MyD88抑制乙型肝炎病毒复制依赖活化NF-κB信号通路

目的 研究髓样细胞分化蛋白(MyD88)抗乙型肝炎病毒(HBV)效应的作用机制。方法 构建MyD88的截短突变体,获得核因子kappa B(NF-κB)超抑制剂IkBa-SR或者NF-κB信号通路激活剂IKKα/IKKβ的表达质粒,分别与HBV复制型质粒瞬时转染Huh7细胞,检测细胞上清液中HBeAg,HBsAg的表达以及胞质中HBV复制中间体DNA的含量,并以NF-κB依赖的荧光素酶报道系统检测它们活化NF-κB的程度。结果 MyD88全长蛋白和2个截短突变体M(1-151)、M(151-296)活化NF-κB的程度与其抑制HBV蛋白以及复制中间体DNA合成的能力相一致。与空载相比,表达NF-κB信号通路激活剂IKKα/IKKβ的质粒共同瞬转细胞后,转染MyD88和HBV表达质粒的细胞中NF-κB的通路明显活化,同时HBV core蛋白的合成显著降低;而NF-κB的超抑制剂IκBα-SR共同瞬转的细胞中core蛋白的表达量显著增加,检测细胞培养上清液中HBeAg和HBsAg及胞质中HBV复制中间体DNA的合成,得到相似结果。结论 NF-κB信号通路的活化在MyD88抑制HBV复制中发挥了关键作用     

Selective recruitment of endothelial progenitor cells to ischemic tissues with increased neovascularization

Tissue ischemia remains a common problem in plastic surgery and one for which proangiogenic approaches have been investigated. Given the recent discovery of circulating endothelial stem or progenitor cells that are able to form new blood vessels, the authors sought to determine whether these cells might selectively traffic to regions of tissue ischemia and induce neovascularization. Endothelial progenitor cells were isolated from the peripheral blood of healthy human volunteers and expanded ex vivo for 7 days. Elevation of a cranially based random-pattern skin flap was performed in nude mice, after which they were injected with fluorescent-labeled endothelial progenitor cells (5 x 10(5); n = 15), fluorescent-labeled human microvascular endothelial cells (5 x 10(5); n = 15), or media alone (n = 15). Histologic examination demonstrated that endothelial progenitor cells were recruited to ischemic tissue and first appeared by postoperative day 3. Subsequently, endothelial progenitor cell numbers increased exponentially over time for the remainder of the study [0 cells/mm2 at day 0 (n = 3), 9.6 +/- 0.9 cells/mm2 at day 3 (n = 3), 24.6 +/- 1.5 cells/mm2 at day 7 (n = 3), and 196.3 +/- 9.6 cells/mm2 at day 14 (n = 9)]. At all time points, endothelial progenitor cells localized preferentially to ischemic tissue and healing wound edges, and were not observed in normal, uninjured tissues. Endothelial progenitor cell transplantation led to a statistically significant increase in vascular density in ischemic tissues by postoperative day 14 [28.7 +/- 1.2 in the endothelial progenitor cell group (n = 9) versus 18 +/- 1.1 in the control media group (n = 9) and 17.7 +/- 1.0 in the human microvascular endothelial cell group (n = 9; p < 0.01)]. Endothelial progenitor cell transplantation also showed trends toward increased flap survival [171.2 +/- 18 mm2 in the endothelial progenitor cell group (n = 12) versus 134.2 +/- 10 mm2 in the media group (n = 12) and 145.0 +/- 13 mm2 in the human microvascular endothelial cell group (n = 12)], but this did not reach statistical significance. These findings indicate that local tissue ischemia is a potent stimulus for the recruitment of circulating endothelial progenitor cells. Systemic delivery of endothelial progenitor cells increased neovascularization and suggests that autologous endothelial progenitor cell transplantation may have a role in the salvage of ischemic tissue.     

Inhibition of hepatitis B virus replication by MyD88 is mediated by nuclear factor-kappaB activation

In our previous paper, we reported that myeloid differential primary response protein (MyD88), a key adaptor in the signaling cascade of the innate immune response, inhibits hepatitis B virus (HBV) replication. The MyD88 activated nuclear factor-kappaB (NF-kappaB) signaling pathway and the intracellular upregulation of NF-kappaB signaling can induce an antiviral effect. Therefore, the association between the inhibition of HBV replication by MyD88 and NF-kappaB activation was investigated further. The results show that NF-kappaB activation was moderately increased after MyD88 expression. The strong activation of NF-kappaB by the IkappaB kinase complex IKKalpha/IKKbeta dramatically suppressed HBV replication; the MyD88 dominant negative mutant that abrogated NF-kappaB activity did not inhibit HBV replication. Furthermore, the IkappaBalpha dominant negative mutant restored the inhibition of HBV replication by MyD88. These results support a role for NF-kappaB activation in the inhibition of HBV replication and suggest a novel mechanism for the inhibition of HBV replication by MyD88 protein.     

Superinfection exclusion in duck hepatitis B virus infection is mediated by the large surface antigen

Superinfection exclusion is the phenomenon whereby a virus prevents the subsequent infection of an already infected host cell. The Pekin duck hepatitis B virus (DHBV) model was used to investigate superinfection exclusion in hepadnavirus infections. Superinfection exclusion was shown to occur both in vivo and in vitro with a genetically marked DHBV, DHBV-ClaI, which was unable to establish an infection in either DHBV-infected ducklings or DHBV-infected primary duck hepatocytes (PDHs). In addition, exclusion occurred in vivo even when the second virus had a replicative advantage. Superinfection exclusion appears to be restricted to DHBV, as adenovirus, herpes simplex virus type 1, and vesicular stomatitis virus were all capable of efficiently infecting DHBV-infected PDHs. Exclusion was dependent on gene expression by the original infecting virus, since UV-irradiated DHBV was unable to mediate the exclusion of DHBV-ClaI. Using recombinant adenoviruses expressing DHBV proteins, we determined that the large surface antigen mediated exclusion. The large surface antigen is known to cause down-regulation of a DHBV receptor, carboxypeptidase D (CPD). Receptor down-regulation is a mechanism of superinfection exclusion seen in other viral infections, and so it was investigated as a possible mechanism of DHBV-mediated exclusion. However, a mutant large surface antigen which did not down-regulate CPD was still capable of inhibiting DHBV infection of PDHs. In addition, exclusion of DHBV-ClaI did not correlate with a decrease in CPD levels. Finally, virus binding assays and confocal microscopy analysis of infected PDHs indicated that the block in infection occurs after internalization of the second virus. We suggest that superinfection exclusion may result from the role of the L surface antigen as a regulator of intracellular trafficking.     

Autopresentation of hepatitis B virus envelope antigens by T cells.

Processing and presentation by T cells appear to be limited to antigens that can directly interact with the T-cell surface, thereby overcoming the T-cell inefficiency in antigen capture and internalization. Our study provides evidence that the hepatitis B virus (HBV) envelope proteins can also be efficiently processed and presented by CD4+ and CD8+ T cells to other T cells in a human leukocyte antigen class II-restricted fashion. This phenomenon suggests a receptor-mediated interaction between T cells and the HBV envelope and defines a system that can, we hope, be exploited for the identification of the receptor binding site within the HBV envelope and for the characterization of the putative cellular HBV receptor.     

血管内皮细胞介导的纤溶作用

血管内皮细胞可合成和释放tPA、uPA和PAI-1,也提供纤溶成份之间相互作用的活性表面,在纤溶的启动和调节中起重要作用。内皮细胞介导的纤溶作用受到神经激素,生长因子、血浆成份、血细胞、血管壁基质和许多外源性物质的调控。     

Infection of resistant avian cells by subgroup B Rous sarcoma virus.

Chicken fibroblasts derived from the H & N flock, which have been characterized as resistant to subgroup B avian oncornaviruses in focus assays, can be infected in suspension shortly after trypsinization by subgroup B sarcoma and leukosis viruses. Once cells are plated, resistance to infection reappears rapidly. C/BE cell suspensions obtained by treatment with EDTA instead of trypsin are not as sensitive to infection. Late interference established by preinfection with subgroup B leukosis viruses is not overcome by trypsinization. In addition to C/BE H & N chicken cells, C/ABE RPRL line 7 cells can also be infected by subgroup B viruses shortly after trypsinization; however, none of the cell types can be made sensitive to subgroup E infection. These results are discussed in relation to current information on the genetic control of resistance to avian oncornaviruses.     

Conditional replication of duck hepatitis B virus in hepatoma cells

To facilitate investigations of replication and host cell interactions in the hepadnavirus system, we have developed cell lines permitting the conditional replication of duck hepatitis B virus (DHBV). With the help of this system, we devised conditions for core particle isolation that preserve replicase activity, which was not found in previous preparations. Investigations of the stability of viral DNA intermediates indicated that both encapsidated DNA and covalently closed circular DNA (cccDNA) were turned over independently of cell division. Moreover, we showed that alpha interferon reduced the accumulation of RNA-containing viral particles. The availability of a synchronized replication system will permit the biochemical analysis of individual steps of the viral replication cycle, including the mechanism and regulation of cccDNA formation.     

DNA of the hepatitis B virus in human generative cells

In spermatozoa of certain patients nucleotide sequences of hepatitis B virus were revealed in an integrated (chromosomal) and free states by means of dot- and blot-hybridization with a cloned HBV-probe. These observations support the conclusion made earlier on the lack of strict hepatotropism for HBV and, in addition, point to a possible involvement of HBV genomes in some molecular pathologies of men.     

Enhanced expression of matrix metalloproteinase-9 by hepatitis B virus infection in liver cells

The hepatitis B virus (HBV) is a major cause of human liver disease, including hepatocellular carcinoma (HCC). The prognosis for HCC is largely dependent on the clinicopathological characteristics regarding invasion and metastasis. Enhanced matrix metalloproteinase-9 (MMP-9) expression has been implicated as playing an important role in metastasis and invasion of HCC. However, the relationship between HBV infection and MMP-9 expression in HCC is currently poorly understood. We report here on a study of the levels of MMP-9 and MMP-2 expression in human fetal liver tissue, rat liver tissue, and Chang, HepG2, and Hep3B cells by gelatin zymography. Among these sources, Hep3B cells, which contain the integrated hepatitis B viral genome, continuously secrete the hepatitis B viral surface antigen, and express HBV genomic RNA, expressed high levels of proMMP-9, and a small amount of active MMP-9 was detected in Hep3B cells as assayed by zymography. We investigated the issue of whether HBV infection affects MMP-9 expression, which is known to play an important role in HCC invasion and metastasis. As a first step, human fetal hepatocyte (HFH) and HepG2 (HCC origin, HBV not detected) cells were subjected to infection with HBV, and the resulting infected cells successfully established are hereafter referred to as HFH-T2 and HepG2-HBV. The expression of MMP-9 was upregulated by the infected HBV in HFH-T2 and HepG2-HBV cells, as assayed by zymography, Northern blot, and Western blot analysis, and small amounts of active MMP-9 were detected in HFH-T2 and HepG2-HBV cells as assayed by zymography. The activation of the immature proMMP-9 to the mature MMP-9 could be induced by plasmin treatment. The activation of proMMP-9 was increased to a greater extent with plasmin treatment than without plasmin in HFH-T2 and HepG2-HBV cells but the addition of recombinant TIMP-1 inhibited the activation of proMMP-9. Finally, the addition of plasmin to the invasion assay using Matrigel resulted in an increase in invasiveness of HFH-T2 and HepG2-HBV cells, as well as MMP-9 activation, but the treatment with TIMP-1 inhibited the invasiveness of HFH-T2 and HepG2-HBV cells as well as MMP-9 activation. We conclude from these findings that HBV infection of hepatocytes and HepG2 cells affected the upregulation of MMP-9 expression and MMP-9 activation and, thus, increased the invasion potential by plasmin. To our knowledge, this is a first report showing that an HBV infection is linked to the upregulation of MMP-9 in HCC.     

Prevalence of hepatitis B virus in chronic hepatitis B patients

The aim of the current study was to detect HBV by Real time - PCR in chronic hepatitis B patients. Fifty-eight sera of chronic hepatitis B patients were subjected during the period March 2009 to April 2010 in Ilam cities in West of Iran. Sera assayed by real-time PCR and ELISA methods. Twenty serum samples from healthy volunteers and non-hepatitis B patients and negative for hepatitis B seromarkers served as negative controls for the study. Among fifty-eight sera, ELISA showed fifty-five (94.8%) of the samples were positive for HBsAg and three (5.2%) negative results obtained while real-time PCR specified fifty-eight (100%) positive results in chronic hepatitis B patients. HBsAg status did not necessarily reflect HBV DNA level in the serum, as 5.2% of chronic Hepatitis B patients were positive for HBV DNA but negative for HBsAg. HBV DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV DNA quantization.     

Multiplication of hepatitis B virus in fulminant hepatitis B.

The presence in serum of hepatitis B e antigen (HBeAg) and hepatitis B virus DNA, which are each regarded as reflecting multiplication of hepatitis B virus, were looked for one to five days after the onset of hepatic encephalopathy in 64 patients with fulminant hepatitis B. HBeAg and hepatitis B virus DNA were found in the serum of only 24 (37%) and six (9%) patients, respectively. Hepatitis B virus DNA was absent from the serum in all 13 patients positive for anti-HBs. These findings indicate that replication of hepatitis B virus stopped after the onset of hepatic encephalopathy in most of the patients and support the view that an enhanced immune response stops the replication. Agents that inhibit viral multiplication would probably not have any effect at this stage of the disease.     

Impaired therapeutic vasculogenesis by transplantation of OxLDL-treated endothelial progenitor cells

Previous in vitro studies have revealed that oxidized low density lipoprotein (OxLDL) has negative effects on the proliferation and activity of endothelial progenitor cells (EPCs). Here, we evaluated the effect of OxLDL on the therapeutic potential of EPCs in ischemia-induced neovascularization. EPCs derived from mobilized human peripheral blood mononuclear cells were cultured without or with OxLDL before transplantation. Hindlimb ischemia models were surgically induced in athymic nude mice, which then received an intracardiac injection of 3 x 10(5) EPCs. By laser Doppler perfusion image and ischemia damage score, we found that blood perfusion and ischemia damage were less well recovered in the OxLDL-treated EPC transplantation group than in controls. Histological examination showed fewer transplanted EPCs and lower capillary density in ischemic tissue. Local delivery of Stromal cell-derived factor (SDF-1) restored this defect and improved blood perfusion by recruiting OxLDL-treated EPCs to the ischemic area and increasing host capillary density. These results provide for the first time direct evidence that OxLDL impaired the therapeutic potential of EPCs in ischemia-induced neovascularization through an inhibitory effect on the migration, adhesion, and incorporation of EPCs into vasculature and/or entrapment in the perivascular region in vivo. A therapeutic strategy based on SDF-1 administration ameliorated such defects and improved postischemic neovascularization.     

高同型半胱氨酸血症与内皮祖细胞凋亡

内皮祖细胞在内皮损伤后的修复中起重要作用.高同型半胱氨酸血症作为动脉粥样硬化的一个独立危险因素,可影响外周血内皮祖细胞的数量和功能,导致内皮功能障碍.在其引起内皮祖细胞损伤的机制中,凋亡扮演了重要角色.本文就高同型半胱氨酸血症对内皮祖细胞凋亡的影响及机制的研究进展进行了综述.