line may provide a useful tool for studying signal pathways related to C5a and C5aR interplay and antibody development specific for C5aR. Cellular ＆ Molecular Immunology.     

Research of the Effect of the Shear Stress on Endothelial Cells

1 IntroductionCellular mechanism is one of the foundations of regenerating medicine and tissue engineering, which is also an advanced subject in cell mechanism in recent years~([1]). The form and function of a cell, and the growing, reproducing and death, even canceration are related to the characteristics of cell mechanism. While the research of the shear stress on endothelial cells is an important field in cell mechanism. The main bio-functions of endothelial cells are as follows: anti-cruor…     

高糖诱导内皮细胞氧化型低密度脂蛋白受体表达

目的:研究高浓度葡萄糖对内皮细胞凝集素样氧化型低密度脂蛋白受体(LOX-1) mRNA和蛋白表达的影响,探讨糖尿病易合并血管病变的可能机制。方法:用倒置相差显微镜观察细胞生长情况, RT-PCR检测不同浓度D-葡萄糖(7.5、15、30 mmol/L)作用48 h及15 mmol/L浓度作用不同时间0、12、24、48、72、96 h内皮细胞LOX-1 mRNA表达;免疫组化观察葡萄糖15 mmol/L作用48 h LOX-1蛋白表达。结果:高糖组细胞粗糙,轮廓增强,细胞内有许多发亮的颗粒;不同浓度高糖均可诱导LOX-1 mRNA表达,30 mmol/L作用最强(P＜0.01);同一浓度下作用不同时间,LOX-1 mRNA表达随时间延长而增加,48 h达高峰(P＜0.01),之后下降;免疫组化显示葡萄糖组细胞棕色着色颗粒位于胞膜和胞浆,与对照组差异明显(P＜0.01)。结论:高浓度D-葡萄糖呈浓度依赖性和时间依赖性地诱导内皮细胞LOX-1 mRNA的表达;也可诱导LOX-1蛋白表达,表达的蛋白存在于细胞浆中和细胞膜上。葡萄糖的这种作用可能是糖尿病容易合并血管病变的原因之一。     

Herpes simplex virus 1 induced LOX-1 expression in an endothelial cell line, ECV 304

Infections, such as by Chlamydophilia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori, have been shown to be involved in atherogenesis. Herpes simplex virus I (HSV-1) could infect vascular endothelial cells, and it has been shown that, when endothelial cells were activated with oxidized LDL (oxLDL), a number of cellular events are occurred, leading to endothelial cell dysfunction. Since LOX-1 is a major receptor for oxLDL on endothelial cells and its expression was increased in atherosclerosis, we investigated whether HSV1 infection can lead to the increase expression of LOX-1 in endothelial cells. LOX-1 mRNA expression determined by RT-PCR and LOX-1 promoter activity measured by luciferase assay were increased in endothelial cells following HSV-1 infection. This suggests that one of the mechanisms by which HSV-1 is involved in atherogenesis maybe the enhanced uptake of oxLDL via the increased expression of LOX-1 in endothelial cells.     

The expression and down stream effect of lectin like-oxidized low density lipoprotein 1 (LOX-1) in hyperglycemic state

The lesions of atherosclerosis represent a series of highly specific cellular and molecular responses. Low density lipoprotein (LDL), which may be modified by oxidation, glycation, aggregation, association with proteoglycans, or incorporation into immune complexes, is a major cause of injury to the endothelium and vascular smooth muscle cells (VSMC).The major major cell types involved in atherogenesis, macrophages and VSMC, are activated by pro-inflammatory stimuli including modified LDL. Modified LDL induces inflammatory responses in macrophages, migration and proliferation of SMC, and triggers foam cell formation. Scavenger receptors, including LOX-1, play a key role in foam cell formation by mediating the uptake of modified LDL. LOX-1 expression is detected in endothelial cells of early atherosclerosis lesions of human carotid arteries. Advanced lesions showed LOX-1 expression not only in endothelial cells but also in macrophages and more frequently in VSMC, and may be involved in foam cell transformation in macrophages and VSMC. The metabolic abnormalities that characterize diabetes, particularly hyperglycemia, free fatty acids, and insulin resistance, provoke molecular mechanisms that alter the function and structure of blood vessels. These include increased oxidative stress, intracellular signal transduction disturbances, and activation of the receptor for advanced glycation end products (R-AGE). Data showed that LOX-1 expression is enhanced by proatherogenic factors relevant to human diabetes, including high glucose, oxLDL, advance glycation end products, and C-reactive protein. LOX-1 expression increased also through oxygen species (ROS), endothelin-1 (ET-1), tumor necrosis factor-alpha (TNF-alpha), shear stress, activation of protein kinase-C (PKC), angiotensin-II (ANG-II), and through inflammatory pathways.     

APOPTOSIS IN HUMAN MONOCYTE-MACROPHAGES EXPOSED TO OXIDIZED LOW DENSITY LIPOPROTEIN

This study has demonstrated the toxicity to human monocyte-macrophages of low-density lipoprotein (LDL) which had been artificially oxidized using copper sulphate. The assays of cell damage used were tritiated adenine release, neutral red staining, lactate dehydrogenase leakage, and MTT dye reduction. Toxicity was concentration- and time-dependent. Exposure to native LDL under the same conditions did not result in toxicity. Transmission electron microscopy of cells exposed to oxidized LDL showed characteristic changes of apoptosis, including chromatin condensation and a decrease in cell volume. There was extensive loss of cell surface protrusions and evidence of the phagocytosis of apoptotic cells by neighbouring monocyte-macrophages. Apoptotic features preceded the increased membrane permeability revealed by the release of radioactivity from cells preloaded with tritiated adenine and by lactate dehydrogenase leakage. DNA fragmentation was indicated by nick end-labelling using the terminal transferase enzyme (TUNEL). The number of TUNEL-positive cells was markedly greater in cells exposed to oxidized LDL, compared with those incubated as no-additions controls. Inhibition of de novo protein synthesis with cycloheximide and of Ca²⁺/Mg²⁺-activated endonuclease activity with aurintricarboxylic acid or zinc ion did not inhibit the toxicity produced by oxidized LDL.     

Oxidative stress and lectin-like ox-LDL-receptor LOX-1 in atherogenesis and tumorigenesis

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been identified as a major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, monocytes, platelets, cardiomyocytes, and vascular smooth muscle cells. Its expression is minimal under physiological conditions but can be induced under pathological conditions. The upregulation of LOX-1 by ox-LDL appears to be important for physiologic processes, such as endothelial cell proliferation, apoptosis, and endothelium remodeling. Pathophysiologic effects of ox-LDL in atherogenesis have also been firmly established, including endothelial cell dysfunction, smooth muscle cell growth and migration, monocyte transformation into macrophages, and finally platelet aggregation-seen in atherogenesis. Recent studies show a positive correlation between increased serum ox-LDL levels and an increased risk of colon, breast, and ovarian cancer. As in atherosclerosis, ox-LDL and its receptor LOX-1 activate the inflammatory pathway through nuclear factor-kappa B, leading to cell transformation. LOX-1 is important for maintaining the transformed state in developmentally diverse cancer cell lines and for tumor growth, suggesting a molecular connection between atherogenesis and tumorigenesis.     

LOX-1 deletion improves neutrophil responses, enhances bacterial clearance, and reduces lung injury in a murine polymicrobial sepsis model

Inflammatory tissue injury and immunosuppression are the major causes of death in sepsis. Novel therapeutic targets that can prevent excessive inflammation and improve immune responses during sepsis could be critical for treatment of this devastating disease. LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a membrane protein expressed in endothelial cells, has been known to mediate vascular inflammation. In the present study, we demonstrated that LOX-1 deletion markedly improved the survival rate in a murine model of polymicrobial sepsis. Wild-type (LOX-1(+/+)) and LOX-1 knockout (LOX-1(-/-)) mice were subjected to cecal ligation and puncture (CLP) to induce sepsis. LOX-1 deletion significantly reduced systemic inflammation and inflammatory lung injury during sepsis, together with decreased production of proinflammatory cytokines and reduced lung edema formation. Furthermore, LOX-1 deletion improved host immune responses after the induction of sepsis, as indicated by enhanced bacterial clearance. Interestingly, we were able to demonstrate that LOX-1 is expressed in neutrophils. LOX-1 deletion prevented neutrophil overreaction and increased neutrophil recruitment to infection sites after sepsis induction, contributing at least partly to increased immune responses in LOX-1 knockout mice. Our study results indicate that LOX-1 is an important mediator of inflammation and neutrophil dysfunction in sepsis.     

血管紧张素II对巨噬细胞(THP-1细胞)凝集素样氧化低密度脂蛋白受体表达的影响

目的:研究AngII对人单核/巨噬细胞(THP-1细胞)凝集素样氧化低密度脂蛋白受体(LOX-1)蛋白表达和基因转录的影响,从细胞蛋白、分子水平探讨AngII和巨噬细胞LOX-1相互之间的关系,以进一步了解两者在动脉粥样硬化中的地位。方法:将不同浓度AngII(1×10^-9-1×10^-5mol/L)与经0.1μmol/L佛波酯(PMA)诱导分化后的THP-1细胞共孵育24h,以及将1×10^-6mol/L浓度的AngII与诱导分化后的THP-1细胞作用不同时间0、3、6、12、24、48h后,用细胞酶联免疫法和半定量RT-PCR分别检测LOX-1蛋白和mRNA表达的情况。结果:未经诱导的THP-1细胞不表达LOX-1mRNA;而经PMA诱导后,THP-1细胞停止增殖,由单核细胞分化成为巨噬细胞,并表达LOX-1mRNA。不同浓度的AngII作用诱导分化后的THP-1细胞24h,细胞LOX-1蛋白和mRNA的表达呈浓度依赖性显著增加。同一浓度的AngII作用THP-1细胞,可呈时间依赖性诱导LOX-1蛋白和mRNA表达,其趋势是3h左右开始增加,24h左右至最高峰,之后逐渐减低。结论:经PMA诱导分化后的THP-1细胞表达LOX-1;AngII能明显增强分化后的THP-1细胞表达LOX-1蛋白和mRNA,并呈浓度和时间依赖性。AngII这种作用可能是促进动脉粥样硬化发生、发展的机制之一。     

Variation in the human lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) gene is associated with plasma soluble LOX-1 levels

The lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) expressed on vascular cells plays a major role in atherogenesis by internalizing and degrading oxidized low-density lipoprotein. LOX-1 can be cleaved from the cell surface and released as soluble LOX-1 (sLOX-1), and elevated sLOX-1 levels may be indicative of atherosclerotic plaque instability. We examined associations between the LOX-1 gene 3'UTR-C/T and G501C polymorphisms and plasma sLOX-1 levels in 97 healthy older men and women. The frequencies for the 3'UTR-T and 501C alleles were 46 and 10%, respectively. Plasma sLOX-1 levels were significantly higher in the 3'UTR CC genotype group compared with both the CT (P = 0.02) and TT genotype groups (P = 0.002). Plasma sLOX-1 levels were also significantly higher in the 501GC genotype group compared with the GG genotype group (P = 0.004). In univariate analyses, sLOX-1 levels were significantly associated with both the 3'UTR-C/T and G501C polymorphisms. These associations remained significant after adjusting for age, sex, race and body mass index. In conclusion, variation in the LOX-1 gene is associated with plasma sLOX-1 levels in older men and women.     

Surface expression and rapid internalization of macrosialin (mouse CD68) on elicited mouse peritoneal macrophages

Macrosialin, the mouse homolog of human CD68, is a heavily glycosylated transmembrane protein found almost exclusively in macrophages. Its function remains uncertain. It has a high affinity for oxidized low-density lipoprotein (LDL) in ligand blots and antibodies against the human homolog, CD68, inhibit the binding of oxidized LDL to a human monocyte-derived cell line (THP-1). However, there is still controversy as to whether macrosialin, found predominantly in late endosomes, is expressed at all on the plasma membrane. The present studies, done in thioglycollate-elicited peritoneal macrophages, confirm that macrosialin is predominantly intracellular but show clearly that 10-15% of it is expressed on the cell surface. Exchange with intracellular pools occurs at an extremely high rate. The results are compatible with a surface function, including internalization of bound ligands or adhesion to surfaces.     

Role of LOX-1 in Ang II-induced oxidative functional damage in renal tubular epithelial cells

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), plays an important role in angiotension II (Ang II)-induced hypertensive renal injury associated with pro-inflammatory responses, tubular functional damage and cellular apoptosis. In this study, we report on the role of LOX-1 in Ang II-induced oxidative functional damage and underlying signaling in human renal proximal tubular epithelial cells (HRPTEpiCs). The exposure to Ang II enhanced the expression of the NADPH oxidases (the p22phox, p47phox and Nox4 subunits), LOX-1 and the adhesion molecule, ICAM-1. It also promoted monocytic U937 cell adherences to HRPTEpiCs, increased reactive oxygen species formation and stimulated apoptosis, which was concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). Furthermore, the Ang II treatment disturbed the balance of the Bcl-2 family proteins, destabilized mitochondrial membrane potential, and subsequently triggered the release of cytochrome c into the cytosol, causing the activation of caspase-3. The NADPH oxidase inhibitors and LOX-1 small interfering RNA markedly ameliorated these detrimental effects by reducing LOX-1 expression and MAPK activation. The p38 and p44/42MAPK inhibitors also inhibited the Ang II-induced functional damage without affecting LOX-1 expression in the HRPTEpiCs. These observations suggest that LOX-1 mediates Ang II-induced renal tubular epithelial dysfunction. In addition, MAPK pathway activation occurs downstream of the Ang II/reactive oxygen species/LOX-1 cascade.