While several clinical studies have shown that HIV-1 infection is associated with increased permeability of the intestinal tract, there is very little understanding of the mechanisms underlying HIV-induced impairment of mucosal barriers. Here we demonstrate that exposure to HIV-1 can directly breach the integrity of mucosal epithelial barrier, allowing translocation of virus and bacteria. Purified primary epithelial cells (EC) isolated from female genital tract and T84 intestinal cell line were grown to form polarized, confluent monolayers and exposed to HIV-1. HIV-1 X4 and R5 tropic laboratory strains and clinical isolates were seen to reduce transepithelial resistance (TER), a measure of monolayer integrity, by 30–60% following exposure for 24 hours, without affecting viability of cells. The decrease in TER correlated with disruption of tight junction proteins (claudin 1, 2, 4, occludin and ZO-1) and increased permeability. Treatment of ECs with HIV envelope protein gp120, but not HIV tat, also resulted in impairment of barrier function. Neutralization of gp120 significantly abrogated the effect of HIV. No changes to the barrier function were observed when ECs were exposed to Env defective mutant of HIV. Significant upregulation of inflammatory cytokines, including TNF-α, were seen in both intestinal and genital epithelial cells following exposure to HIV-1. Neutralization of TNF-α reversed the reduction in TERs. The disruption in barrier functions was associated with viral and bacterial translocation across the epithelial monolayers. Collectively, our data shows that mucosal epithelial cells respond directly to envelope glycoprotein of HIV-1 by upregulating inflammatory cytokines that lead to impairment of barrier functions. The increased permeability could be responsible for small but significant crossing of mucosal epithelium by virus and bacteria present in the lumen of mucosa. This mechanism could be particularly relevant to mucosal transmission of HIV-1 as well as immune activation seen in HIV-1 infected individuals.

The gene encoding the proprotein convertase subtilisin/kexin type 9 (PCSK9) is linked to familial hypercholesterolemia, as are those of the low-density lipoprotein receptor (LDLR) and apolipoprotein B. PCSK9 enhances LDLR degradation, resulting in low-density lipoprotein accumulation in plasma. To analyze the role of hepatic PCSK9, total and hepatocyte-specific knockout mice were generated. They exhibit 42% and 27% less circulating cholesterol, respectively, showing that liver PCSK9 was responsible for two thirds of the phenotype. We also demonstrated that, in liver, PCSK9 is exclusively expressed in hepatocytes, representing the main source of circulating PCSK9. The data suggest that local but not circulating PCSK9 regulates cholesterol levels. Although transgenic mice overexpressing high levels of liver and circulating PCSK9 led to the almost complete disappearance of the hepatic LDLR, they did not recapitulate the plasma cholesterol levels observed in LDLRdeficient mice. Single LDLR or double LDLR/PCSK9 knockout mice exhibited similar cholesterol profiles, indicating that PCSK9 regulates cholesterol homeostasis exclusively through the LDLR. Finally, the regenerating liver of PCSK9-deficient mice exhibited necrotic lesions, which were prevented by a high-cholesterol diet. However, lipid accumulation in hepatocytes of these mice was markedly reduced under both chow and high-cholesterol diets, revealing that PCSK9 deficiency confers resistance to liver steatosis. Conclusion: Although PCSK9 is a target for controlling hypercholesterolemia, our data indicate that upon hepatic damage, patients lacking PCSK9 could be at risk. (HEPATOLOGY 2008;48:646-654.) P roprotein convertase subtilisin/kexin type 9 (PCSK9) 1 is the ninth member of the proprotein convertase family. 2 The first seven members, including furin, cleave protein precursors of hormones, growth factors, receptors, or surface glycoproteins at basic sites (after Arg or Lys residues). The eighth member, SKI-1 3 or S1P, 4 is known to cleave membrane-bound transcription factors such as the SREBPs 5 in their luminal domains, resulting in the release of their DNA-binding domain. Proprotein convertases can also inactivate secreted substrates, such as endothelial lipase 6 and PCSK9. 7 PCSK9 is synthesized as a precursor that undergoes autocatalytic cleavage of its N-terminal prosegment in the ER, 1 a step required for its exit from this compartment and its efficient secretion. Secreted PCSK9 remains associated with its prosegment. 1 Different from the other proprotein convertases, this serine protease has no known substrate other than itself. In addition, the tight association of the prosegment with the active site 8 raises the question of the existence of an in trans PCSK9 protease

Background: Drug resistance can be complex, and several mutations responsible for it can coexist in a resistant cell. Transcriptional profiling is ideally suited for studying complex resistance genotypes and has the potential to lead to novel discoveries. We generated full genome 70-mer oligonucleotide microarrays for all protein coding genes of the human protozoan parasites Leishmania major and Leishmania infantum. These arrays were used to monitor gene expression in methotrexate resistant parasites.

The interaction between human immunodeficiency virus type 1 (HIV-1) and RNA silencing pathways is complex and multifaceted. Essential for efficient viral transcription and supporting Tat-mediated transactivation of viral gene expression, the trans-activation responsive (TAR) element is a structured RNA located at the 5′ end of all transcripts derived from HIV-1. Here, we report that this element is a source of microRNAs (miRNAs) in cultured HIV-1-infected cell lines and in HIV-1-infected human CD4+ T lymphocytes. Using primer extension and ribonuclease (RNase) protection assays, we delineated both strands of the TAR miRNA duplex deriving from a model HIV-1 transcript, namely miR-TAR-5p and miR-TAR-3p. In vitro RNase assays indicate that the lack of a free 3′ extremity at the base of TAR may contribute to its low processing reactivity in vivo. Both miR-TAR-5p and miR-TAR-3p down-regulated TAR miRNA sensor activity in a process that required an integral miRNA-guided RNA silencing machinery. miR-TAR-3p exerted superior gene downregulatory effects, probably due to its preferential release from HIV-1 TAR RNA by the RNase III Dicer. Our study suggests that the TAR element of HIV-1 transcripts releases functionally competent miRNAs upon asymmetrical processing by Dicer, thereby providing novel insights into viral miRNA biogenesis.

The proprotein convertase subtilisin kexin-9 (PCSK9) circulates in plasma as mature and furin-cleaved forms. A polyclonal antibody against human PCSK9 was used to develop an ELISA that measures total plasma PCSK9 rather than only the mature form. A cross-sectional study evaluated plasma levels in normal (n = 254) and hypercholesterolemic (n = 200) subjects treated or untreated with statins or statin plus ezetimibe. In controls, mean plasma PCSK9 (89.5 ± 31.9 ng/ml) correlated positively with age, total cholesterol, LDL-cholesterol (LDL-C), triglycerides, and fasting glucose. Sequencing PCSK9 from individuals at the extremes of the normal PCSK9 distribution identifi ed a new loss-of-function R434W variant associated with lower levels of circulating PCSK9 and LDL-C. In hypercholesterolemic subjects, PCSK9 levels were higher than in controls (99.3 ± 31.7 ng/ml, P < 0.04) and increased in proportion to the statin dose, combined or not with ezetimibe. In treated patients (n = 139), those with familial hypercholesterolemia (FH; due to LDL receptor gene mutations) had higher PCSK9 values than non-FH (147.01 ± 42.5 vs . 127.2 ± 40.8 ng/ml, P < 0.005), but LDL-C reduction correlated positively with achieved plasma PCSK9 levels to a similar extent in both subsets ( r = 0.316, P < 0.02 in FH and r = 0.275, P < 0.009 in non-FH). The detection of circulating PCSK9 in both FH and non-FH subjects means that this assay could be used to monitor response to therapy in a wide range of patients.-Dubuc, G., M. Tremblay, G. Paré, H. Jacques, J. Hamelin, S. Benjannet, L. Boulet, J. Genest, L. Bernier, N. G. Seidah, and J. Davignon. A new method for measurement of total plasma PSCK9: clinical applications.

Human immunodeficiency virus type 1 (HIV-1) incorporates several host proteins. Earlier studies have indicated that such foreign constituents can modulate the virus life cycle, although the potential roles that these proteins might play in the viral pathology in vivo remain unclear. In an attempt to shed light on this issue, we first exposed explants of human lymphoid tissue to isogenic viruses except for the presence or absence of host-derived ICAM-1. Incorporation of ICAM-1 alone increased HIV-1 infectivity for human tonsillar tissue cultured ex vivo. This observation was made for viruses bearing distinct coreceptor utilization profiles. Conversion of LFA-1 to a high-affinity-high-avidity state for ICAM-1 further augmented the susceptibility of human tonsillar histocultures to infection by ICAM-1-bearing virions. A more massive depletion of CD4(+) T lymphocytes was seen with X4 ICAM-1/POS viruses than with isogenic ICAM-1/NEG virions. Exposure of X4 and R5 primary isolates of HIV-1 to a blocking anti-ICAM-1 antibody resulted in a decrease of virus infection. Finally, X4 and R5 virions derived from a natural human lymphoid tissue microenvironment incorporated high levels of ICAM-1. Altogether, these results indicate that the incorporation of host ICAM-1 can significantly modulate the biology of HIV-1 in a cellular milieu recognized as the major site of replication in vivo and suggest that host proteins found in HIV-1 particles may participate in the pathogenesis of this disease.

The establishment of HIV type 1 (HIV-1) infection is initiated by the stable attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between virus-encoded gp120 and cell surface CD4, a number of distinct interactions influence binding of HIV-1 to host cells. In this study, we report that galectin-1, a dimeric β-galactoside-binding protein, promotes infection with R5, X4, and R5X4 variants. Galectin-1 acts as a soluble adhesion molecule by facilitating attachment of HIV-1 to the cell surface. This postulate is based on experiments where galectin-1 rendered HIV-1 particles more refractory to various agents that block HIV-1 adsorption and coreceptor binding (i.e., a blocking anti-CD4, soluble CD4, human anti-HIV-1 polyclonal Abs; stromal cell-derived factor-1α; RANTES). Experiments performed with the fusion inhibitor T-20 confirmed that galectin-1 is primarily affecting HIV-1 attachment. The relevance of the present findings for the pathogenesis of HIV-1 infection is provided by the fact that galectin-1 is abundantly expressed in the thymus and lymph nodes, organs that represent major reservoirs for HIV-1. Moreover, galectin-1 is secreted by activated CD8+ T lymphocytes, which are found in high numbers in HIV-1-positive patients. Therefore, it is proposed that galectin-1, which is released in an exocrine fashion at HIV-1 replication sites, can cross-link HIV-1 and target cells and promote a firmer adhesion of the virus to the cell surface, thereby augmenting the efficiency of the infection process. Overall, our findings suggest that galectin-1 might affect the pathogenesis of HIV-1 infection.

No abstract