Novel immunosuppressive effect of FK506 by augmentation of T cell apoptosis

We have recently reported the accumulation of oligoclonal activated T cells in the spontaneously developed autoimmune pancreatitis in aly/aly mouse. In this study, we examined the effects of FK506 in this mouse model in preventing autoimmune pancreatitis and investigated its action on calcium signalling apoptosis of alymphoplasia (aly) lymphocytes in vitro. Mice were treated with FK506 from 8 to 25 weeks of age. At the age of 15 weeks, minimal mononuclear cell infiltration was observed in the pancreas in both the FK506 treated group and the control group. Furthermore, a marked cell infiltration associated with destruction of acini and partial fatty changes were observed in 25-week-old control mice. In contrast, FK506 treated mice showed almost no tissue destruction or mononuclear cell infiltration at the age of 25 weeks. Furthermore, at 15 weeks of age, most mononuclear cells in FK506-treated mice were TUNEL positive, whereas only a few were positive in control mice. This augmentation of T cell apoptosis by FK506 was confirmed using naive splenocytes activated by PMA and ionomycin in vitro. Finally, a suppressive effect of FK506 on Bcl-2 production but not on Bax production was confirmed by Western blotting. This unique effect of FK506 on the augmentation of T cell apoptosis is probably one of the mechanisms explaining its beneficial effect on aly autoimmune pancreatitis.     

Effect of VEGF on Mouse Thymocyte Proliferation and Apoptosis <Emphasis Type="Italic">In Vitro</Emphasis>

Vascular endothelial growth factor is not only angiogenic, but also immunoregulatory factor. For evaluation of the possibility of its direct interaction with mouse thymocytes we studied the effect of vascular endothelial growth factor on proliferation and apoptosis of thymocytes and expression of genes for the corresponding receptor on these cells. Vascular endothelial growth factor modulated mitogen-induced proliferation of thymocytes and stimulated spontaneous apoptosis in intact thymus cells. Thymocytes express mRNA of type 2, but not type 1 vascular endothelial growth factor receptors.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 5, pp. 533–537, May, 2005     

Immunohistochemical analysis of cleaved caspase-3 detects high level of apoptosis frequently in diffuse large B-cell lymphomas of the central nervous system

The purpose of the present paper was to examine the level of apoptosis and the relationships among apoptosis, apoptosis-associated proteins, and proliferating potential in lymphoma tissues to clarify the characteristics of apoptosis in diffuse large B-cell lymphomas (DLBCL) of the central nervous system (CNS). The formalin-fixed, paraffin-embedded tissues of CNS and non-CNS DLBCL (20 cases each) were studied by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) and immunohistochemistry, using antibodies against single-stranded DNA (ssDNA), cleaved caspase-3, bcl-2, bax, p53, Fas and Ki-67. The cleaved caspase-3 immunohistochemistry detected apoptosis of the lymphoma cells most sensitively compared to TUNEL and ssDNA immunohistochemistry. High expression (grade + + or + + +) of cleaved caspase-3 was found more frequently in CNS DLBCL (11 cases, 55%) than non-CNS DLBCL (three cases, 15%; P = 0.009). Bax-positivity of lymphoma cells was increased in six cases of CNS DLBCL, which also showed high positivity of cleaved caspase-3. There was no significant correlation between the cleaved caspase-3-positivity and the Ki-67 positivity. The present study indicates that the number of apoptotic cells and expression level of cleaved caspase-3 were significantly higher in CNS DLBCL than non-CNS DLBCL, and that the correlation of bax and cleaved caspase-3 expression was often present in CNS DLBCL.     

Correction of lipid composition of the lymph and blood during immediate type hypersensitivity

The survival of neurons is a key condition for complete posttraumatic regeneration of the peripheral nerve. In experiments on rats we studied survival capacity of different neuronal subpopulations in L_IV-L_V dorsal root ganglia after ligation or transection and suturing of the sciatic nerve. Experiments with nerve ligation showed that IB4⁺ neurons are more sensitive to the injury than NF200⁺ neurons. By day 90 after ligation of the sciatic nerve IB4⁺ neurons were virtually not detected in the dorsal root ganglia. By day 90 after nerve transection the number of surviving NF200⁺ and IB4⁺ neurons decreased by 26.1 and 21.4%, respectively, in comparison with intact animals. Treatment with xymedon, a regeneration stimulator, led to a 48.5% increase in the number of surviving NF200⁺ neurons by day 30 after ligation of the nerve and a 50.7% increase by day 90. The number of surviving IB4⁺ neurons increased more than 8-fold by this term after ligation of the nerve and drug stimulation. Xymedon had a neuroprotective effect towards both neuron subpopulations, more intensely preventing apoptosis of IB4⁺ neurons.     

诱骗受体DcR3对佐剂型关节炎大鼠模型的作用分析

目的 研究诱骗受体DcR3 对佐剂型关节炎(AA)大鼠模型的作用及其机理.方法 注射弗式完全佐剂建立大鼠佐剂型关节炎(AA)模型,尾静脉注射DcR3蛋白,观察大鼠关节肿胀度、间接 ELISA 检测血清和滑膜液中细胞因子IL-1 β、TNF-α、IFN-γ的变化.RT-PCR检测滑膜和淋巴细胞中DcR3、Fas、FasL mRNA的表达以及脾脏中TGF-β、IFN-γ、TNF-α、IL-4、IL-10 mRNA的表达.Western blot分析滑膜细胞中Caspase-8、Caspase-3、Caspase-9、Bcl-2蛋白的表达.结果 DcR3 治疗AA大鼠后,足肿胀度降低;血液和滑膜液的IL-1β、TNF-α、IFN-γ水平下降;脾脏中TGF-β、IFN-γ、TNF-α mRNA 表达下调,IL-4、IL-10 mRNA表达上调;滑膜细胞中Caspase-8、Caspase-3蛋白表达上调,Bcl-2蛋白表达下调. 结论 DcR3 可以用于实验性大鼠AA的治疗,其治疗机制与调节滑膜细胞FasL、Fas mRNA的表达和血液淋巴细胞中 Fas mRNA的表达,促进滑膜细胞和自身反应性淋巴细胞的凋亡;调节脾脏细胞Th1/Th2细胞因子平衡相关.本研究为进一步阐明RA的发病机理奠定了重要基础,为有效治疗RA提供了新思路.     

Morphological changes in mesothelial cells induced by shed menstrual endometrium in vitro are not primarily due to apoptosis or necrosis

In a previous study on the pathogenesis of endometriosis, we observed that constituents of menstrual effluent induce morphological alterations in human mesothelial cells. In this study, we investigated whether these alterations were associated with apoptosis or necrosis or were the result of cellular remodelling. After overnight incubation of confluent monolayers of human omental mesothelial cells (HOMEC) with conditioned media prepared from menstrual effluent shed anterogradely, severe alterations in morphology were observed. Typical polygonal mesothelial cell cultures at confluency acquired elongated spindle morphology, resulting in gaps between the cells. In contrast, mesothelial cells from the control groups receiving culture medium only, retained a normal morphology. Immunofluorescence staining revealed that cytokeratin, vimentin and actin filaments were still present, homogeneously distributed in the cell cytoplasm following changes in morphology. To evaluate whether the morphological alterations were associated with apoptosis and/or necrosis, the cells were stained with the M30 CytoDeath antibody or annexin V with propidium iodide and analysed using flow cytometry. The results showed that only a small percentage (1-7%) of the affected HOMEC were undergoing apoptosis or necrosis. We conclude that the profoundly altered morphology of HOMEC is a result of cellular remodelling and that the role of apoptosis and necrosis is negligible. Soluble paracrine factors released by cells isolated from menstrual effluent shed anterogradely may induce a reorganization of the cytoskeleton. As a result, the underlying basement membrane will be exposed and the mesothelium may no longer prevent implantation of endometrium shed retrogradely into the peritoneum, thus facilitating the development of endometriosis.     

凋亡肿瘤细胞负载的树突状细胞对抗原特异性CTL的激活

为探讨凋亡Daudi细胞负载的树突状细胞 (DC )激发诱导肿瘤抗原特异性CTL及其生物学特性 ,采用常规方法从人外周血单个核细胞 (PBMC )诱导DC ,激发型CD40mAb联合 50Gyγ射线辐照诱导Daudi凋亡后负载DC ,然后与自体T细胞共育 ,并联合IL 2激发诱导肿瘤特异性CTL ;采用免疫荧光标记和流式细胞仪测定分析膜分子的表达 ;ELISA检测细胞因子的产生 ;Dextran FITC内吞实验分析DC的抗原摄取能力 ;3H掺入试验和51 Cr释放试验分别测定CTL的增殖和细胞毒效应。结果 :(1 )细胞因子序贯体外诱导 7d的DC ,对Dextran吞噬能力最强 ;(2 )CD40mAb诱导联合γ射线辐照 ,能有效介导Daudi细胞的凋亡 ;(3 )抗原负载联合CD40mAb激发可使DC上调表达CD1a、CD80、CD86和HLA DR ,并能促进IL 1 2的分泌 ;(4)凋亡Daudi负载后的DC在激发型CD40mAb作用下 ,能激发和扩增对Daudi细胞具有高效和特异杀伤作用的细胞毒性T细胞。因而认为凋亡肿瘤细胞负载联合CD40mAb刺激的DC可有效激活和扩增肿瘤特异性CTL。     

Antigen presentation by macrophages is enhanced by the uptake of necrotic, but not apoptotic, cells

The aim of this study was to determine whether phagocytosis of necrotic or apoptotic cells affects antigen presentation by murine bone marrow-derived macrophages. After uptake of necrotic neutrophils, macrophages were able to stimulate significantly higher T cell proliferation in vitro against both the recall antigen albumin and the mitogen concanavalin A. No such effect was seen following phagocytosis of apoptotic neutrophils. Flow cytometry revealed that, within 4h of ingestion, macrophages that had taken up the necrotic cells expressed higher levels of CD40 than those that had phagocytosed apoptotic cells. Macrophage cultures pulsed with apoptotic, but not necrotic, neutrophils contained higher levels of transforming growth factor beta1, but lower concentrations of tumour necrosis factor alpha, compared to untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic neutrophils co-stimulate T cells with greater efficiency due to rapid CD40 up-regulation, whereas those that have ingested apoptotic cells are not only ineffective in co-stimulation, but also secrete inhibitory cytokine.     

三取代型钛钨硅酸盐体内抑瘤效应的免疫机制探讨

目的:探讨三取代型钛钨硅酸盐(WT)体内抑瘤效应的免疫机制。方法:建立荷H22肝癌小鼠模型,WT连续灌胃10d,取出肿瘤称重测定抑瘤率。用MTT比色法测定荷H22肝癌小鼠淋巴细胞转化的活性及NK细胞的杀伤活性。通过形态学观察和流式细胞仪检测WT诱导BEL-7402细胞的凋亡。结果:WT可显著抑制荷瘤小鼠肿瘤生长(P<0.05),提高荷瘤小鼠淋巴细胞转化的活性及NK细胞的杀伤活性(P<0.05),并诱导肿瘤细胞发生凋亡。结论:WT的抑瘤作用与机体免疫功能的增强有关。