Effect of a short-term HAART on SIV load in macaque tissues is dependent on time of initiation and antiviral diffusion

<p>Abstract</p> <p>Background</p> <p>HIV reservoirs are rapidly established after infection, and the effect of HAART initiated very early during acute infection on HIV reservoirs remains poorly documented, particularly in tissue known to actively replicate the virus. In this context, we used the model of experimental infection of macaques with pathogenic SIV to assess in different tissues: (i) the effect of a short term HAART initiated at different stages during acute infection on viral dissemination and replication, and (ii) the local concentration of antiviral drugs.</p> <p>Results</p> <p>Here, we show that early treatment with AZT/3TC/IDV initiated either within 4 hours after intravenous infection of macaques with SIVmac251 (as a post exposure prophylaxis) or before viremia peak (7 days post-infection [pi]), had a strong impact on SIV production and dissemination in all tissues but did not prevent infection. When treatment was initiated after the viremia peak (14 days pi) or during early chronic infection (150 days pi), significant viral replication persists in the peripheral lymph nodes and the spleen of treated macaques despite a strong effect of treatment on viremia and gut associated lymphoid tissues. In these animals, the level of virus persistence in tissues was inversely correlated with local concentrations of 3TC: high concentrations of 3TC were measured in the gut whereas low concentrations were observed in the secondary lymphoid tissues. IDV, like 3TC, showed much higher concentration in the colon than in the spleen. AZT concentration was below the quantification threshold in all tissues studied.</p> <p>Conclusions</p> <p>Our results suggest that limited antiviral drug diffusion in secondary lymphoid tissues may allow persistent viral replication in these tissues and could represent an obstacle to HIV prevention and eradication.</p

Purification and Characterization of Naturally Occurring Post-Translationally Cleaved Ara h 6, an Allergen That Contributes Substantially to the Allergenic Potency of Peanut

The 2S albumin Ara h 6 is one of the most important peanut allergens. A post-translationally cleaved Ara h 6 (pAra h 6) was purified from Virginia type peanuts, and the cleavage site was mapped using high-resolution mass spectrometry. Compared to intact Ara h 6, pAra h 6 lacks a 5-amino acid stretch, resembling amino acids 43−47 (UniProt accession number Q647G9) in the nonstructured loop. Consequently, pAra h 6 consists of two chains: an N-terminal chain of approximately 5 kDa and a C-terminal chain of approximately 9 kDa, held together by disulfide bonds. Intermediate post-translationally cleaved products, in which this stretch is cleaved yet still attached to one of the subunits, are also present. The secondary structure and immunoglobulin E (IgE) binding of pAra h 6 resembles that of intact Ara h 6, indicating that the loss of the nonstructured loop is not critical for maintaining the protein structure. Commercially available monoclonal and polyclonal immunoglobulin G (IgG) antibodies directed to Ara h 6 react with both intact Ara h 6 and pAra h 6, suggesting that the involved epitopes are not located in the area that is post-translationally cleaved. No differences between intact Ara h 6 and pAra h 6 in terms of IgE binding were found, suggesting that the area that is post-translationally cleaved is not involved in IgE epitopes either. For all main cultivars Runner, Virginia, Valencia, and Spanish, intact Ara h 6 and pAra h 6 occur in peanut at similar levels, indicating that pAra h 6 is a consistent and important contributor to the allergenic potency of peanut

Dosages immunoenzymatiques plasmatiques et intracellulaires des antirétroviraux anti-VIH (synthèses d'haptènes, développements et applications)

Le développement de méthodes de dosage sensibles et facilement utilisable en milieu hospitalier offre un moyen de mieux comprendre les mécanismes d action des antiviraux anti-VIH, d optimiser l utilisation de ces molécules afin de limiter les effets secondaires et à plus long terme individualiser les traitements. Ce manuscrit présente le développement de dosages immunoenzymatiques de trois classes d antiviraux : l AZT-TP (un INTI-TP), l efavirenz (un INNTI) et l atazanavir (un IP). Nous avons ainsi synthétisé les immunogènes et traceurs nécessaires à l obtention d anticorps spécifiques par couplage des molécules concernées avec la BSA et l AChE. Quatre haptènes, analogues stables de l AZT-TP, ont été synthétisés. Les anticorps obtenus reconnaissent l AZT-TP mais présentent de forts taux de réactions croisées avec l AZT-MP et l AZT-DP. Le dosage n a donc pas pu être finalisé. La synthèse énantioselective en sept étapes d un haptène de l EFV nous a permis d obtenir des anticorps spécifique anti-EFV, et de développer un dosage EIA de l EFV assez sensible (limite de détection < 150 pg/mL) pour être utilisé en intracellulaire. Nous avons mis en évidence l accumulation intracellulaire de l EFV (rapport intra/extra = 3) et la possible implication des P-gp dans son efflux. Nous avons aussi développé le dosage EIA plasmatique et intracellulaire de l ATV (limite de détection < 200 pg/mL). Le dosage intracellulaire nous a permis de mettre en évidence la faible accumulation de l ATV à l intérieur des CEM, l implication des P-gp dans son efflux ainsi qu une interaction significative avec l EFV dans son accumulation intracellulaire.Development of sensitive and inexpensive quantification methods may increase antiretroviral efficacy by reducing toxicity and managing drug-drug interactions. Moreover, a better understanding of action of antiretroviral (ARV) drugs requires an intracellular quantification which is considered to be the true active concentration. This manuscript describes the development of immunoassay of three antivirals classes: AZT-TP (NRTI-TP), atazanavir (PI) and efavirenz (NNRTI). We have synthetized immunogens and tracers by covalent coupling of molecules with BSA and AChE. Four haptens, stable analogs of AZT-TP, had been synthesized. Antibodies obtained after immunizations, recognized AZT-TP but also AZT-MP and AZT-DP. The immunoassay could not be fully developed. The seven steps enantioselective synhesis of EFV-hapten had been used to obtain specific anti-EFV antibodies and to develop a sensitive EIA of EFV (limit of detection < 150 pg/mL) which allows the intracellular quantification. We have observed a low intracellular accumulation of EFV and the possible implication of P-gp in its efflux. We have developed a new EIA for measuring plasma and intracellular ATV. This EIA was fully validated. Moreover, in vitro results suggested a weak intracellular accumulation of ATV and P-gp and MRP1 may be involved in the efflux of the drug. An interesting interaction with EFV had been observed.NICE-BU Sciences (060882101) / SudocSudocFranceF

Quantitative Immunoassay To Measure Plasma and Intracellular Atazanavir Levels: Analysis of Drug Accumulation in Cultured T Cells

We have developed an enzyme immunoassay to measure atazanavir (ATV) levels in plasma and cells. Anti-ATV polyclonal antibodies were raised in rabbits by using a synthetic ATV derivative coupled to bovine serum albumin as the immunogen, and the enzyme tracer was prepared by chemically coupling the ATV derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates, and the lowest limit of quantification was 150 pg/ml, which is about 10 times more sensitive than previously published techniques. The plasma assay was performed, after a simple methanol extraction, with a minimum of 30 μl of plasma. This assay showed good precision and efficiency, since the rates of recovery from human plasma and cell extracts spiked with ATV ranged form 93 to 113%, with coefficients of variation of less than 10%. ATV concentrations were measured in peripheral blood mononuclear cells incubated with various ATV concentrations and in CEM cells in the absence or presence of antiretroviral drugs and drug transporter inhibitors. The results indicated a dose-dependent uptake (intracellular concentration/extracellular concentration ratio range, 0.04 to 19). A significant increase in the accumulation of ATV was noticed in the presence of P-glycoprotein and MRP1 inhibitors (dipyridamole, inter alia). Interestingly, efavirenz significantly increased the baseline accumulation of ATV, whereas nevirapine induced a marked reduction. This new enzyme immunoassay for measuring plasma and intracellular ATV levels was fully validated and provides an inexpensive and useful tool for routine therapeutic drug monitoring. Moreover, in vitro results suggested the implication of drug transporters and interactions with other antiviral drugs that should be further explored in human immunodeficiency virus-infected patients

Microenvironment tailors nTreg structure and function.

Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFβ, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts