Analysis of temporal expression of HTLV-2 reveals similarities and functional differences from HTLV-1

In the present study, we developed a robust splice site-specific real-time RT-PCR method to quantitate all HTLV-2 transcripts. Results of this analysis conducted on three different infected cell lines (HTLV-2A Mo-T , C344 and HTLV-2B BJAB-Gu) showed that the most abundant mRNA was gag/pol followed by the accessory transcript 1-3, coding for the p28 and for p22/p20 proteins. The third most abundant mRNA was tax/rex. To investigate if different mRNAs produced by HTLV-2 are expressed at different levels upon viral reactivation, we studied the kinetics of viral expression in PBMCs from three subjects infected with HTLV-2B and cultured in vitro for 48 hours. The level of expression of the full length gag/pol transcript was the highest in all samples. The tax/rex mRNA was detected already at time zero and increased very rapidly following in vitro culture, reaching the highest copy number between zero and 2-4 hours. The minus-strand APH-2 mRNA, was expressed at high level. As observed in the infected cell lines, the 1-3 mRNA was expressed at high levels in all subjects. This finding is particularly intriguing, as it encodes two proteins that were shown to exert a powerful control on Tax and Rex function. This peculiar pattern of expression, which is in striking contrast with that of HTLV-1, might in part explain the differential pathogenicity of the two viruses

Comparison of Tax-1 and Tax-2B post-translational modifications using specific lysine mutants in relation to activation of NF-κB and intracellular localization

ost-translational modifications of HTLV-1 and HTLV-2 Tax-1 and Tax-2 proteins have been shown to play a critical role in their cellular localization, transactivation and protein interactions. Five of ten lysine residues were found to be major targets for Tax-1 modifications: Lys189(K4); Lys197(K5), Lys263(K6), Lys280(K7) and Lys284(K8), are essential for ubiquitination, while sumoylation takes place on Lys280 (K7) and Lys284(K8). Tax-2 contains four additional lysine residues, namely at position Lys100(K2i), Lys149(K3i), Lys185(K3ii), and Lys356(K10i).Very few studies have been so far performed on Tax-2 lysine mutants. We have previously demonstrated that Tax-2B is ubiquitinated and sumoylated similarly to Tax-1. To identify the Tax-2 lysine residues which are directly involved in post-translational modifications, we have constructed a series of Tax-2B mutants with substitutions of lysine (K) residues by arginines (R) and analyzed them for NF-kB and CREB/ATF transactivation, intracellular distribution and extent of ubiquitination and sumoylation. We have found that Tax-2 K7-8R mutant, contrary to its Tax-1 homologue, is only partially affected in its capacity to transactivate NF-\u3baB pathway, is regularly sumoylated and presents formation of nuclear bodies by confocal analysis. However, Tax-2 mutants with extended (K3ii-8R) and/or total (K1-10iR) mutation rate were severely affected for NF-kB transactivation and sumoylation. By comparing Tax-2 WT with mutants K7-8R and K3ii-8R, we observed that the reduction of NF-\u3baB activity is correlated to a parallel decrease in sumoylation. These results suggest that the target for Tax-2 ubiquitination and sumoylation differs from that described for Tax-1

The class A macrophage scavenger receptor attenuates CXC chemokine production and the early infiltration of neutrophils in sterile peritonitis

The macrophage scavenger receptor (SR-A) is a multifunctional receptor that is associated with several important pathological conditions, including atherosclerosis. In this study, we show, using a sterile peritonitis model, that it can regulate the inflammatory response. SR-A null mice display an increased initial granulocytic infiltration because of overproduction of the CXC chemokines, MIP-2 and keratinocyte-derived cytokine. This differential response is dependent upon particle internalization and can be mimicked by advanced glycation end product-BSA-conjugated latex beads. Thus SR-A is a nonactivating receptor, which is the first example of a pattern recognition receptor that serves to counter the activities of proinflammatory receptors and attenuates the production of specific chemokines to ensure an inflammatory response of the appropriate magnitude

Temporal regulation of HTLV-2 expression in infected cell lines and patients: evidence for distinct expression kinetics with nuclear accumulation of APH-2 mRNA

Background: Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are delta retroviruses with similar genetic organization. Although both viruses immortalize T-cells in vitro, they exhibit distinct pathogenic potential in vivo. To search for possible differences in its expression strategy with respect to HTLV-1, we investigated the pattern of HTLV-2 expression in infected cell lines and peripheral blood mononuclear cells (PBMCs) from infected patients using splice site-specific quantitative RT-PCR. Findings: A novel alternative splice acceptor site for exon 2 was identified; its usage in env transcripts was found to be subtype-specific. Time-course analysis revealed a two-phase expression kinetics in an infected cell line and in PBMCs of two of the three patients examined; this pattern was reminiscent of HTLV-1. In addition, the minus-strand APH2 transcript was mainly detected in the nucleus, a feature that was similar to its HTLV-1 orthologue HBZ. In contrast to HTLV-1, expression of the mRNA encoding the main regulatory proteins Tax and Rex and that of the mRNAs encoding the p28 and truncated Rex inhibitors is skewed towards p28/truncated Rex inhibitors in HTLV-2. Conclusion: Our data suggest a general converging pattern of expression of HTLV-2 and HTLV-1 and highlight peculiar differences in the expression of regulatory proteins that might influence the pathobiology of these viruse

Coregulation in human leukocytes of the long pentraxin PTX3 and TSG-6

The prototypic long PTX3 is a multifunctional protein involved in innate resistance to pathogens and in controlling inflammation. TSG-6 is a hyaluronan-binding protein that is involved in ECM remodeling and has anti-inflammatory and chondroprotective functions. PTX3 and TSG-6 are coregulated by growth differentiation factor-9 in granulosa cells, where they are produced during the periovulatory period and play essential roles in the incorporation of hyaluronan into the ECM during cumulus expansion. The present study was designed to assess whether PTX3 and TSG-6 are coregulated in leukocytes, in particular, in phagocytes and DC. Monocytes, macrophages, and myeloid DC were found to produce high levels of TSG-6 and PTX3 in response to proinflammatory mediators (LPS or cytokines). Unstimulated neutrophil polymorphonuclear granulocytes expressed high levels of TSG-6 mRNA, but not PTX3 transcript, and stored both proteins in granules. In contrast, endothelial cells expressed substantial amounts of PTX3 mRNA and low levels of TSG-6 transcript under the conditions tested. Anti-inflammatory cytokines, such as IL-4, dampened LPS-induced TSG-6 and PTX3 expression. Divergent effects were observed with IL-10, which synergizes with TLR-mediated PTX3 induction but inhibits LPS-induced TSG-6 transcription. Immunohistochemical analysis confirms the colocalization of the two proteins in inflammatory infiltrates and in endothelial cells of inflamed tissues. Thus, here we show that myelomonocytic cells and MoDC are a major source of TSG-6 and that PTX3 and TSG-6 are coregulated under most of the conditions tested. The coordinated expression of PTX3 and TSG-6 may play a role in ECM remodeling at sites of inflammatio

PTX3 predicts severe disease in febrile patients at the emergency department

Objectives: The long pentraxin PTX3 is a promising marker of disease severity in severely ill patients. In order to identify patients warranting critical care as quickly as possible, we investigated the value of PTX3 as a biomarker for disease severity in patients presenting with fever at the emergency department. Methods: Levels of PTX3 were measured in 211 febrile patients at the emergency and the levels were linked to markers of disease severity including admittance to a special care unit, bloodstream infection and congestive heart failure. Results: In comparison to median baseline levels of 2.30 ng/ml (interquartile range 1.66-3.67 ng/ml), levels of PTX3 were significantly elevated in patients admitted to the intensive/medium care unit (median value 44.4 ng/ml, interquartile range 13.6-105.9 ng/ml) and in patients referred to the ward (median value 14.2 ng/ml, interquartile range 7.01-25.1 ng/ml). In addition, PTX3 was associated with duration of hospital stay and acute congestive heart failure. The levels were predictive for bloodstream infection (AUC = 0.71; 95% CI 0.62-0.81). Conclusions: PTX3 may be a useful marker for differentiation of patients with severe disease in patients presenting with fever to the emergency department. (C) 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserve