Human T-cell leukemia virus type I Tax-protein-mediated activation of NF-kappa B from p100 (NF-kappa B2)-inhibited cytoplasmic reservoirs.

The human T-cell leukemia virus type I Tax protein transforms T cells through induced expression of many cellular genes, including those encoding the growth-related proteins interleukin 2 and the alpha chain of its receptor. Induction of these genes is mediated, at least in part, through Tax-dependent posttranslational activation of NF-kappa B, typically heterodimers of p50 (NF-kappa B1) and p65 (RelA). The preexisting NF-kappa B proteins are retained in the cytoplasm of cells by association with inhibitory ankyrin-motif-containing I kappa B proteins, primarily I kappa B-alpha but also including the precursor proteins p105 (NF-kappa B1) and p100 (NF-kappa B2). Here we demonstrate the existence of a previously undescribed multimeric cytoplasmic complex in which NF-kappa B dimers are associated with the p100 inhibitor in a manner dependent on the precursor protein's ankyrin domain. We also demonstrate an antagonistic effect of the Tax protein on the cytoplasmic sequestration function of p100; this in turn leads to nuclear translocation of NF-kappa B dimers liberated from multimeric complexes. Tax may exert these effects through the physical association with p100. Tax also relieves the p100-mediated inhibition of DNA binding by p50-p65 heterodimers in vitro. The results demonstrate a mechanism by which Tax may activate NF-kappa B in T cells.     

Up-regulation of TLR2 and TLR4 in dendritic cells in response to HIV type 1 and coinfection with opportunistic pathogens

The ability to trigger an innate immune response against opportunistic pathogens associated with HIV-1 infection is an important aspect of AIDS pathogenesis. Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens, but in HIV-1 patients coinfected with opportunistic infections, the regulation of TLR expression has not been studied. In this context, we have evaluated the expression of TLR2 and TLR4 in monocytes, plasmacytoid dendritic cells, and myeloid dendritic cells of HIV-1 patients with or without opportunistic infections. Forty-nine HIV-1-infected individuals were classified according to viral load, highly active antiretroviral therapy (HAART), and the presence or absence of opportunistic infections, and 21 healthy subjects served as controls. Increased expression of TLR2 and TLR4 was observed in myeloid dendritic cells of HIV-1 patients coinfected with opportunistic infections (without HAART), while TLR4 increased in plasmacytoid dendritic cells, compared to both HIV-1 without opportunistic infections and healthy subjects. Moreover, TLR2 expression was higher in patients with opportunistic infections without HAART and up-regulation of TLR expression in HIV-1 patients coinfected with opportunistic infections was more pronounced in dendritic cells derived from individuals coinfected with Mycobacterium tuberculosis. The results indicate that TLR expression in innate immune cells is up-regulated in patients with a high HIV-1 load and coinfected with opportunistic pathogens. We suggest that modulation of TLRs expression represents a mechanism that promotes HIV-1 replication and AIDS pathogenesis in patients coinfected with opportunistic pathogens.     

The contribution of NF-kappa B activity to spontaneous proliferation and resistance to apoptosis in human T-cell leukemia virus type 1 Tax-induced tumors

Human T-cell leukemia virus type I is the etiologic agent of adult T-cell leukemia/lymphoma. The Tax protein of this virus is thought to contribute to cellular transformation and tumor development. In this report, we have used a Tax transgenic mouse model of tumorigenesis to study the contribution of nuclear factor (NF)-kappa B activity to spontaneous tumor cell proliferation and resistance to apoptosis. We have demonstrated elevated expression levels of NF-kappa B--inducible cytokines, including interleukin (IL)-6, IL-10, IL-15, and interferon (IFN)-gamma, in freshly isolated primary tumors from Tax transgenic mice. Inhibitors of NF-kappa B activity, sodium salicylate and cyclopentenone prostaglandins (prostaglandin A(1) and 15-deoxy-Delta(12,14)-prostaglandin J(2)), blocked spontaneous proliferation of Tax transgenic mouse spleen cells. In addition, Tax-induced tumor cells, which are resistant to irradiation-induced apoptosis, became sensitive to apoptosis in the presence of sodium salicylate and prostaglandins. These results strongly suggest that Tax-mediated induction of NF-kappa B activity contributes to tumorigenesis in vivo. (Blood. 2001;98:1200-1208)     

TLR4 activation and IL-6-mediated cross talk between adipocytes and mononuclear cells synergistically stimulate MMP-1 expression

Obesity is associated with increased monocyte infiltration into adipose tissue and hence increased interaction between adipocytes and monocytes. Although it has been shown that matrix metalloproteinases (MMP) play a critical role in adipose tissue development, the effect of adipocyte and monocyte interaction on MMP production remains largely unknown. Furthermore, although it has been shown that Toll-like receptor 4 (TLR4), a receptor mediating innate immune response, plays an important role in the obesity-associated inflammation and insulin resistance, the effect of TLR4 activation in coculture of adipocytes and monocytes on MMP production has not been investigated. In this study, we cocultured adipocytes with U937 mononuclear cells in a Transwell coculture system and activated TLR4 with lipopolysaccharide or palmitic acid. We found that TLR4 activation and the coculture had a synergistic effect on MMP-1 production. In our further investigation on the underlying mechanisms, it was indicated that adipocyte-derived IL-6 and TLR4 activation acted in concert to synergistically stimulate MMP-1 expression by U937 cells. Taken together, this study has uncovered a novel mechanism potentially involved in MMP-1 up-regulation in adipose tissue, which may facilitate adipose tissue development and obesity.     

PreS1、HBV-DNA、HBeAg反映乙肝病毒复制的临床应用研究

目的 比较PreS1、HBV-DNA、HBeAg反映病毒复制的临床应用价值.方法 对疑有HBV病毒复制的乙型肝炎患者血清标本分别采用实时荧光定量PCR检测HBV-DNA、时间分辨荧光免疫分析技术(TRFIA)定量检测HBV-e抗原和HBV PreS1抗原.结果 PreS1抗原阳性率87.5%(460/526)高于HBV-DNA 71.1%(374/526),有显著性差异(X<'2>=46.3,P<0.001),PreS1抗原阳性率高于HBeAg 37.1%(195/526),有显著性差异(X<'2>=251.6,P<0.001),HBV-DNA阳性率高于HBeAg(X<'2>=161.8,P<0.001),有显著性差异.结论 TRFIA方法 定量检测PreS1抗原,灵敏度高,特异性强,能够准确及时敏感地反映患者体内乙型肝炎病毒的复制情况.     

Cooperative inhibition of NF-kappa B and Tat-induced superactivation of human immunodeficiency virus type 1 long terminal repeat.

Human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-regulated gene expression is stimulated independently by the cellular trans-activator NF-kappa B and the viral protein Tat. Noncytotoxic concentrations of the drug pentoxifylline (PTX) inhibited interaction of NF-kappa B with its motif and the stimulation of HIV-1 LTR-driven gene expression in Jurkat cells. Tat protein (from a cotransfected Tat-expression vector) also induced activation of HIV-1 LTR-driven gene expression. This activation was unaffected by PTX when NF-kappa B sites in the HIV-1 LTR were mutated, suggesting that this drug does not directly influence Tat function, which, however, was inhibited by the Tat-inhibitor Ro 24-7429. Transient reporter gene expression regulated by HIV-1 LTR with wild-type NF-kappa B motifs in the presence of Tat protein was 10- to 60-fold higher than in the presence of either of the trans-activators alone, demonstrating superactivation of HIV-1 LTR by the concerted action of both the trans-activators. Treatment of cells with either PTX or Ro 24-7429 inhibited this superactivation of the HIV-1 LTR. The inhibitory effect of these two drugs in combination, at concentrations that alone did not significantly influence viral promoter activity, was far more than additive. A cooperative action of PTX (NF-kappa B inhibitor) and Ro 24-7429 (Tat inhibitor) on HIV-1 LTR-regulated gene expression is suggested. Concentrations of the drugs that induced maximum inhibition of HIV-1 LTR through their cooperative action are far below cytotoxic levels. Thus, the combination of these two inhibitors could be very effective for anti-HIV therapy.     

血清乙型肝炎病毒前S1抗原对判定HBV DNA复制的临床价值

目的通过分析HBV Pre-S1抗原与HBeAg和HBV DNA的相关性,以探讨其在诊断HBV复制的临床价值。方法采用ELISA法、荧光定量PCR法检测450例HBsAg阳性及50例健康对照血清标本的HBV Pre-S1抗原、血清HBV标志物、HBV DNA及肝功能。结果在450例HBsAg阳性血清中,HBeAg、Pre-S1抗原和HBV DNA阳性率分别为40.0%、57.3%和61.6%;Pre-S1抗原、HBV DNA阳性率在HBeAg阴性与阳性组间差异有统计学意义（x2=84.2,x2=110.7,P〈0.01）;PreS1抗原与HBeAg和HBV DNA均存在相关性（x2=86.5x,2=272.1,P〈0.01）;Pre-S1抗原阳性组AST、ALT、TBl、γ-GT均高于阴性组（P〈0.01）;当HBV DNA拷贝数的对数值〉2.7lgcopies/ml时,Pre-S1抗原诊断HBV复制的敏感度为87.7%,特异度为91.3%,准确性为89.1%,阳性预测值为94.2%,阳性似然比为10.1,优势比为75.3,而HbeAg则分别为59.2%、90.8%、71.3%、91.1%、6.43和14.2。结论 HBV Pre-S1抗原与HBeAg和HBV DNA均有较好的相关性,可作为一项新的病毒复制的指标。     

Pivotal role of cyclic nucleoside phosphodiesterase 4 in Tat-mediated CD4+ T cell hyperactivation and HIV type 1 replication

We show here that HIV type 1 (HIV-1) Tat protein, in combination with anti-CD3/CD28 mAbs, promotes IL-2 production and proliferation of primary CD4(+) T lymphocytes, obtained from HIV-1-seronegative donors. This effect was observed when Tat was immobilized on a solid support, but it was not observed with soluble Tat. Such hyperactivation was accomplished by recruiting the rolipram-sensitive cyclic nucleoside phosphodiesterase 4 and resulted in increased susceptibility to HIV-1 infection. Accordingly, rolipram potently inhibited HIV-1 replication in cultures stimulated by anti-CD3/CD28 +/- Tat. These results add to the concept that decreasing Tat activity is an important addition to anti-HIV-1 therapy, and they suggest a target for anti-HIV-1 chemotherapy, phosphodiesterase 4.     

高迁移率族蛋白1对肝星状细胞Toll样受体4信号途径的激活作用

目的 探讨高迁移率族蛋白1 (HMGB1)对肝星状细胞Toll样受体4(TLR4)信号途径的激活作用及其下游表达产物的影响.方法 培养永生化小鼠肝星状细胞株野生型(JS1)、TLR4基因敲除TLR4-/-(JS2)、MyD88基因敲除MyD88-/- (JS3),用脂质体介导虫荧光酶标记的NF-κB(NF-κB-luc)或AP-1 (AP-1-luc)反应性报告质粒与内参照质粒(R-Luc)共转染JS1、JS2和JS3细胞.每种细胞均分为阴性对照组(NC组)、脂多糖(LPS)处理组、高迁移率族蛋白1(HMGB1)处理组,检测HMGB1、LPS处理后细胞NF-κB及AP-1的活性、各组细胞单核细胞趋化蛋白(MCP-1)mRNA及蛋白的表达.数据经正态性及方差齐性检验,两组间样本均数比较采用t检验.结果 JS1细胞LPS处理组和HMGB1处理组的NF-κB活性分别为(12.72±5.06)、(1.97±0.29)与NC组的(0.85±0.08)相比,t值分别为4.06和6.27,P值均＜0.05,差异均有统计学意义; JS1细胞LPS处理组和HMGB1处理组的AP-1活性分别为(2.01±0.21)、(1.07±0.17)与NC组的(0.61±0.11)相比,t值分别为7.93和3.32,P值均＜0.05,差异均有统计学意义;JS1细胞LPS处理组和HMGB1处理组MCP-1的mRNA相对表达量分别为4.44±0.38、2.42±0.26,与NC组(值为1)相比,t值分别为15.54和9.29,P值均＜0.05,差异均有统计学意义; JS1细胞LPS处理组和HMGB1处理组的MCP-1的蛋白相对表达量分别为(765.57±10.23)、(550.46±15.97)与NC组的(437.14±3.68)相比,t值分别为52.32和11.97,P值均＜0.05,差异均有统计学意义.在JS2和JS3细胞中,LPS和HMGB1处理后上述观测指标与NC组相比,P值均＞0.05,差异无统计学意义.结论 HMGB1作为一种内源性TLR4配体,能激活肝星状细胞株JS1细胞的TLR4信号,促进TLR4介导的炎症表型.     

Coxsackievirus B4 and type 1 diabetes pathogenesis: contribution of animal models

The role of enteroviruses, in particular type B coxsackieviruses (CV‐B), in type 1 diabetes (T1D) pathogenesis is supported by epidemiological, clinical and experimental observations. The investigation of T1D pathogenesis benefits from the contribution of animal models called spontaneously diabetic. Among these animals the non‐obese diabetic (NOD) mouse and the bio‐breeding diabetes‐prone (BBDP) rat present a genetic susceptibility manifested by the expression of an autoimmune diabetes similar to the pathology observed in human beings. Other models whose genetic predisposition is less known are of considerable contribution as well. Numerous major observations relative to several aspects of T1D pathogenesis in the context of CV‐B infections, such as susceptibility, diabetogenicity, pancreatotropism, mechanisms of β cells destruction and others, have been deduced thanks to investigations with animal models. Despite their limits, these models are necessary in improving our knowledge of the role of enteroviruses, like CV‐B4, in the pathogenesis of T1D, and the recent advances ensuing from their contribution may have important therapeutic and preventive spin‐offs. Copyright © 2009 John Wiley & Sons, Ltd.     

The hematopoietic cell-specific Rho GTPase inhibitor ARHGDIB/D4GDI limits HIV type 1 replication

Rho GTPases are able to influence the replication of human immunodeficiency virus type 1 (HIV-1). However, little is known about the regulation of HIV-1 replication by guanine nucleotide dissociation inhibitors (GDIs), one of the three major regulators of the Rho GTPase activation cycle. From a T cell-based cDNA library screening, ARHGDIB/RhoGDIβ, a hematopoietic lineage-specific GDI family protein, was identified as a negative regulator of HIV-1 replication. Up-regulation of ARHGDIB attenuated the replication of HIV-1 in multiple T cell lines. The results showed that (1) a significant portion of RhoA and Rac1, but not Cdc42, exists in the GTP-bound active form under steady-state conditions, (2) ectopic ARHGDIB expression reduced the F-actin content and the active forms of both RhoA and Rac1, and (3) HIV-1 infection was attenuated by either ectopic expression of ARHGDIB or inhibition of the RhoA signal cascade at the HIV-1 Env-dependent early phase of the viral life cycle. This is in good agreement with the previous finding that RhoA and Rac1 promote HIV-1 entry by increasing the efficiency of receptor clustering and virus-cell membrane fusion. In conclusion, the ARHGDIB is a lymphoid-specific intrinsic negative regulator of HIV-1 replication that acts by simultaneously inhibiting RhoA and Rac1 functions.     

TLR2 and TLR4 agonists stimulate unique repertoires of host resistance genes in murine macrophages: interferon-beta-dependent signaling in TLR4-mediated responses

That TLRs share a common MyD88-dependent signaling pathway which results in the generation of nuclear DNA-binding proteins, such as NF-kappaB, is a well-accepted paradigm. However, studies from our laboratories and others suggested that TLR4 agonists elicit a more diverse pattern of gene expression in murine macrophages than TLR2 agonists. The data presented show that activation of TLR4 by Escherichia coli LPS results in an MyD88-independent, TIRAP/Mal-dependent signaling pathway that, in turn, leads to early induction of interferon-beta (IFN-beta). IFN-beta, in turn, acts in an autocrine/paracrine fashion on the macrophage to activate STAT1-containing DNA binding complexes that participate in the induction of genes not expressed in response to natural or synthetic TLR2 agonists. These data support the hypothesis that the host response to microbes is controlled by TLRs at two levels: (i) the "sensing" of differences in microbial structures through the TLR extracellular domain; and (ii) signaling pathways that are initiated via interactions through unique intracytoplasmic regions of different TLRs with adaptor proteins.     

HIV Type 1 Infection Up-Regulates TLR2 and TLR4 Expression and Function in Vivo and in Vitro

Abstract Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens. Their stimulation induces the activation of NF-κB, an important inducer of HIV-1 replication. In recent years, an increasing number of studies using several cells types from HIV-infected patients indicate that TLRs play a key role in regulating the expression of proinflammatory cytokines and viral pathogenesis. In the present study, the effect of HIV-1 stimulation of monocyte-derived macrophage (MDM) and peripheral blood mononuclear cell (PBMC) subpopulations from healthy donors on the expression and functions of TLR2 and TLR4 was examined. In addition, and to complete the in vitro study, the expression pattern of TLR2 and TLR4 in 49 HIV-1-infected patients, classified according to viral load and the use of HAART, was determined and compared with 25 healthy subjects. An increase of TLR expression and production of proinflammatory cytokines were observed in MDMs and PBMCs infected with HIV-1 in vitro and in response to TLR stimulation, compared to the mock. In addition, an association between TLR expression and up-regulation of CD80 in plasmacytoid dendritic cells (pDCs) was observed. The ex vivo analysis indicated increased expression of TLR2 and TLR4 in myeloid dendritic cells (mDCs), but only of TLR2 in monocytes obtained from HIV-1-infected patients, compared to healthy subjects. Remarkably, the expression was higher in cells from patients who do not use HAART. In monocytes, there was a positive correlation between both TLRs and viral load, but not CD4(+) T cell numbers. Together, our in vitro and ex vivo results suggest that TLR expression and function can be up-regulated in response to HIV-1 infection and could affect the inflammatory response. We propose that modulation of TLRs represents a mechanism to promote HIV-1 replication or AIDS progression in HIV-1-infected patients.