自噬在晚期糖基化终产物诱导的内皮细胞凋亡中的作用

目的: 研究晚期糖基化终产物(AGEs)对人脐静脉内皮细胞(HUVECs)自噬水平的影响并探讨自噬在AGEs诱导的内皮细胞凋亡中的作用。方法: 用AGEs处理HUVECs,相同条件牛血清白蛋白处理为对照组,Western blotting检测相应蛋白表达的变化,电镜观察细胞自噬体的出现,流式细胞术检测细胞凋亡,MTT比色法测定细胞活性。结果: AGEs处理HUVECs后,自噬相关蛋白LC3-Ⅱ的表达显著上调并呈时间和浓度依赖性,电镜观察到细胞胞浆内自噬体数量增加;与对照组比较,AGEs处理组内皮细胞凋亡率增加,活性下降,自噬抑制剂3-甲基腺嘌呤预处理的AGEs组细胞活性较AGEs组进一步下降,凋亡率继续增加。AGEs处理HUVECs后,蛋白激酶B(Akt)和哺乳动物雷帕霉素靶蛋白(mTOR)的磷酸化水平也明显下调, Akt激活剂胰岛素样生长因子1预处理后,Akt的磷酸化水平增加,自噬相关蛋白LC3-Ⅱ的增高表达被抑制。结论: AGEs通过PI3K/Akt/mTOR介导的信号通路诱导HUVECs自噬水平升高。自噬在AGEs诱导的内皮细胞凋亡中对细胞起保护作用。     

氧化应激在乙醛引起的心肌细胞凋亡中的作用

目的： 探讨活性氧（ROS）的氧化损伤机制在经乙醛诱导心肌细胞凋亡过程中的作用，阐明氧化应激水平增高导致细胞凋亡可能是乙醇性心肌病（AHMD）的主要原因之一。 方法: 体外培养大鼠心肌细胞，分别用乙醇（100 μmol/L）和乙醛（100 μmol/L）干预24 h，比较细胞凋亡程度；检测48 h内细胞胞内ROS水平变化、胞外分泌SOD活性变化；并用Western blotting技术检测ROS介导MAPK信号途径相关蛋白磷酸化水平变化；并与氧化剂H2O2处理组和预先加入抗氧化剂乙酰半胱氨酸（NAC）的乙醛处理组比较。 结果: 乙醇和乙醛分别干预心肌细胞24 h后，凋亡途径激活因子caspase 3均被激活(P<0.05)，乙醛较乙醇具有更强的诱导凋亡作用（P<0.05）。乙醛诱导心肌细胞ROS水平增高呈时间依赖性，相应抗氧自由基SOD酶活性也随之增高，分别于18-24 h达到峰值。同时，ROS通过JNK和ERK磷酸化水平增高而激活MAPK途径，诱导心肌细胞凋亡，这种效应可以被NAC逆转，等浓度乙醛弱于H2O2的诱导作用。结论: 乙醛通过增高胞内ROS水平损伤心肌细胞，激活ROS介导JNK途径诱导细胞凋亡。其作用弱于H2O2等强氧化剂，提示降低细胞ROS 水平，阻断凋亡信号通路可有效抑制乙醛诱导的心肌凋亡，对于AHMD临床预防和治疗具有指导意义。     

Increased autophagy in CD4+ T cells of rheumatoid arthritis patients results in T‐cell hyperactivation and apoptosis resistance

Rheumatoid arthritis (RA) is an autoimmune disease hallmarked by aberrant cellular homeostasis, resulting in hyperactive CD4⁺ T cells that are more resistant to apoptosis. Both hyperactivation and resistance to apoptosis may contribute to the pathogenicity of CD4⁺ T cells in the autoimmune process. A better knowledge of the mechanisms determining such impaired homeostasis could contribute significantly to both the understanding and the treatment of the disease. Here we investigated whether autophagy, is dysregulated in CD4⁺ T cells of RA patients, resulting in disturbed T‐cell homeostasis. We demonstrate that the rate of autophagy is significantly increased in CD4⁺ T cells from RA patients, and that increased autophagy is also a feature of in vitro activated CD4⁺ T cells. The increased apoptosis resistance observed in CD4⁺ T cells from RA patients was significantly reversed upon autophagy inhibition. These mechanisms may contribute to RA pathogenesis, as autophagy inhibition reduced both arthritis incidence and disease severity in a mouse collagen induced arthritis mouse model. Conversely, in Atg5^flox/flox‐CD4‐Cre⁺ mice, in which all T cells are autophagy deficient, T cells showed impaired activation and proliferation. These data provide novel insight into the pathogenesis of RA and underscore the relevance of autophagy as a promising therapeutic target.     

黄嘌呤氧化酶在激素诱导成骨细胞凋亡过程中的作用及机制

目的探究黄嘌呤氧化酶在糖皮质激素诱导小鼠胚胎成骨细胞前体细胞(MC3T3-E1)细胞凋亡过程中的作用及机制。方法将细胞分为空白组、模型组、5 mol/L组、10 mol/L组和15 mol/L组,接种完成后培养24 h,确认细胞贴壁后换液。空白组加入10%FBS培养基2 mL,模型组加入1 10-6 mol/L DEX培养基2 mL, 5 mol/L组加入5 mol/L别嘌醇DEX培养基2 mL,10 mol/L组加入10 mol/L别嘌醇DEX培养基2 mL,15 mol/L组加入15 mol/L别嘌醇DEX培养基2 mL。培养72 h后检测Caspase-3、STAT-1、Bax、Bcl-2等蛋白表达情况,检测细胞凋亡情况以及活性氧簇含量。检测细胞内黄嘌呤氧化酶活性、丙二醛(MDA)含量及线粒体膜电位。结果模型组中Caspase-3、STAT-1和Bax蛋白表达量明显升高,Bcl-2蛋白表达量降低,细胞凋亡比例增高,细胞内活性氧簇含量增高,细胞内黄嘌呤氧化酶活性、丙二醛含量增高,而线粒体膜电位降低,与空白组、5 mol/L组、10mol/L组和15 mol/L组相比,差异有统计学意义(P 0.05)。结论黄嘌呤氧化酶通过产生过量的活性氧簇从而导致成骨细胞氧化应激损伤,通过激活STAT-1和Bax蛋白来诱导成骨细胞凋亡,同时诱导线粒体膜电位稳态崩溃触发内源性线粒体凋亡途径。别嘌醇可以较好地抑制黄嘌呤氧化酶的活性,减少活性氧簇的产生,通过减少Caspase-3、STAT-1和Bax蛋白表达,稳定线粒体膜电位,从而减少成骨细胞凋亡。     

The kiwi fruit peptide kissper displays anti‐inflammatory and anti‐oxidant effects in in‐vitro and ex‐vivo human intestinal models

Literature reports describe kiwi fruit as a food with significant effects on human health, including anti‐oxidant and anti‐inflammatory activity. Fresh fruit or raw kiwi fruit extracts have been used so far to investigate these effects, but the molecule(s) responsible for these health‐promoting activities have not yet been identified. Kissper is a kiwi fruit peptide displaying pore‐forming activity in synthetic lipid bilayers, the composition of which is similar to that found in intestinal cells. The objective of this study was to investigate the kissper influence on intestinal inflammation using cultured cells and ex‐vivo tissues from healthy subjects and Crohn's disease (CD) patients. The anti‐oxidant and anti‐inflammatory properties of kissper were tested on Caco‐2 cells and on the colonic mucosa from 23 patients with CD, by challenging with the lipopolysaccharide from Escherichia coli (EC‐LPS) and monitoring the appropriate markers by Western blot and immunofluorescence. EC‐LPS challenge determined an increase in the intracellular concentration of calcium and reactive oxygen species (ROS). The peptide kissper was highly effective in preventing the increase of LPS‐induced ROS levels in both the Caco‐2 cells and CD colonic mucosa. Moreover, it controls the calcium increase, p65‐nuclear factor (NF)‐kB induction and transglutaminase 2 (TG2) activation inflammatory response in Caco‐2 cells and CD colonic mucosa. Kissper efficiently counteracts the oxidative stress and inflammatory response in valuable model systems consisting of intestinal cells and CD colonic mucosa. This study reports the first evidence supporting a possible correlation between some beneficial effects of kiwi fruit and a specific protein molecule rather than generic nutrients.     

Adoptive T‐cell therapy for cancer: The era of engineered T cells

Tumors originate from a number of genetic events that deregulate homeostatic mechanisms controlling normal cell behavior. The immune system, devoted to patrol the organism against pathogenic events, can identify transformed cells, and in several cases cause their elimination. It is however clear that several mechanisms encompassing both central and peripheral tolerance limit antitumor immunity, often resulting into progressive diseases. Adoptive T‐cell therapy with either allogeneic or autologous T cells can transfer therapeutic immunity. To date, genetic engineering of T cells appears to be a powerful tool for shaping tumor immunity. In this review, we discuss the most recent achievements in the areas of suicide gene therapy, and TCR‐modified T cells and chimeric antigen receptor gene‐modified T cells. We provide an overview of current strategies aimed at improving the safety and efficacy of these approaches, with an outlook on prospective developments.     

Dissociated expression of natural killer 1.1 and T‐cell receptor by invariant natural killer T cells after interleukin‐12 receptor and T‐cell receptor signalling

Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with α-galactosylceramide (α-GalCer) or interleukin (IL)-12. Although down-modulation of surface T-cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after stimulation with α-GalCer, it is unclear whether this also holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL-12 in the disappearance of iNKT cells after stimulation with α-GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR-P1C by iNKT cells after stimulation with α-GalCer or IL-12, and the influence of IL-12 neutralization on the down-modulation of sTCR/sNKR-P1C expression by iNKT cells after stimulation with α-GalCer were examined. The s/cTCR⁺s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of α-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of IL-12, s/cTCR⁺ iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by α-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. By contrast, cTCR⁺cNKR-P1C⁺ sTCR⁻ sNKR-P1C⁻ iNKT cells and cNKR-P1C⁺ sNKR-P1C⁻ iNKT cells were detectable after in vitro stimulation with α-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after stimulation with α-GalCer by down-modulating not only sNKR-P1C, but also sTCR.     

Induced arginine transport via cationic amino acid transporter‐1 is necessary for human T‐cell proliferation

Availability of the semiessential amino acid arginine is fundamental for the efficient function of human T lymphocytes. Tumor‐associated arginine deprivation, mainly induced by myeloid‐derived suppressor cells, is a central mechanism of tumor immune escape from T‐cell‐mediated antitumor immune responses. We thus assumed that transmembranous transport of arginine must be crucial for T‐cell function and studied which transporters are responsible for arginine influx into primary human T lymphocytes. Here, we show that activation via CD3 and CD28 induces arginine transport into primary human T cells. Both naïve and memory CD4⁺ T cells as well as CD8⁺ T cells specifically upregulated the human cationic amino acid transporter‐1 (hCAT‐1), with an enhanced and persistent expression under arginine starvation. When hCAT‐1 induction was suppressed via siRNA transfection, arginine uptake, and cellular proliferation were impaired. In summary, our results demonstrate that hCAT‐1 is a key component of efficient T‐cell activation and a novel potential target structure to modulate adaptive immune responses in tumor immunity or inflammation.     

Piperine impairs cell cycle progression and causes reactive oxygen species-dependent apoptosis in rectal cancer cells

Piperine, an alkaloid phytochemical found in the fruit of black and long pepper plants, is reported to inhibit the growth of cancer cells; however, the mechanism of action in human cancer cells is not clear. In this study we investigated the effect of piperine on the growth of HRT-18 human rectal adenocarcinoma cells. MTT assays showed that piperine inhibited the metabolic activity of HRT-18 cells in a dose- and time-dependent fashion, suggesting a cytostatic and/or cytotoxic effect. Flow cytometric analysis of Oregon Green 488-stained and propidium iodide-stained HRT-18 cells showed that piperine inhibited cell cycle progression. Piperine also caused HRT-18 cells to die by apoptosis, as determined by Annexin-V-FLUOS staining and characteristic changes in cell morphology. Flow cytometric analysis of dihydroethidium- and 2′,7′-dichlorofluorescein diacetate-stained HRT-18 cells showed increased production of reactive oxygen species in piperine-treated cells. Furthermore, the antioxidant N-acetylcysteine reduced apoptosis in cultures of piperine-treated HRT-18 cells, indicating that piperine-induced cytotoxicity was mediated at least in part by reactive oxygen species. The cytostatic and cytotoxic effects of piperine on rectal cancer cells suggest that this dietary phytochemical may be useful in cancer treatment.     

A stromal cell free culture system generates mouse pro‐T cells that can reconstitute T‐cell compartments in vivo

T‐cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage^?Sca1⁺c‐kit⁺ BM hematopoietic progenitors very efficiently differentiate into pro‐T cells. This culture system consists of plate‐bound Delta‐like 4 Notch ligand and the cytokines SCF and IL‐7. The pro‐T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D–J rearranged. They could be expanded for over 3 months and used to reconstitute the T‐cell compartments of sublethally irradiated T‐cell‐deficient CD3ε^?/? mice or lethally irradiated WT mice. Pro‐T cells generated in this system could partially correct the T‐cell lymphopenia of pre‐Tα^?/? mice. However, reconstituted CD3ε^?/? mice suffered from a wasting disease that was prevented by co‐injection of purified CD4⁺ CD25^high WT Treg cells. In a T‐cell‐sufficient or T‐lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro‐T cells for transplantation and possibly with clinical applications.     

LKB1 is essential for the proliferation of T‐cell progenitors and mature peripheral T cells

The serine/threonine kinase LKB1 has a conserved role in Drosophila and nematodes to co‐ordinate cell metabolism. During T lymphocyte development in the thymus, progenitors need to synchronize increased metabolism with the onset of proliferation and differentiation to ensure that they can meet the energy requirements for development. The present study explores the role of LKB1 in this process and shows that loss of LKB1 prevents thymocyte differentiation and the production of peripheral T lymphocytes. We find that LKB1 is required for several key metabolic processes in T‐cell progenitors. For example, LKB1 controls expression of CD98, a key subunit of the L ‐system aa transporter and is also required for the pre‐TCR to induce and sustain the regulated phosphorylation of the ribosomal S6 subunit, a key regulator of protein synthesis. In the absence of LKB1 TCR‐β‐selected thymocytes failed to proliferate and did not survive. LBK1 was also required for survival and proliferation of peripheral T cells. These data thus reveal a conserved and essential role for LKB1 in the proliferative responses of both thymocytes and mature T cells.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

Defective T‐cell receptor‐induced apoptosis of T cells and rejection of transplanted immunogenic tumors in p53−/− mice

Mice lacking the tumor suppressor gene p53 spontaneously develop T‐cell lymphomas at a high rate, suggesting that in these mice lymphomas arise due to defective apoptosis mechanisms in T cells mediated by p53. However, a role of p53 in regulation of T‐cell responses or apoptosis has been poorly defined. TCR‐mediated signaling in the absence of CD28 costimulation induces both apoptosis and proliferation of naïve T cells from WT mice. In this report we show that, in response to TCR stimulation, T cells from naïve p53‐deficient mice exhibited higher proliferation and drastically reduced apoptosis than WT T cells. CD28 costimulation enhanced the proliferation of TCR‐stimulated WT and p53^?/? T cells, suggesting that p53 uncouples CD28‐mediated antiapoptotic and proliferative signals. To evaluate the physiological significance of these findings, we transplanted OVA expressing‐EG.7 tumor cells into WT and p53^?/? mice. Unlike WT mice, p53^?/? mice exhibited a robust tumor‐resistant phenotype and developed cytotoxic T‐cell responses against OVA. Collectively, these data support the hypothesis that p53 is an essential factor in negative regulation of T‐cell responses and have implication for immunomodulation during treatment of cancers and other inflammatory conditions.     

A novel T‐cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice

We used a newly generated T‐cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10‐mer) or SSVWNATTA (9‐mer) in complex with class I MHC antigen H‐2D^b, were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8⁺ T cells can detect 10‐ and 9‐mer length variants similarly. This result varies from the idea that a given T‐cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10‐mer was presented on the surface of uninfected and infected CD8α⁺CD11c⁺ dendritic cells, which suggests the use of direct and cross‐presentation pathways. In contrast, CD11b⁺CD11c^? cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell‐type specific mechanisms of antigen presentation in vivo.     

T‐bet regulates differentiation of forkhead box protein 3+ regulatory T cells in programmed cell death‐1‐deficient mice

Programmed cell death‐1 (PD‐1) plays an important role in peripheral T cell tolerance, but whether or not it affects the differentiation of helper T cell subsets remains elusive. Here we describe the importance of PD‐1 in the control of T helper type 1 (Th1) cell activation and development of forkhead box protein 3 (FoxP3⁺) regulatory T cells (T_regs). PD‐1‐deficient T cell‐specific T‐bet transgenic (P/T) mice showed growth retardation, and the majority died within 10 weeks. P/T mice showed T‐bet over‐expression, increased interferon (IFN)‐γ production by CD4⁺ T cells and significantly low FoxP3⁺ T_reg cell percentage. P/T mice developed systemic inflammation, which was probably induced by augmented Th1 response and low FoxP3⁺ T_reg count. The study identified a unique, previously undescribed role for PD‐1 in Th1 and T_reg differentiation, with potential implication in the development of Th1 cell‐targeted therapy.     

CD4 and CD8 double‐negative adult T‐cell leukemia/lymphoma with monomorphic cells expressing CD99: A diagnostic challenge in a country non‐endemic for human T‐cell leukemia virus

Adult T‐cell leukemia/lymphoma (ATLL) is a peripheral T‐cell neoplasm caused by human T‐cell lymphotropic virus type I (HTLV‐I). The neoplastic cells are highly pleomorphic and are usually CD4+ and CD8? phenotypically. We reported the case of a 46‐year‐old woman presenting with fever, abdominal distention, lymphadenopathy, leukocytosis and hypercalcemia. Nodal biopsy showed diffuse infiltration by monomorphic small to medium‐sized atypical lymphocytes expressing CD3, CD25, CD30 and CD99, but not CD1a, CD4, CD8, CD34, terminal deoxynucleotidyl transferase or ALK. An initial diagnosis of T‐lymphoblastic leukemia/lymphoma was made based on cytomorphology, CD4 and CD8 double negativity, and the expression of CD99. The diagnosis was later revised to ATLL based on the positive serology study for anti‐HTLV I/II antibody and confirmation by the clonal integration of HTLV‐I proviral DNA into the tumor tissues by Southern blotting analysis. The patient had a stage IVB disease and died of septic shock after 2 courses of chemotherapy 3 months after diagnosis. Immunohistochemical staining for CD99 in archival ATLL tissues showed a positive rate of 67% (4 of 6 tumors). Our case showed that ATLL with atypical morphology and immunophenotype in HTLV non‐endemic areas might pose a diagnostic challenge and CD99 expression is frequent in ATLL.     

One cause for the apparent inability of human T cell clones to function as professional superantigen-presenting cells is autoactivation

Human T cell clones (TCC) are antigen-presenting cells (APC) able to present peptides and superantigens (SAg) and to process and present intact proteins. TCC express major histocompatibility complex (MHC) class II antigens and molecules involved in the accessory signal delivery, such as B7.1 and B7.2/B70. Notwithstanding these observations, the role of professional APC has been often denied to T cells because anergy of responder T cells rather than proliferation has been observed following the TCC presentation in the absence of added professional APC. Here, we show that upon stimulation with free SAg, TCC undergo proliferative responses followed, after a 1-week culture, by an SAg-dependent unresponsiveness to T cell receptor (TCR)-mediated stimuli, but not to interleukin-2. The anergy induced by the SAg can not be prevented by the addition of autologous Epstein-Barr virus (EBV)-transformed B cells, indicating that the induction of anergy occurs also in the presence of conventional APC. Conversely, if the TCC are stimulated by SAg-prepulsed irradiated APC, either EBV and TCC, the induction of anergy is not observed. After a 1-week culture, in fact, TCC stimulated with APC-bound SAg responded to TCR-mediated stimuli, irrespective of the APC (EBV or TCC) used for the SAg presentation. Stimulation of TCC with free SAg in a semisolid medium that prevents T-T cell contacts resulted in an activation followed by a state of anergy, suggesting that anergy is the consequence of SAg recognition at the single T cell level. These data indicate that the anergy observed in TCC upon a 1-week culture in the presence of soluble SAg is not the result of an inherent inability of TCC to act as professional APC. Rather the phenomenon depends on the presence of soluble SAg, leading to T cell autostimulation.