BackgroundMacrophages, which are CD4 and CCR5 positive, can sustain HIV-1 replication for long periods of time. Thus, these cells play critical roles in the transmission, dissemination and persistence of viral infection. Of note, current antiviral therapies do not target macrophages efficiently. Previously, it was demonstrated that interactions between CCR5 and gp120 stimulate PKC. However, the PKC isozymes involved were not identified.ResultsIn this study, we identified PKC-delta as a major cellular cofactor for HIV-1 replication in macrophages. Indeed, PKC-delta was stimulated following the interaction between the virus and its target cell. Moreover, inhibition of PKC-delta blocked the replication of R5-tropic viruses in primary human macrophages. However, this inhibition did not have significant effects on receptor and co-receptor expression or fusion. Additionally, it did not affect the formation of the early reverse transcription product containing R/U5 sequences, but did inhibit the synthesis of subsequent cDNAs. Importantly, the inhibition of PKC-delta altered the redistribution of actin, a cellular cofactor whose requirement for the completion of reverse transcription was previously established. It also prevented the association of the reverse transcription complex with the cytoskeleton.ConclusionThis work highlights the importance of PKC-delta during early steps of the replicative cycle of HIV-1 in human macrophages.

The objective of this project was to study the interaction between HR1 and HR2, the stability of the complex formed, and to characterize the antibodies produced against monomeric HR1 and HR2 peptides as well as the HR1-HR2 complex. In this work, HR1 was mimicked by peptide N36, and HR2 was mimicked by peptide C34L and its analogues C34M2, C34M3, and C34D. Whereas C34M2 and C34M3 are partially composed of D-amino acids, C34D has same sequence as C34L, but is assembled entirely of D-amino acids. Using CD analysis, SPR assays, and gel filtration chromatography, we demonstrate the physical interaction between N36 and C34L and its analogues C34M2 and C34M3, but not C34D. We show that the HR1-HR2 complex is formed rapidly (<1 min) and remains stable, as demonstrated by its inability, in contrast to each free peptide, to inhibit the formation of syncytia. To generate antibodies with predetermined specificity against the transiently exposed intermediate that corresponds to the six-helix bundle structure, purified preformed HR1-HR2 complex was used, in parallel with monomeric HR1 and HR2 peptides, as immunogens in mice. Although the produced antibodies recognize total HIV-1 envelope glycoproteins in ELISA, they are unable to neutralize HIV-1-mediated fusion at 37 °C. However, if the incubation with these antibodies is carried out at 27 °C, a temperature that allows stabilization of the transient intermediate complex, anti-peptide antibodies are able to bind their corresponding domains in HeLa cells expressing HIV-1 gp41 in co-culture with HeLa CD4-CCR5/CXCR4 during the dynamic mechanism of membrane fusion. In agreement with the latter results, these antibodies, if previously incubated for 2 h at 27 °C, are able to strongly neutralize HIV-1 entry by membrane fusion, as shown by their ability to block the formation of syncytia.

The trimeric heptad repeat domains HR1 and HR2 of the human immunodeficiency virus 1 (HIV-1) gp41 play a key role in HIV-1-entry by membrane fusion. To develop efficient inhibitors against this step, the corresponding trimeric-N36 and C34 peptides were designed and synthesized. Analysis by circular dichroism of monomeric and trimeric N36 and C34 peptides showed their capacities to adopt α-helical structures and to establish physical interactions. At the virological level, while trimeric-C34 conserves the same high anti-fusion activity as monomeric-C34, trimerization of N36-peptide induced a significant increase, reaching 500-times higher in anti-fusion activity, against R5-tropic virus-mediated fusion. This result was associated with increased stability of the N36 trimer peptide with respect to the monomeric form, as demonstrated by the comparative kinetics of their antiviral activities during 6-day incubation in a physiological medium. Collectively, our findings demonstrate that while the trimerization of C34 peptide had no beneficial effect on its stability and antiviral activity, the trimerization of N36 peptide strengthened both stability and antiviral activity. This approach, promotes trimers as new promising HIV-1 inhibitors and point to future development aimed toward innovative peptide fusion inhibitors, microbicides or as immunogens.

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A495outcomes evaluated included change from baseline in trough forced expiratory volume in 1 second (FEV1) and COPD exacerbations leading to hospitalizations. Efficacy of tiotropium was presented as the mean difference to placebo. Results: Difference in change from baseline in FEV1 versus placebo was evaluated in 21 RCTs. The relative effect of tiotropium increased over time, ranging from 0.084 to 0.150 in 2000 (launch) and from 0.120 to 0.189 in 2012. Difference in rates of exacerbations versus placebo was reported in 24 studies. The relative effect of tiotropium was found to decrease over time for this outcome. This trend was partly explained by an increase in placebo effect. ConClusions: We identified variations in the relative efficacy of tiotropium over time on two major outcomes for assessing drug relativeness effectiveness in COPD. Further research is required to understand these changes and the implications for evaluating the relative effectiveness of a new drug versus already marketed ones. Time of marketing authorization may not be the appropriate time to assess the relative effectiveness of a new drug.

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