Tenofovir diphosphate and emtricitabine triphosphate concentrations in blood cells compared with isolated peripheral blood mononuclear cells: a new measure of antiretroviral adherence?

BACKGROUND: The active metabolites of tenofovir (TFV) and emtricitabine (FTC) in peripheral blood mononuclear cells (PBMCs) have been used as markers of long-term antiretroviral (ARV) adherence. However, the process of isolating PBMCs is expensive, complex, and not feasible in many settings. We compared concentrations of TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) in the upper layer packed cells (ULPC) obtained after whole blood centrifugation to isolated PBMCs as a possible alternative marker of adherence. METHODS:: Ten HIV+ adults with HIV RNA <50 copies/mL on a TDF/FTC-containing regimen provided five paired PBMC and ULPC samples over 6h. TFV-DP and FTC-TP concentrations were analyzed by liquid chromatography/mass spectrometry (LC-MS/MS). Partial areas under the curve (AUC) were calculated using noncompartmental methods and Spearman Rank Correlations (rho) between PBMC and ULPC were determined. RESULTS:: The median (25-75 percentile) concentration of TFV-DP in PBMCs was 143 (103-248) fmol/10 cells and in ULPC was 227 (160-394) fmol/10 cells (rho=0.65;p <0.0001). The concentration of FTC-TP in PBMCs was 6660 (5650-10000) fmol/10 cells and in ULPC was 19.0 (12.0-27.8) fmol/10 cells (rho 0.55;p<0.0001). Compared to PBMCs, ULPC TFV-DP was 64% higher and FTC-TP was 99.7% lower. ULPC concentrations of TFV-DP and FTC-TP in 1 additional subject receiving a single dose of TDF/FTC were only 0.05% and 25%, of the other 10 subjects, respectively. CONCLUSIONS:: ULPC concentrations significantly correlated with PBMC concentrations. Preliminary single-dose data suggest some discrimination between intermittent vs. consistent dosing. ULPC concentrations of TFV-DP and FTC-TP should be further investigated as a simply-collected, surrogate measure of ARV adherence

Type I IFN counteracts the induction of antigen-specific immune responses by lipid-based delivery of mRNA vaccines

The use of DNA and viral vector-based vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to the lack of their genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study, we evaluated the potency of antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanola mine (DOTAP/DOPE ) as a novel vaccination approach. We demonstrate that subcutaneous immunization of mice with mRNA encoding the HIV-1 antigen Gag complexed with DOTAP/DOPE elicits antigen-specific, functional T cell responses resulting in specific killing of Gag peptide-pulsed cells and the induction of humoral responses. In addition, we show that DOTAP/DOPE complexed antigen-encoding mRNA displays immune-activating properties characterized by secretion of type I interferon (IFN) and the recruitment of proinflammatory monocytes to the draining lymph nodes. Finally, we demonstrate that type I IFN inhibit the expression of DOTAP/DOPE complexed antigen-encoding mRNA and the subsequent induction of antigen-specific immune responses. These results are of high relevance as they will stimulate the design and development of improved mRNA-based vaccination approaches.Molecular Therapy (2012); doi:10.1038/mt.2012.202

Lipoplexes carrying mRNA-encoding Gag protein modulate dendritic cells to stimulate HIV-specific immune responses

Aim: Cationic lipids (Lipofectamine [Invitrogen, Merelbeke, Belgium] and 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanol amine) and polymers (jetPEI and in vivo-jetPEI [Polyplus-transfection, Illkirch, France]) were evaluated for their potential to deliver mRNA to monocyte-derived dendritic cells. Materials & methods: Lipoplexes and polyplexes, containing mRNA-encoding GFP or Gag protein, were incubated with human monocyte-derived dendritic cells and transfection efficiencies were assessed by flow cytometry. Results: Lipofectamine was by far the most efficient in mRNA delivery, therefore it was used in further experiments. Incubation of monocyte-derived dendritic cells isolated from HIV-1-positive donors with mRNA-encoding Gag protein complexed to Lipofectamine resulted in 50% transfection. Importantly, coculture of these Gag-transfected dendritic cells with autologous T cells induced an over tenfold expansion of IFN-gamma- and IL-2-secreting CD4(+) and CD8(+) T cells. Conclusion: Cationic lipid-mediated mRNA delivery may be a useful tool for therapeutic vaccination against HIV-1. This approach can be applied to develop vaccination strategies for other infectious diseases and cancer. Original submitted 26 January 2012; Revised submitted 17 April 2012

Antimony-resistant but not antimony-sensitive Leishmania donovani up-regulates host IL-10 to overexpress multidrug-resistant protein 1

The molecular mechanism of antimony-resistant Leishmania donovani (Sb(R)LD)-driven up-regulation of IL-10 and multidrug-resistant protein 1 (MDR1) in infected macrophages (Ms) has been investigated. This study showed that both promastigote and amastigote forms of Sb(R)LD, but not the antimony-sensitive form of LD, express a unique glycan with N-acetylgalactosamine as a terminal sugar. Removal of it either by enzyme treatment or by knocking down the relevant enzyme, galactosyltransferase in Sb(R)LD (KD Sb(R)LD), compromises the ability to induce the above effects. Infection of Ms with KD Sb(R)LD enhanced the sensitivity toward antimonials compared with infection with Sb(R)LD, and infection of BALB/c mice with KD Sb(R)LD caused significantly less organ parasite burden compared with infection induced by Sb(R)LD. The innate immune receptor, Toll-like receptor 2/6 heterodimer, is exploited by Sb(R)LD to activate ERK and nuclear translocation of NF-kappaB involving p50/c-Rel leading to IL-10 induction, whereas MDR1 up-regulation is mediated by PI3K/Akt and the JNK pathway. Interestingly both recombinant IL-10 and Sb(R)LD up-regulate MDR1 in M with different time kinetics, where phosphorylation of PI3K was noted at 12 h and 48 h, respectively, but Ms derived from IL-10(-/-) mice are unable to show MDR1 up-regulation on infection with Sb(R)LD. Thus, it is very likely that an IL-10 surge is a prerequisite for MDR1 up-regulation. The transcription factor important for IL-10-driven MDR1 up-regulation is c-Fos/c-Jun and not NF-kappaB, as evident from studies with pharmacological inhibitors and promoter mapping with deletion constructs

Traditional nets interfere with the uptake of long-lasting insecticidal nets in the peruvian Amazon: the relevance of net preference for achieving high coverage and use

BACKGROUND: While coverage of long-lasting insecticide-treated nets (LLIN) has steadily increased, a growing number of studies report gaps between net ownership and use. We conducted a mixed-methods social science study assessing the importance of net preference and use after Olyset(R) LLINs were distributed through a mass campaign in rural communities surrounding Iquitos, the capital city of the Amazonian region of Peru. METHODS: The study was conducted in the catchment area of the Paujil and Cahuide Health Centres (San Juan district) between July 2007 and November 2008. During a first qualitative phase, participant observation and in-depth interviews collected information on key determinants for net preference and use. In a second quantitative phase, a survey among recently confirmed malaria patients evaluated the acceptability and use of both LLINs and traditional nets, and a case control study assessed the association between net preference/use and housing structure (open vs. closed houses). RESULTS: A total of 10 communities were selected for the anthropological fieldwork and 228 households participated in the quantitative studies. In the study area, bed nets are considered part of the housing structure and are therefore required to fulfil specific architectural and social functions, such as providing privacy and shelter, which the newly distributed Olyset(R) LLINs ultimately did not. The LLINs' failure to meet these criteria could mainly be attributed to their large mesh size, transparency and perceived ineffectiveness to protect against mosquitoes and other insects, resulting in 63.3% of households not using any of the distributed LLINs. Notably, LLIN usage was significantly lower in houses with no interior or exterior walls (35.2%) than in those with walls (73.8%) (OR = 5.2, 95CI [2.2; 12.3], p<0.001). CONCLUSION: Net preference can interfere with optimal LLIN use. In order to improve the number of effective days of LLIN protection per dollar spent, appropriate quantitative and qualitative methods for collecting information on net preference should be developed before any LLIN procurement decision is made