Dexamethasone inhibits apoptosis of human neutrophils induced by reactive oxygen species

Neutrophils are completely differentiated cells that die in tissues a few days after they migrate from the vascular compartment as a consequence of a rigouous apoptotic program. Many of the mediators produced during an inflammatory response delay neutrophil apoptosis allowing a more efficient removal of microorganisms but also favoring the tissue damage by reactive oxygen species (ROS) and lysosomal proteins released by neutrophils. Glucocorticoids delay the apoptosis of neutrophils but the mechanisms are not completely understood. To investigate the inhibition of glucocorticoids on neutrophil apoptosis we have used the glucose/glucose oxidase (G/GO) system as a constant source of hydrogen peroxide. When neutrophils are incubated in the presence of the G/GO system, a significant acceleration of their apoptotic response is observed. Preincubation with 10^–6 M, 10^–7 M, 10^–8 M or 10^–9 M of dexamethasone, negatively modulated the spontaneous and G/GO induced apoptosis of neutrophils. Then the G/GO system is a useful model to simulate the oxidative stress of neutrophils, and that the effect of DXM on neutrophil apoptosis depends, at least in part, on blocking the proapoptotic effect of ROS.     

Commercial potting soils as an alternative infection source of Legionella pneumophila and other Legionella species in Switzerland

Legionella spp. are pathogens that can cause Legionnaires' disease in humans through inhalation of contaminated aerosols. The principal reservoir for these microorganisms is water, but Legionella spp. have been isolated from composted vegetable and plant material, and from many potting mixes as well. In Australia, there have been several cases of Legionnaires' disease in which Legionella longbeachae has been isolated from potting soils. In Switzerland, the source of infection cannot always be identified as water or cooling towers: therefore, we have investigated 46 commercially available potting soils in Switzerland to determine the presence of Legionella spp. We were able to detect Legionella spp. in 45.7% (21/46) of the potting soil samples analysed by culture. Legionella pneumophila was present in 19.6% (9/46) of the samples and L. pneumophila serogroup  1 in 6.5% (3/46). Quantification by both culture and quantitative real-time PCR revealed high concentrations of legionellae in potting soils, ranging between 10³ CFU/g and 10⁵ CFU/g and 10⁴ genomic units (GU)/g and 10⁶ GU/g, respectively. Thus, potting soils may represent an alternative reservoir for Legionella spp. in Switzerland.     

Mycobacterial 65-kD heat shock protein induces release of proinflammatory cytokines from human monocytic cells.

Monocytes having phagocytosed mycobacteria are known to present the bacterial 65-kD heat shock protein (hsp) on their cell surface to alpha beta and gamma delta T lymphocytes. Cytotoxic CD4+ cells may then lyse monocytes expressing mycobacterial 65-kD hsp. However, it is not known whether 65-kD hsp directly stimulates monocyte functions other than antigen presentation. This study has demonstrated that following extraction of bacterial lipopolysaccharide, purified recombinant mycobacterial 65-kD hsp may directly activate THP-1 cells, a human monocytic line, to accumulate mRNA for and secrete tumour necrosis factor (TNF), a cytokine important in granuloma formation, the characteristic host immune response to mycobacterial infection. TNF gene expression and secretion following stimulation by hsp was dose-dependent and abolished by heat-induced proteolysis. Subsequently, THP-1 cells secreted IL-6 and IL-8, cytokines involved in recruitment and differentiation of T lymphocytes. The data indicate that secretion of proinflammatory cytokines from monocytes activated by mycobacterial 65-kD hsp may be important in the host immune response and in the development of antigen-specific T cell-mediated immunity.     

Heme-induced heme oxygenase-1 (HO-1) in human monocytes inhibits apoptosis despite caspase-3 up-regulation

Monocyte activation, apoptosis and differentiation are hallmarks of most inflammatory vascular disorders. We studied the effects of heme oxygenase-1 (HO-1) induced by its substrate hemin on apoptosis, caspase-3 expression and the differentiation of freshly isolated human monocytes. Hemin induced HO-1 in a dose- and time-dependent fashion as measured by semi-quantitative RT-PCR and flow cytometry. Apoptosis was markedly suppressed by hemin in cells rendered apoptotic by serum deprivation or dexamethasone as determined by flow cytometric detection of annexin V binding or transmission electron microscopy (TEM). The specific HO-1 inhibitor zinc protoporphyrin (ZnPP) reversed the effects of hemin on monocyte apoptosis and diminished cell lifespan. Surprisingly, the cytoprotective effects of hemin were positively correlated with caspase-3 up-regulation. Hemin-induced apoptosis suppression was enhanced by the caspase-3 inhibitor DEVD-CHO, indicating that caspase-3 was active in a pro-apoptotic fashion. Hemin inhibited CD95 as a putative cytoprotective mechanism. Morphological studies and detection of CD86 showed that monocytes differentiated into macrophages in response to hemin after relatively long incubation times, a phenomenon that might be provoked by caspase-3-regulated pathways. Our results confirm a similar cytoprotective effect of hemin/HO-1 for monocytes as has been shown for other cells, despite caspase-3 up-regulation. The fact that HO-1 may adversely affect monocyte survival and differentiation could be of particular significance in future therapies for occlusive vascular diseases or transplant rejection.     

Significance of non-pneumophila legionella species in adult community-acquired and nosocomial pneumonias

Summary The number of different Legionella species is increasing at an impressive rate. In two prospective studies, one involving 110 intensive-care unit (ICU) patients with mainly nosocomial pneumonias and the other 105 patients with community-acquired pneumonias, we investigated the incidence and significance ofLegionella pneumophila and non-pneumophila pneumonias on the basis of 17 different main serogroups. In the first study, 14 ICU patients had 15 (13.6%) Legionella pneumonias, which, in 5 cases (33%), were of non-pneumophila etiology. In the second study, 9 patients with community-acquired pneumonias had 10 (9.5%) Legionella pneumonias. Leading this study were 6L. gormanii infections, followed by 2L. dumoffii and only 1L. pneumophila and 1L. longbeachae pneumonia. Of the total, 22 of 23 patients with Legionnaires' disease suffered from severe basic diseases and complications (acute renal failure, respiratory insufficiency, etc.) predominant among the nosocomial pneumonias. The mortality rate was significant in these patients at 33% (5 patients) in the ICU group and 10% (1 patient) in the group with community-acquired pneumonias. We conclude that non-pneumophila Legionella species should receive more diagnostic and therapeutic consideration in patients with nosocomial or community-acquired pneumonias.     

Phenotypic and immunoregulatory characteristics of monocytic iris cells

The introduction of antigen into the anterior chamber of an eye induces the antigen-specific suppression of cell-mediated immunity and the antigen-induced production of immunoglobulin G2 antibodies. To define further the role of iris monocytic cells in the systemic suppression of cell-mediated immunity that follows the entry of foreign antigen into the anterior chamber, murine iris wholemounts or cell suspensions of iris cells were stained with fluorescent anti-F4/80 and/or anti-CD11c, anti-CD11b antibodies and examined by confocal microscopy or flow cytometry, respectively. Monocytic cells in iris cell suspensions were recovered from mice receiving an injection of trinitrophenylated bovine serum albumin (TNP-BSA) into an anterior chamber and Percoll-enriched iris cells separated into cells expressing F4/80 or CD11c were injected intravenously into TNP-BSA-immunized or naive recipients. The recipients were challenged to induce delayed-type hypersensitivity (DTH) or were provided with splenocytes or thymocytes that transfer the suppression of DTH. The homing of monocytic bone marrow cells to the iris was determined by the intravenous injection of bone marrow cells from green fluorescent protein (GFP)-transgenic donors into C57 mice, and the staining of recipient iris wholemounts with anti-F4/80 antibodies. Iris cells with a dendritic morphology expressing both F4/80 and/or CD11c and CD11b, some cells expressing only F4/80 or CD11c, were detected. The irides of irradiated GFP- mice that received intravenous GFP+ bone marrow cells contained GFP+ F4/80+ cells. F4/80+ and CD11c+ cells from the irides of donors that received intracameral TNP-BSA transferred the suppression of DTH when injected intravenously into TNP-BSA-immunized recipients, activated immunoregulatory thymocytes and activated antigen-specific splenic regulatory effector cells. These results support the hypothesis that iris monocytic cells may participate in the systemic induction of regulatory T cells.     

Knockdown of WISP1 inhibit proliferation and induce apoptosis in ALL Jurkat cells

WISP1, a Wnt-induced secreted protein, has been found to have anticancer activity. ALL is a leading cause of death. Here we investigate the WISP1 effects on ALL Jurkat cells. Cell viability was assessed by CCK-8. Cell cycle and apoptosis were detected by flow cytometry. Mitochondrial membrane potential (MMP) was monitored using TMRM. Generation of reactive oxygen species (ROS) was quantified using DCFH-DA. Western blot was used to detect the expression of cell proliferation and apoptosis related genes. The results showed that knockdown of WISP1 significantly inhibited proliferation of Jurkat cells. Parallelly, cell cycle distribution was increased at G1 phase and apoptotic rate was induced after WISP1 knockdown. Furthermore, knockdown of WISP1 induced apoptosis of Jurkat cells was also associated with loss of MMP and generation of ROS. Western blot results showed that the protein expression p-AKT, PCNA, CDK1, P-ERK, CDK2, VEGF, VEGFR2 and Bcl2 were decreased, while the expression of Bax was up-regulated. In conclusion, WISP1 plays an important role in proliferation and apoptosis of Jurkat cells in mitochondria dependent pathway, the specific mechanisms need further study.     

Emodin induces apoptosis of human osteosarcoma cells via mitochondria- and endoplasmic reticulum stress-related pathways

Aim: Emodin showed anti-cancer activity against multiple human malignant tumors by inducing apoptosis. However, the apoptotic inducing effect against human osteosarcoma and related mechanism are still not studied. This study was aimed to investigate them. Methods: Emodin was used to incubate human OS cell U2OS cells at serially diluted concentrations. Hoechst staining was used to evaluate apoptosis; flow cytometry was applied to assess the collapse of mitochondrial membrane potential (MMP); intracellular ROS generation was detected by DCFH-DA staining; endoplasmic reticulum stress activation was examined by western blotting. Results: Cell apoptosis of U2OS cells was induced by emodin incubation in a concentration-dependent manner; MMP collapse and ROS generation were identified at starting concentration of 80 μmol/L of emodin in a concentration-dependent manner. ER stress activation was found at beginning concentration of 40 μmol/L of emodin. The MMP collapse was inhibited while the ER stress was not inhibited by NAC administration. Conclusions: Emodin induces death of human osteosarcoma cells by initiating ROS-dependent mitochondria-induced and ROS-independent ER stress-induced apoptosis.     

抗Fas抗体诱导人肾小球系膜细胞凋亡

研究抗Ｆａｓ抗体对人肾小球系膜细胞的致凋亡作用，探讨Ｆａｓ－ＦａｓＬ在肾脏损务中的作用。方法：常规方法分离，培养人肾小球系膜细胞，并传代鉴定，用不同浓度的抗Ｆａｓ抗体刺激４－６代Ｍｃｓ１６ｈ后，荧光染色观察Ｍｃｓ凋亡变化，二苯胺法和ＤＮＡ凝胶电泳方法定量和定性分析ＭｃｓＤＮＡ片段化变化。     

大黄酸及大黄素诱导高糖大鼠肾小球系膜细胞的凋亡

背景：课题组前期实验证实中药复方消可宁能有效防治早期糖尿病肾病。 目的：比较大黄酸与大黄素对高糖培养的大鼠肾小球系膜细胞凋亡的影响程度。 方法：分别用不同浓度20,40,80 µmol/L的大黄酸和大黄素刺激高糖培养的肾小球系膜细胞,苏木精-伊红染色观察凋亡细胞形态,DAPI荧光染色观察细胞核凋亡情况,流式细胞仪观察细胞凋亡率。 结果与结论：苏木精-伊红染色及DAPI染色结果显示大黄酸对髙糖培养的肾小球系膜细胞凋亡的影响程度强于大黄素的影响程度。细胞凋亡率的对比显示大黄酸对髙糖培养的肾小球系膜细胞的早期凋亡率与晚期凋亡率均强于大黄素。说明低中高浓度的大黄酸、大黄素均可诱导肾小球系膜细胞凋亡,但大黄酸的药效强于大黄素。     

Evaluation of the SAS Legionella Test, a new immunochromatographic assay for the detection of Legionella pneumophila serogroup 1 antigen in urine

This study evaluated a new immunochromatographic assay (SAS Legionella Test) for its ability to detect Legionella pneumophila serogroup 1 antigen in urine. Results were compared with those obtained using the Binax Now urinary antigen test. Sensitivity and specificity were estimated as 82.9% and 99.0%, respectively, for the SAS Legionella Test, and 91.4% and 100%, respectively, for the Binax Now urinary antigen test. The sensitivity of both tests increased to 97.1% (p 0.009) and 94.2% (p 0.7), respectively, if the tests were examined after 1 h.     

Pseudomonas aeruginosa induces apoptosis in human endothelial cells

Pseudomonas aeruginosa has been shown to enter into human endothelial cells in vitro. To ascertain the effects of bacterial intracellular (IC) infection, endothelial cells were exposed to PAK and PAO-1 strains for 1 h and treated with gentamicin in culture medium for different periods. P. aeruginosa induced a significant production of superoxide and hydrogen peroxide by endothelial cells. Concentrations of IC bacteria were reduced progressively with time and no viable PAO-1 was detected at 24 h after infection. However, IC infection led to killing of 32.2%±2.9 and 51.8%±3.5 of the cells infected with PAK and PAO-1, respectively, as determined by the MTT assay. By three criteria (transmission electron microscopy, DNA electrophoresis and reactivity with annexin V) infected cells exhibited features of apoptosis. Treatment of infected cells with anti-oxidants (catalase, tocopherol and N -acetyl-L-cysteine) significantly decreased the percentage of cell death. In contrast, treatment with aminoguanidine, an inhibitor of inducible NO synthase, increased significantly the killing of PAO-1 infected cells. Based on these results we speculate that in response to P. aeruginosa infection, endothelial cells increase the production of reactive oxygen intermediates to eliminate IC pathogens, but cells do not resist the oxidative stress and die by apoptosis.     

Early apoptosis of monocytes contributes to the pathogenesis of systemic inflammatory response and of bacterial translocation in an experimental model of multiple trauma

The objective of this study was to investigate the occurrence of apoptosis of monocytes in an experimental model of multiple trauma and its probable correlation to bacterial translocation. Thirty-two rabbits were applied in three groups: A, controls; B, myotomy of the right femur; and C, myotomy and fracture of the right femur. Blood was sampled for the estimation of endotoxins [lipopolysaccharide (LPS)], tumour necrosis factor (TNF)-alpha, malondialdehyde (MDA) and isolation of peripheral blood mononuclear cells (PBMCs). PBMCs, derived after centrifugation over Ficoll, were incubated in flasks and apoptosis of non-adherent lymphocytes and adherent monocytes was estimated after staining for Annexin-V and flow cytometry. TNF-alpha of supernatants of cultured monocytes was also determined. Tissue segments were cultured after death. Median survival of groups A, B and C was > 14, > 14 and 9.00 days, respectively. Apoptosis of lymphocytes in group C was higher than group A at 2, 4 and 48 h and of monocytes in group C higher than group A at 2 and 4 hours. LPS in group C was higher than group A at 2, 4 and 48 h. Apoptosis of lymphocytes and monocytes was correlated positively with serum TNF-alpha and negatively with TNF-alpha of monocyte supernatants. Cultures of organ segments of group A were sterile. Pseudomonas aeruginosa was isolated from liver, lung and spleen in five animals in group B (45.45%) and in six in group C (54.54%). Early apoptosis of blood monocytes supervened after multiple trauma; the phenomenon was accompanied by apoptosis of blood lymphocytes and subsequent bacterial translocation.     

Regulation of FcεRI expression on human monocytic cells by ligand and IL-4

BACKGROUND: The Fc epsilonRI subunit composition and kinetic of expression differ between antigen-presenting cells and mast cells. Up to now, there has been no human in vitro model available that mimics the characteristics on monocytes. OBJECTIVE: The characterization of a natural human monocytic cell line (THP1), which expresses Fc epsilonRI, and the comparison to primary human monocytes and other monocytic cell lines, which only express Fc epsilonRI after transfection with the human Fc epsilonRI alpha-chain gene. METHODS: Surface receptor expression was characterized by flow cytometry, the human Fc epsilonRI alpha-chain gene was introduced by electroporation, and induction of Fc epsilonRI alpha-chain message was detected by semiquantitative RT PCR. RESULTS: Here we show that the parental human cell line THP1, but none of the other cell lines tested, displays surface Fc epsilonRI in response to IL-4 or incubation with receptor ligand (IgE, antibody). Transfection of Fc epsilonRI alpha-chain resulted in receptor expression on all cell lines, all of which increased surface Fc epsilonRI in the presence of IgE. Only the THP1-alpha transfectant, however, further increased receptor levels in response to IL-4, resulting from mRNA induction for the Fc epsilonRI-alpha, but not the beta- or gamma-subunit. CONCLUSION: Based on THP1, U937 and HL60 and their alpha-chain transfectants we present a model system for the study of Fc epsilonRI regulation and signalling on human cells. THP1 in particular, due to its responsiveness to both ligand and IL-4, even without prior manipulation, is ideally suited to address questions on Fc epsilonRI modulation in an 'allergic environment'.     

Proteasome inhibitors induce p53-independent apoptosis in human cancer cells

Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.     

青蒿琥酯通过增加活性氧簇生成诱导人肝癌HepG2细胞凋亡

目的:探讨青蒿琥酯诱导人肝癌Hep G2细胞凋亡的机制及活性氧簇(ROS)在青蒿琥酯诱导Hep G2细胞凋亡中的作用。方法:采用MTT法观查青蒿琥酯对人肝癌Hep G2细胞存活的影响,Hoechst 33258荧光染色法观察细胞凋亡形态的变化,流式细胞术检测Hep G2细胞的凋亡率,DCFH-DA检测细胞凋亡过程中ROS的变化。Western blot检测细胞内凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3和细胞色素C(Cyt C)蛋白水平的变化。采用NADPH氧化酶抑制剂夹竹桃麻素(apocynin)预处理Hep G2细胞,Western blot检测NADPH氧化酶亚基p47~(phox)和p22~(phox)蛋白表达水平,流式细胞术检测ROS变化。结果:与对照组相比,青蒿琥酯作用于Hep G2细胞24 h后,细胞存活率明显减少(P0.05);细胞核呈致密浓染色,细胞凋亡比例升高(P0.05);ROS明显升高(P0.05);Western blot结果显示,青蒿琥酯作用后细胞内Bcl-2蛋白表达下调,Bax蛋白表达上调,Bax/Bcl-2蛋白表达比例升高,cleaved caspase-3和Cyt C蛋白水平升高。Apocynin预处理能降低青蒿琥酯给药组细胞内p47~(phox)和p22~(phox)蛋白表达及ROS的生成。结论:青蒿琥酯能诱导Hep G2细胞凋亡,其凋亡过程可能与ROS的生成增加相关。     

麦芽酚可抑制活性氧引起的SH-SY5Y细胞凋亡

近年来研究表明，氧化应激在阿尔茨海默病等神经退行性疾病发生发展中具有重要作用。本文研究了麦芽酚对过氧化氢(H2O2)引起的人神经瘤细胞株(SH-SY5Y)凋亡时细胞形态、磷脂酰丝氨酸(PS)外翻和DNA损伤的保护作用。结果发现麦芽酚浓度为2mmol／L，作用2h后，可以保护细胞免受H2O2引起的氧化损伤。实验结果表明麦芽酚可作为一种抗氧化剂，有效保护H2O2引起的SH-SY5Y的凋亡。