回转对离体大鼠成骨细胞中骨粘连蛋白及骨桥素mRNA的影响

为研究模拟失重对成骨细胞细胞外基质mRNA的影响,实验采用离体人鼠成骨细胞水平轴回转模拟失重效应,用RT-PCR技术分别检测成骨细胞中骨桥素(osteopontin,OPN)及骨粘连蛋白(osteonectin,ON)mRNA的水平,并观察细胞培养液中碱性磷酸酶(alkaline phosphatase,ALP)的活性和骨钙素(osteocalcim, BGP)含量的变化。结果观察到,分别回转24、48、72h后,OPN、ON的mRNA含量及细胞培养液中BGP含量均显著下降,细胞培养液中ALP活性也呈下降趋势。上述结果表明,模拟失重后成骨细胞OPN及ON的表达下调,进而使BGP及ALP的分泌量减少,从而导致骨钙化能力降低,提示模拟失重导致的细胞外基质蛋白基因表达下降可能是模拟火重引起骨丢失的原因之一。     

TNF and IL-10 are major factors in modulation of the phagocytic cell environment in lung and lymph node in tuberculosis: A next-generation two-compartmental model

Tuberculosis (TB) is one of the earliest recorded human diseases and still one of the deadliest worldwide. Its causative agent is the bacteria Mycobacterium tuberculosis (Mtb). Cytokine-mediated macrophage activation is a necessary step in control of bacterial growth, and early immunologic events in lymph node and lung are crucial to the outcome of infection, although the factors that influence these environments and the immune response are poorly understood.Our goal is to build the next-generation two-compartmental model of the immune response to provide a gateway to more spatial and mechanistic investigations of M. tuberculosis infection in the LN and lung. Crucial immune factors emerge that affect macrophage populations and inflammation, namely TNF-dependent recruitment and apoptosis, and IL-10 levels. Surprisingly, bacterial load plays a less important role than TNF in increasing the population of infected macrophages and inflammation.Using a mathematical model, it is possible to distinguish the effects of pro-inflammatory (TNF) and anti-inflammatory (IL-10) cytokines on the spectrum of phagocyte populations (macrophages and dendritic cells) in the lung and lymph node. Our results suggest that TNF is a major mediator of recruitment of phagocytes to the lungs. In contrast, IL-10 plays a role in balancing the dominant macrophage phenotype in LN and lung.     

TLR2 mediates immunity to experimental cysticercosis

Information concerning TLR-mediated antigen recognition and regulation of immune responses during helminth infections is scarce. TLR2 is a key molecule required for innate immunity and is involved in the recognition of a wide range of viruses, bacteria, fungi and parasites. Here, we evaluated the role of TLR2 in a Taenia crassiceps cysticercosis model. We compared the course of T. crassiceps infection in C57BL/6 TLR2 knockout mice (TLR2^-/-) with that in wild type C57BL/6 (TLR2^+/+) mice. In addition, we assessed serum antibody and cytokine profiles, splenic cellular responses and cytokine profiles and the recruitment of alternatively activated macrophages (AAMφs) to the site of the infection. Unlike wild type mice, TLR2^-/- mice failed to produce significant levels of inflammatory cytokines in either the serum or the spleen during the first two weeks of Taenia infection. TLR2^-/- mice developed a Th2-dominant immune response, whereas TLR2^+/+ mice developed a Th1-dominant immune response after Taenia infection. The insufficient production of inflammatory cytokines at early time points and the lack of Th1-dominant adaptive immunity in TLR2^-/- mice were associated with significantly elevated parasite burdens; in contrast, TLR2^+/+ mice were resistant to infection. Furthermore, increased recruitment of AAMφs expressing PD-L1, PD-L2, OX40L and mannose receptor was observed in TLR2^-/- mice. Collectively, these findings indicate that TLR2-dependent signaling pathways are involved in the recognition of T. crassiceps and in the subsequent activation of the innate immune system and production of inflammatory cytokines, which appear to be essential to limit infection during experimental cysticercosis.     

SPARC antagonizes the effect of basic fibroblast growth factor on the migration of bovine aortic endothelial cells.

Migration of endothelial cells is requisite to wound repair and angiogenesis. Since the glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is associated with remodeling, cellular migration, and angiogenesis in vitro, we questioned whether SPARC might influence the motility of endothelial cells. In this study we show that, in the absence of serum, exogenous SPARC inhibits the migration of bovine aortic endothelial cells induced by bFGF. Similar results were obtained from two different assays, in which cell migration was measured in a Boyden chamber and in monolayer culture after an experimental wound. Without bFGF, the migration of endothelial cells was unaffected by SPARC. The inhibitory effect of SPARC on cell motility was dose-dependent, required the presence of Ca2+, was mimicked by synthetic peptides from the N- and C-terminal Ca(2+)-binding domains of the protein, and was not seen in the presence of serum. Modulation of the activities of secreted and cell-associated proteases, including plasminogen activators and metalloproteinases, appeared not to be responsible for the effects that we observed on the motility of endothelial cells. Moreover, a molecular interaction between SPARC and bFGF was not detected, and SPARC did not interfere with the binding of bFGF to high-affinity receptors on endothelial cells. Finally, in culture medium that contained serum, SPARC inhibited the incorporation of [3H]-thymidine into newly synthesized DNA, both in the absence and presence of bFGF. However, DNA synthesis was not affected by SPARC when the cells were plated on gelatin or fibronectin in serum-free medium. We propose that the combined action of a serum factor and SPARC regulates both endothelial cell proliferation and migration and coordinates these events during morphogenetic processes such as wound repair and angiogenesis.     

Expression and characterization of recombinant NH2-terminal cell binding fragment of vitronectin in E. coli

A recombinant peptide fragment of vitronectin (rVN143), that includes the Arg-Gly-Asp (RGD) cell recognition site, was expressed in Escherichia coli using a prokaryotic expression system. The addition of recombinant rVN143 peptide enhances cell adhesion and proliferation similar (approximately 70%) to those of native VN.     

The membrane form of the DNA repair protein Ku interacts at the cell surface with metalloproteinase 9

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks repair. Ku is also expressed on the cell surface of different types of cells where its function remains poorly understood. From a yeast two-hybrid screen, we have identified a specific interaction between the core region of Ku80 and the hemopexin domain of metalloproteinase 9 (MMP-9), a key enzyme involved in the degradation of extracellular matrix (ECM) components. Ku associates with MMP-9 on the surface of leukemic cells as demonstrated by co-immunoprecipitation experiments in membrane extracts and double-label immunofluorescence studies. In normal and tumoral migratory cells, Ku80 and MMP-9 colocalize at the periphery of leading edge of cells and cellular invasion of collagen IV matrices was blocked by antibodies directed against Ku70 or Ku80 subunits as well as by Ku80-specific antisense oligonucleotides. Our results indicate that Ku and MMP-9 interact at the cell membrane of highly invasive hematopoietic cells of normal and tumoral origin and document the unexpected importance of the membrane-associated form of Ku in the regulation of ECM remodelling.     

降钙素基因相关肽对LPS诱导肺泡巨噬细胞分泌MMP-9的影响

目的：探讨降钙素基因相关肽（CGRP）对经脂多糖（LPS）诱导大鼠肺泡巨噬细胞分泌基质金属蛋白酶-9（MMP-9）的影响及其机制。方法：对经LPS诱导的大鼠肺泡巨噬细胞给予不同浓度的CGRP干预,并同时设置对照,分别收集上清液,采用明胶酶谱法测定LPS、CGRP或二者联合干预后大鼠肺泡巨噬细胞分泌MMP-9的变化。结果：①正常肺泡巨噬细胞仅分泌少量MMP-9,各浓度CGRP对其分泌无影响,但经LPS诱导后MMP-9的分泌均明显升高（P〈0．01）;②不同浓度的CGRP干预呈剂量依赖方式降低LPS诱导的肺泡巨噬细胞MMP-9的分泌（P〈0.01）。③CGRP下调LPS诱导的肺泡巨噬细胞MMP-9分泌的作用可为蛋白激酶C阻断剂H-7及钙调蛋白阻断剂W-7部分逆转（P〈0.05）。结论：CGRP可明显下调LPS诱导的大鼠肺泡巨噬细胞MMP-9活性,其机制与蛋白激酶C及钙调蛋白信号途径有关。     

巨噬细胞的分类及其调节性功能的差异

巨噬细胞在固有免疫和适应性免疫反应中具有重要的作用,它可将加工后的抗原提呈给相应的T细胞,活化后的T细胞通过细胞膜上的分子或分泌的细胞介素进一步活化巨噬细胞。此时的巨噬细胞吞噬杀伤能力大大加强,并释放各种活性物质,因此巨噬细胞是主要的炎性反应调节细胞。巨噬细胞可分为经典活化和选择性活化的巨噬细胞,其在炎性反应过程中分泌不同的细胞因子、趋化因子等,然后间接或直接地参与各种炎症性疾病的反应过程。该文介绍了不同型巨噬细胞在胰岛素抵抗、HIV感染和肿瘤等疾病中的调节功能。     

Podosomes in space: Macrophage migration and matrix degradation in 2D and 3D settings

Migration of macrophages is a key process for a variety of physiological functions, such as pathogen clearance or tissue homeostasis. However, it can also be part of pathological scenarios, as in the case of tumor-associated macrophages. This review presents an overview of the different migration modes macrophages can adopt, depending on the physical and chemical properties of specific environments, and the constraints they impose upon cells. We discuss the importance of these environmental and also of cellular parameters, as well as their relative impact on macrophage migration and on the formation of matrix-lytic podosomes in 2D and 3D. Moreover, we present an overview of routinely used and also newly developed assays for the study of macrophage migration in both 2D and 3D contexts, their respective advantages and limitations, and also their potential to reliably mimic in vivo situations.     

SPARC/Osteonectin Functions to Maintain Homeostasis of the Collagenous Extracellular Matrix in the Periodontal Ligament

Expression of secreted protein acidic and rich in cysteine (SPARC)/osteonectin, a collagen-binding matricellular protein, is frequently associated with tissues with high rates of collagen turnover, such as bone. In the oral cavity, expression of SPARC/osteonectin has been localized to the periodontal ligament (PDL), a collagen-rich tissue with high rates of collagen turnover. The PDL is critical for tooth position within the alveolar bone and for absorbing forces generated by chewing. To characterize the function of SPARC/osteonectin in PDL, SPARC/osteonectin expression in murine PDL was evaluated by immunochemistry at 1, 4, 6, and >18 months. Highest levels of SPARC/osteonectin were detected at 1 and >18 months, with decreased levels associated with adult (4–6 months) PDL. To determine whether the absence of SPARC/osteonectin expression influenced cellular and fibrillar collagen content in PDL, PDL of SPARC-null mice was evaluated using histological stains and compared with that of wild-type (WT). Our results demonstrated decreased numbers of nuclei in PDL of SPARC-null mice at 1 month. In addition, decreased collagen volume fractions were found at 1 and >18 months and decreases in thick collagen fiber volume fraction were detected at 4, 6, and >18 months in SPARC-null PDL. The greatest differences in cell number and in collagen content between SPARC-null and WT PDL coincided with ages at which levels of SPARC/osteonectin expression were highest in WT PDL, at 1 and >18 months. These results support the hypothesis that SPARC/osteonectin is critical in the control of tissue collagen content and indicate that SPARC/osteonectin is necessary for PDL homeostasis. (J Histochem Cytochem 58:871–879, 2010)