Induction of VEGF and MMP-9 expression by toll-like receptor 2/4 in human endothelial cells infected with Chlamydia pneumoniae

Matrix metalloproteinases (MMP) are a family of host-derived enzymes involved in the turnover of extracellular matrix (ECM) molecules, and, in particular, it is demonstrated that the 92 KDa gelatinase MMP-9 is often expressed in atherosclerotic plaques by macrophages and smooth muscle cells. Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerosis lesions. In this study, we analyzed the TLR2/TLR4 expression in HUVEC infected with C. pneumoniae and correlated it to the production of VEGF and MMP-9. The results obtained showed an increased VEGF and MMP-9 production correlated with a time-dependent increase in cellular proliferation in HUVEC infected with C. pneumoniae at a multiplicity of infection (MOI) of 2 IFU/cell. HUVEC preincubated with VEGF antibody did not release MMP-9, as detected by zymography assessment and ELISA assay. In addition, we demonstrated that TLR2/TLR4 are expressed in HUVEC infected with viable microorganisms (25% and 17%, respectively), while UV-inactivated microorganisms induced a lesser expression (20% and 11%, respectively) compared to control cells and HUVEC exposed to heat-killed bacteria showed a percentage of TLR-expressing cells similar to the control cells. In addition, the cells preincubated for 60 min with TLR2/TLR4 neutralizing antibodies showed a decrease in C. pneumonia-induced VEGF and MMP-9 production.     

Effect of heparin affin regulatory peptide on the expression of vascular endothelial growth factor receptors in endothelial cells

BACKGROUND: Heparin affin regulatory peptide (HARP) is an 18-kDa secreted protein that has been implicated in tumor growth and angiogenesis, although the mechanisms involved remain largely unknown. In the present work, the effect of human recombinant HARP on the expression of the vascular endothelial growth factor (VEGF) receptors KDR, Flt-1 and neuropilin-1 was studied in cultured human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: The mRNA and protein levels of VEGF receptors were estimated by semi-quantitative RT-PCR and Western blot, respectively. Cell proliferation and migration were measured by MTT, direct counting of the cells and modified Boyden chamber assays. RESULTS: HARP decreased the expression of KDR but increased the expression of Flt-1 and neuropilin-1 at both the mRNA and protein level. The effect reached a maximum 4 h after the addition of HARP into the cell culture medium and was reversed at later time-points. When HARP was added to the culture medium 4 h before the addition of VEGF165, it inhibited VEGF165-induced proliferation and migration of HUVEC. CONCLUSION: These data suggest that HARP affects the expression of VEGF receptors and inhibits VEGF165-induced activation of HUVEC.     

重组VEGF165基因腺相关病毒的包装和鉴定

目的包装重组VEGF165基因的无致病性腺相关病毒,以其作为基因载体感染HUVEC细胞并检测其表达,并在体外检测病毒生物学活性。方法从含有VEGF165基因的pET-32a（＋）-VEGF165原核表达载体中扩增出VEGF165片段,构建重组骨架质粒pAAV-VEGF165。将此质粒和对照质粒pAAV-GFP分别与腺相关病毒包装质粒pAAV-RC、辅助质粒pAAV-Helper用钙-磷共转法转染HEK293细胞,通过同源重组分别产生rAAV-VEGF165和rAAV-GFP重组腺相关病毒。通过real-timePCR法测定病毒滴度后,转染HUVEC细胞并检测其表达,并通过感染HUVEC来检测其促增殖作用。结果扩增出的VEGF165片段成功构建至重组骨架质粒中,pAAV-VEGF165经双酶切和测序鉴定正确。病毒包装效率达90%以上,收获纯化rAAV-VEGF165病毒滴度达6.0×1010pfu/mL。重组腺病毒rAAV-VEGF165能够感染HUVEC细胞并得到显著性表达;与未转染组和GFP组相比,能够显著促进HUVEC细胞的增殖。结论我们成功包装了重组腺相关病毒rAAV-VEGF165,它能够感染HUVEC细胞并高表达VEGF165蛋白,并具有促增殖作用,这为进一步开展VEGF165基因的基因靶向治疗奠定了基础。     

Mechanism of vascular endothelial growth factor on the prevention of restenosis after angioplasty

To evaluate the mechanism of vascular endothelial growth factor (VEGF) on the prevention of restenosis after angioplasty, the recombinant adenovirus vector containing hVEGF₁₆₅ cDNA was constructed and transfected into vascular smooth muscle cells (VSMC) in vitro. The conditioned medium containing VEGF was collected 72 h after the infection. Then, the VSMC and human umbilical vein endothelial cells (HUVEC) were divided into control group, H₂O₂-treated group and H₂O₂+ VEGF-treated group to observe the proliferation and apoptosis by water soluble tetrazolium (WST-1) method, in situ nick end labeling (TUNEL) and flow cytometry (FCM). Compared with the control and H₂O₂+ VEGF-treated groups, the absorbance (A) value of HUVEC was decreased, and apoptosis of HUVEC was significantly increased in H₂O₂-treated group. The changes of A value and apoptosis of VSMC were contrary to those of HUVEC. H₂O₂ could stimulate the proliferation of VSMC and induce the apoptosis of HUVEC, inhibit the proliferation of HUVEC and the apoptosis of VSMC and induce restenosis. VEGF could inhibit the effect of H₂O₂ on HUVEC and VSMC and prevent restenosis. These results offered further theoretical evidence for VEGF on the prevention of restenosis after angioplasty.     

Induction of pro-inflammatory cytokine release by human macrophages during exposure of Streptococcus pneumoniae to penicillin is influenced by minimum inhibitory concentration ratio

beta-Lactam antibiotics cause release of pro-inflammatory bacterial cell wall structures. We determined the effect of penicillin treatment of Streptococcus pneumoniae on the induction of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) genes by human macrophages and the influence of antibiotic concentration and bacterial growth phase upon this induction. Gene expression was measured by real-time polymerase chain reaction (PCR) and protein was measured by enzyme-linked immunosorbent assay (ELISA). Treatment of lag phase S. pneumoniae with one-eighth minimum inhibitory concentration (MIC) penicillin resulted in enhanced expression of TNF-alpha messenger RNA (mRNA), but not TNF-alpha protein at 6h compared with untreated bacteria. IL-1beta mRNA and protein were not affected by these bacteria. MIC treatment of lag or early log phase bacteria induced both protein and mRNA for IL-1beta. Bacteria exposed to concentrations of penicillin that cause lysis (MIC) or no lysis with morphological changes (sub-MIC) induce differential patterns of pro-inflammatory cytokine expression by human macrophages.     

Role of interleukin-1 in bacterial atherogenesis

The high incidence of cardiovascular diseases resulting from atherosclerosis, especially in individuals lacking classic risk factors, has spawned interest in the possibility of unrecognized risk factors, such as chronic bacterial infection. Longstanding low-grade infections, such as periodontal disease, have the potential to affect distant sites in the body by inducing host cells to release inflammatory mediators into the bloodstream. Inflammatory mediators are released by macrophages upon interaction with activated T-helper cells or upon direct recognition of bacterial antigens, and by nonimmune cells upon recognition of antigen through Toll-like receptors. One key mediator, interleukin-1 (IL-1) is released in response to bacterial infection and is known to have specific proatherogenic properties. Increased levels of IL-1 enhance vascular adhesion, vascular permeability, macrophage activation, endothelial and smooth muscle cell proliferation, and protease-induced plaque rupture - all key steps in the progression of atherogenesis. In a recent study, we demonstrated a profound reduction in the progression of atherosclerosis in IL-1 knockout mice. IL-1 holds potential as a target for future antiatherosclerotic therapies, although given its ubiquity in the body, this would not come without unwanted side effects, such as immunosuppression.     

Modulation of cytokine and beta-defensin 2 expressions in human gingival fibroblasts infected with Chlamydia pneumoniae

Human beta-defensin 2 is an antimicrobial peptide that is produced by several epithelial cells after stimulation with micro-organisms and inflammatory mediators. Gram-negative bacteria, which are typically detected in periodontal pockets in periodontitis, elicit a stronger antibacterial peptide response of human beta-defensin 2 by epithelial cells. In this study, we investigated whether Chlamydia pneumoniae is able both to enter and grow in human gingival fibroblasts (HGF), to modify the production of cytokines, and is involved in regulation of beta-defensin 2 expression. Gingival fibroblasts discarded from periodontal procedures on healthy young individuals were infected with viable C. pneumoniae or with heat- or ultraviolet-inactivated organisms at a multiplicity of infection of 4 inclusion-forming units per cell. Our results demonstrate that after 48 h of incubation with viable C. pneumoniae, gingival fibroblasts showed a proliferative response as seen by both colorimetric assay and direct cell count (40% and 45%, respectively). Moreover, cells incubated with viable or ultraviolet light-inactivated C. pneumoniae organisms showed an increase in the levels of interleukin-6, interleukin-10 and human beta-defensin 2 in a time-dependent fashion, while the cells infected with heat-killed bacteria did not show a significant production either of the cytokines or beta-defensin 2 at any time. In conclusion, we demonstrate the correlation between multiplication of C. pneumoniae in human gingival fibroblasts and release of interleukin-6, interleukin-10 and up-regulation of beta-defensin 2, suggesting that gingival fibroblasts may be a periodontium niche for obligate intracellular C. pneumoniae and may play a role in innate gingival immune system and inflammatory response mechanisms of periodontitis.     

Toll-like receptor-positive cells and recognition of pathogens: how human myeloid dendritic cells respond to in vitro infection with Leishmania infantum

Dendritic cells (DCs), instructed by the priming signals from microbial factors, can produce interleukin (IL)-12p70 and promote T helper (Th)1 proliferation and interferon (IFN)-gamma production. This event seems to be critical for the containment of infections caused by intracellular pathogens, even including Leishmania infection. In the present in vitro study we have investigated: 1) phagocytic capacities and IL-12 production by human monocyte-derived DCs and macrophages (M?s), infected with Leishmania infantum promastigotes; 2) IFN-gamma production by human CD4+ T cells co-incubated with DCs or macrophages pulsed with live promastigotes. Monocyte-derived myeloid DCs and M?s from healthy donors were infected with live metacyclic Leishmania infantum (MON-1) promastigotes, previously opsonized with 5% autologous serum, at 1:4 cell/parasite ratio. Percentage and index of phagocytosis were calculated after 2, 24 and 48 h of incubation. IL-12 production was evaluated by an ELISA in supernatants from 48 h Leishmania-infected or lipopolysaccharides (LPS)-stimulated DCs and M?s, also in the presence of phytohemagglutinin-activated or inactivated CD4+ T cells. For IFN-gamma production, CD4+ T cells were repeatedly stimulated with DCs or M?s, pulsed with live Leishmania promastigotes or activated with LPS. The number of IFN-gamma-secreting cells was evaluated by an ELISpot assay. Results showed that M?s have a higher phagocytic capacity towards L. infantum promastigotes than DCs. Moreover, unlike M?s, Leishmania-infected DCs were able to release IL-12p70; this production significantly increased in the presence of activated CD4+ T cells. Finally, DCs pulsed with live parasites and added to autologous CD4+ T cells induced a higher number of IFN-gamma-secreting cells than M?s, thus indicating their ability to polarize Th cells toward the Th1 subset. These data indicate that DCs are able to promote protective Th1 immune responses in our experimental model of Leishmania infantum infection, thus representing the grounds for initiating immunoterapeutic and vaccinal strategies.     

Role of hemin in the modulation of H2O2-mediated endothelial cell injury

Heme oxygenase (HO) has been primarily regarded as the rate-limiting enzyme in the degradation of heme. However it has recently been proposed that the inducible isoform, HO-1 (EC 1.14.99.3), functions as a stress-responsive antioxidant enzyme, with the capacity to protect against oxidant-mediated vascular injury. This study used an in vitro model of endothelial permeability to determine the effects of the HO-1-inducing agent hemin on noncytotoxic endothelial injury mediated by acute oxidant stress. Effects of hemin on oxidant-mediated cytotoxicity in a number of endothelial cell types were also investigated. A 20-min exposure of human umbilical vein endothelial cell (HUVEC) monolayers to H(2)O(2) resulted in a significant concentration-dependent increase in permeability, which was reversible 48 h later. Pretreatment of monolayers with hemin for 2 h followed by 18 h in complete medium resulted in HO-1 induction and the attenuation of H(2)O(2)-mediated increases in endothelial permeability, and significantly improved the restoration of endothelial barrier function 48 h later. In HUVEC and in the human microvascular endothelial cell line HMEC-1, hemin treatment as above resulted in protection against cytotoxicity, but not in bovine aortic endothelial cells (BAECs), where such toxicity was potentiated. This potentiation was inhibited by incubation with the HO inhibitor tin protoporphyrin IX, supporting a role for HO-1 in the potentiation of the cytotoxic response. When the exposure time of BAEC to hemin was extended to 24 h, H(2)O(2)-mediated cytotoxicity was attenuated. We conclude that hemin treatment is cytoprotective against noncytotoxic endothelial injury in vitro, under conditions that may not offer global protection against cytotoxic injury to vascular endothelium. This would indicate that HO-1 induction associated with cytotoxic injury in vivo is not always beneficial and therefore that the use of hemin as a therapeutic agent to offset oxidant injury in vascular endothelium should be undertaken with caution.     

Hypoxia/reoxygenation stimulates endothelial cells to promote interleukin-1 and interleukin-6 production. Effects of free radical scavengers.

Vascular endothelium produces and/or interferes with various cytokines. Previous studies have demonstrated interactions of these inflammatory and immunological mediators with oxygen-derived free radicals. The present work examines the relationship between hypoxia/reoxygenation (H/R) and cytokine production by cultured endothelial cells. Human umbilical vein endothelial cell (HUVEC) monolayers were incubated for 24 h in normoxia or submitted to 5 h hypoxia/19 h reoxygenation. Then, interleukin-1 (IL-1) alpha and beta, and interleukin-6 (IL-6), were measured in culture supernatants by specific enzyme immunoassays and bioassays, respectively. Under these conditions, the spontaneous production of IL-1 and IL-6, detected in normoxic HUVEC, greatly increased after H/R treatment. The observed enhancement was cycloheximide-sensitive and, consequently, reflected a de novo protein synthesis. Superoxide dismutase and glutathione peroxidase prevented H/R-induced IL-1 and IL-6 increase. These results constitute the first demonstration that H/R stimulates HUVEC to promote IL-1 and IL-6 production and strongly suggest a role for oxygen-derived free radicals in the cytokine synthesis.     

Changes in serum levels of cytokines in mice injected with an immunostimulator C3bgp isolated from Cuscuta europea

The effect of C3 binding glycoprotein (C3bgp), isolated from Cuscuta europea seeds on induction of in vivo cytokine synthesis was investigated. Different groups of mice were stimulated with 30 microg C3bgp per mouse, injected intraperitoneally. The quantitative determination of IL-1alpha, IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma was performed in mouse sera by ELISA. The quantities of these cytokines were measured at different hours: 1, 2, 3, 4, 5, 6, 7, and 24 h after injection. No significant changes in serum level of IL-2, IL-4 and TNF-alpha in experimental animal groups were found. A little increase of IL-1alpha, moderate elevation of IL-10 and IFN-gamma (5- to 6-fold more) and strong release, more than 10-fold of IL-6 in sera of C3bgp-treated mice were detected. The results obtained from C3bgp stimulated cultures of mouse peritoneal macrophages and mouse splenocytes suggest that C3bgp binds to mouse peritoneal macrophages and induces production mainly of IL-6, followed by IFN-gamma and in a very low degree of IL-1alpha and IL-10. Based on the results presented, we conclude that the increased level of IL-6 was the basic after injection of C3bgp and that the mouse macrophages were the major cell targets for the C3bgp effect.     

糖化低密度脂蛋白介导的人脐静脉血管内皮细胞增殖及Toll样受体4表达变化与白藜芦醇干预后的影响

目的研究糖化低密度脂蛋白(glycated low density lipoprotein,Gly-LDL)对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVEC)增殖及Toll样受体4(Toll-like receptor 4,TLR4)表达的影响和白藜芦醇干预后的变化。方法通过细胞技术试剂盒(cell counting kit-8,CCK-8)测定不同浓度Gly-LDL处理12、24及48 h后HUVEC增殖能力;实时定量聚合酶链式反应(quantitative real-time polymerase chain reaction,QRT-PCR)检测TLR4的表达量。分别采用CCK-8和QRT-PCR检测Gly-LDL加白藜芦醇干预24 h后HUVEC增殖能力及TLR4水平变化。结果不同浓度Gly-LDL干预12 h后高浓度组HUVEC细胞增殖较正常组下降,组内比较干预24 h、48 h后各浓度Gly-LDL组细胞增值率均较正常组下降,组间比较两者无统计学差异。与普通LDL组比较,TLR4信使 RNA表达水平随着干预时间和Gly-LDL浓度增加。不同浓度白藜芦醇干预24 h后,均能明显提高细胞增殖率,抑制Gly-LDL上调的TLR4信使RNA表达。结论 Gly-LDL可抑制内皮细胞增殖能力,并引起TLR4信号的激活。白藜芦醇能有效改善Gly-LDL介导的HUVEC增殖能力下降和TLR4信号水平的上调,减轻Gly-LDL对内皮细胞的损伤。     

Effect of an oversulfated exopolysaccharide on angiogenesis induced by fibroblast growth factor-2 or vascular endothelial growth factor in vitro

The aim of this study was to determine the angiogenic properties of an oversulfated exopolysaccharide (OS-EPS) derived from a polysaccharide secreted by the mesophilic bacterium Alteromonas infernus. We compared the effect of this OS-EPS with that of a non-oversulfated exopolysaccharide (EPS) on human umbilical vein endothelial cell (HUVEC) proliferation, migration and differentiation induced by basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF). OS-EPS enhanced HUVEC proliferation by 58% when used alone, and by respectively 30% and 70% in the presence of FGF-2 and VEGF. OS-EPS also increased the density of tubular structures on Matrigel in the presence of FGF-2 or VEGF. Vascular tube formation was related to alpha(6) integrin subunit expression, which was enhanced by 50% in the presence of the growth factors. Indeed, a monoclonal anti-alpha(6) blocking antibody abolished this vascular tube formation. EPS had no effect in any of the experimental conditions, underlying the importance of sulfation in the angiogenic effects of exopolysaccharide. By potentiating the angiogenic activity of FGF-2 and/or VEGF, OS-EPS, which possesses low anticoagulant activity and thus a low hemorrhagic risk, could potentially be used to accelerate vascular wound healing or to promote the growth of collateral blood vessels in ischemic tissues.     

MK对人脐静脉内皮细胞增殖及迁移的影响

目的 通过研究midkine（MK）蛋白对人脐静脉内皮细胞（HUVEC）增殖、迁移的影响,阐明MK蛋白在血管生成中的作用.方法 选择HUVEC为研究对象进行细胞常规培养,分为5组分别给予不同浓度MK进行干预,实验组MK浓度分别为0.5、5、50和500 ng/ml,以未处理组作为对照组.利用CCK-8试剂盒检测MK对HUVEC增殖能力的影响,利用Transwell技术检测MK对HUVEC迁移能力的影响.结果 与对照组相比,5 ng/ml的MK作用24h和48 h可以促进细胞增殖,差异有统计学意义（P＜0.05）;5 ng/ml的MK作用24 h可以促进细胞迁移,差异有统计学意义（P＜0.05）.结论 一定浓度的MK作用于内皮细胞,可以促进其增殖和迁移,提示MK在血管生成中具有重要作用.     

Elevated temperature accelerates and amplifies the induction of nitric oxide synthesis in rat macrophages

Evidence has accrued that nitric oxide (NO) is an effector molecule in cell-mediated immunity, and it is generally agreed that fever is beneficial to host defence. Therefore, the role of elevated temperature in the induction of NO synthesis was examined in rat peritoneal macrophages activated by lipopolysaccharide (LPS). When macrophages were incubated in vitro at 40°C, the time between macrophage activation and the induction of NO synthesis, as assessed by nitrite accumulation in the medium, was shortened as compared with incubation at 37°C, and nitrite accumulation was markedly enhanced by 2.6- and 1.8-fold after 6 and 9 h of LPS activation, respectively. These results suggest that elevated temperature may contribute to enhance host defence by accelerating and amplifying the induction of NO synthesis in macrophages.     

Effects of protein tyrosine kinase inhibitors on cytokine-induced adhesion molecule expression by human umbilical vein endothelial cells.

1. Endothelial cells can be stimulated by the pro-inflammatory cytokines interleukin (IL)-1 alpha and tumour necrosis factor (TNF) alpha to express the leukocyte adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine-activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. 2. Maximal E-selectin expression induced by incubation of HUVEC for 4 h with IL-1 alpha (100 u ml-1) and TNF alpha (100 u ml-1) was dose-dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12-myristate, 13-acetate (PMA)-induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31-7549 or Ro31-8220 did not affect IL-1 alpha- or TNF alpha-induced E-selectin expression at concentrations which maximally inhibited PMA-induced expression. 3. Genistein inhibited VCAM-1 expression induced by incubation of HUVEC for 24 h with TNF alpha or IL-1 alpha whereas it did not affect ICAM-1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM-1 and ICAM-1 expression induced by TNF alpha. 4. Basal expression of E-selectin, VCAM-1 and ICAM-1 was dose-dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNF alpha-induced expression of these molecules with maximal E-selectin and ICAM-1 expression being slightly enhanced and VCAM-1 expression dose-dependently reduced. 5. We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre-myeloid cell line U937 to TNF alpha-stimulated HUVEC. Adhesion of U937 cells to HUVEC pretreated for 4 or 24 h with TNF alpha was dose-dependently inhibited by genistein and herbimycin A but unaffected by daidzein. Adhesion of U937 cells after 4 h was partially inhibited by blocking antibodies against both E-selectin and VCAM-1 but after 24 h was only inhibited by anti-VCAM-1. 6. Sodium orthovanadate had no effect on TNF alpha-induced U937 adhesion but dose-dependently enhanced adhesion to unstimulated HUVEC. Vanadate-induced adhesion was inhibited by an antibody against VCAM-1. 7. These results demonstrate that PTK-mediated phosphorylation events are important for the regulation of adhesion molecule expression by human endothelial cells, and additionally show that PTK inhibitors differentially affect upregulation of different adhesion molecules, implicating divergent regulatory pathways for cytokine-induced adhesion molecule expression.     

The effect of a ceramide analog, N-acetylsphingosine on the induction of proliferation and IL-2 synthesis in T cells from young and old F344 rats

Ceramide is a physiological mediator of extracellular signals that control various cellular functions, including proliferation and apoptosis. In the present study, we examined the effects of cell-permeable ceramide analog, N-acetyl-sphingosine (C(2)-ceramide) on the induction of proliferation and interleukin-2 (IL-2) synthesis in T cells from young and old rats. Splenic T cells from 6- and 24-month-old Fischer 344 rats were treated with C(2)-ceramide and then incubated with anti-CD3 antibody for 24 or 48 h. The induction of proliferation and IL-2 production by anti-CD3 was significantly (P<0.001) lower in T cells from old rats compared to T cells from young rats. C(2)-ceramide treatment resulted in suppression of proliferation and IL-2 production in a concentration-dependent manner. The suppressive effect of C(2)-ceramide on proliferation and IL-2 production was greater in T cells from old rats than T cells from young rats. We investigated whether this decreased responsiveness was due to induction of program cell death (apoptosis) and found that there was a significant increase in DNA fragmentation in C(2)-ceramide treated and anti-CD3 stimulated T cells from both young and old rats. The increase in DNA fragmentation was paralleled with an increase in caspase-3 activation. C(2)-ceramide-induced caspase-3 activation and DNA fragmentation was significantly (P<0.5) higher in stimulated T cells from old rats compared to stimulated T cells from young rats. These results suggest that the sphingomyelin-ceramide signaling pathway may play an important regulatory role in the well-documented age-related decline in immune function.     

Effect of Musca domestic maggot polypeptide extract on HUVEC dysfunction induced by early-activated macrophages

Context: Musca domestica Linn. maggot is a traditional Chinese medicine. In our previous studies, Musca domestica maggot protein-enriched fraction and polypeptide extract (molecular weight <30?kD) were found to reverse endothelial cell dysfunction in atherosclerotic lesions.

Objective: This study determines whether and how M. domestica maggot polypeptide extract affects the dysfunction of human umbilical vein endothelial cells (HUVEC) induced by macrophages (M?).

Materials and methods: HUVEC and early-activated THP-1?M? (incubated with LPS of 1?μg/ml for 2?h) were co-cultured in a Transwell system. The effects of Musca domestica maggot polypeptide extract (with increasing concentrations, i.e., 1.0, 2.5, 5.0, 10.0, 20.0, and 40.0?µg/ml) on the proliferation and migration HUVEC and their secretion of vascular endothelial growth factor (VEGF) were determined by flow cytometry, modified Boyden chamber assay, and enzyme-linked immunosorbent assay (ELISA) after incubation for 24?h.

Results: Musca domestica maggot polypeptide extract decreased the proliferation of HUVEC in a concentration-dependent manner, with a 50% effective concentration (EC₅₀) of 22.16?±?1.48?µg/ml, and effectively inhibited HUVEC migration (EC₅₀?=?35.15?±?2.03?µg/ml) and VEGF secretion (EC₅₀?=?28.64?±?1.29?µg/ml).

Discussion and conclusion: Musca domestica maggot polypeptide extract can inhibit the dysfunction of HUVEC induced by early-activated THP-1?M?.     

Syk 参与单增李斯特菌感染中炎症复合体的形成

【摘要】 目的 本研究旨在探讨单增李斯特菌（Listeria monocytogenes, LM）感染过程中炎症复合体形成的分子机制。方法 LM分别感染syk,P38MAPK以及PI3K抑制剂组小鼠腹腔由来巨噬细胞后,利用ELISA方法检测上清液中IL-18的蛋白水平;免疫荧光观察细胞内炎症复合体的关键蛋白组分ASC的变化情况;western blot检测LM感染巨噬细胞不同时间点syk蛋白激酶的磷酸化水平。结果 LM感染后,syk抑制剂组产生的IL-18蛋白水平与未处理组相比显著降低,而其他抑制剂组产生的IL-18的蛋白水平未明显改变,进而免疫荧光结果又显示syk抑制剂可明显降低胞内ASC-speck的形成个数。syk磷酸化水平在LM感染5min即可被检测到,进一步证实syk参与炎症复合体活化的过程。结论 syk参与调控单增李斯特菌感染过程中炎症复合体的活化。