Background: We used a sensitive and specific ␤-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) assay to determine the relationship between BACE1 activity in cerebrospinal fluid (CSF) and markers of APP metabolism and axonal degeneration in early and late stages of Alzheimer disease (AD).

A hallmark of Alzheimer's disease (AD) brain is the amyloid ␤ (A␤) plaque, which is comprised of A␤ peptides. Multiple lines of evidence suggest that A␤ oligomers are more toxic than other peptide forms. We sought to develop a robust assay to quantify oligomers from CSF. Antibody 19.3 was compared in one-site and competitive ELISAs for oligomer binding specificity. A two-site ELISA for oligomers was developed using 19.3 coupled to a sensitive, bead-based fluorescent platform able to detect single photons of emitted light. The two-site ELISA was Ͼ2500ϫ selective for A␤ oligomers over A␤ monomers with a limit of detection ϳ0.09 pg/ml in human CSF. The lower limit of reliable quantification of the assay was 0.18 pg/ml and the antibody pairs recognized A␤ multimers comprised of either synthetic standards, or endogenous oligomers isolated from confirmed human AD and healthy control brain. Using the assay, a significant 3-to 5-fold increase in A␤ oligomers in human AD CSF compared with comparably aged controls was demonstrated. The increase was seen in three separate human cohorts, totaling 63 AD and 54 controls. CSF oligomers ranged between 0.1 and 10 pg/ml. A␤ oligomer levels did not strongly associate with age or gender, but had an inverse correlation with MMSE score. The C statistic for the A␤ oligomer ROC curve was 0.86, with 80% sensitivity and 88% specificity to detect AD, suggesting reasonable discriminatory power for the AD state and the potential for utility as a diagnostic marker.

The persistence of HIV reservoirs, including latently infected, resting CD4+ T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to m ark CD4+ T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4+ T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV+ or SIV+ subjects, we found that most of the circulating memory CD32+ CD4+ T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a TH2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4+ T cells in peripheral blood or lymphoid tissue; isolated CD32+ resting CD4+ T cells accounted for less than 3% of the total H IV DNA in CD4+ T cells. Cell-associated HIV DNA and RNA loads in CD4+ T cells positively correlated with the frequency of CD32+ CD69+ CD4+ T cells but not with CD32 expression on resting CD4+ T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4+ T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4+ T cells enriched for transcriptionally active HIV after long-term ART.

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␤-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid ␤ peptide A␤42, the putative neurotoxic species in Alzheimer's disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC 50 ϳ 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose-and timedependent lowering of brain A␤40 and A␤42. BACE1 inhibition also led to a robust brain secreted (s)APP␤ lowering that was accompanied by an increase in brain sAPP␣ levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (Ϫ/Ϫ) mice, in agreement with previous studies, this effect was also observed in 15-dayold heterozygous (ϩ/Ϫ) mice, but it was not evident in 30-dayold and 2-year-old BACE1 KO (Ϫ/Ϫ) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant A␤ lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major ␤-site cleavage enzyme for APP and that its inhibition can lower brain A␤ and redirect APP processing via the potentially nonamyloidogenic ␣-secretase pathway, without significantly altering NRG-1 processing.

kill' strategy, for future trials significant enhancement of both 'kick' and 'kill' agents will be required. Research in context panel Evidence before this study This randomised clinical trial was designed to test the concept of 'kick and kill' as a strategy to achieve a cure for HIV infection. Prior to this study, there was evidence from in vitro and single arm clinical studies that the histone deacetylase inhibitor (HDACi) class of drugs could induce viral transcription from latently infected cells, potentially creating a target for the immune system. In conjunction with this 'kick' to the latent HIV reservoir there was evidence that T cell immunitywhich determines HIV disease progression-could be enhanced through vaccination-induced responses, providing the 'kill'. Although the strategy of 'kick and kill' looked promising, there had been no powered RCTs to test it. Added value of the study RIVER tested 'kick and kill' using the HDACi vorinostat as the 'kick' combined with a vaccine strategy targeting conserved regions of the HIV genome. The vaccine aimed to produce T cells to kill latently-infected cells in which viral transcription had been induced by the HDACi. RIVER showed that the intervention was safe, with outstanding adherence to the complex trial protocol by the participants. However, even though there was evidence for both increased histone acetylation and potent vaccine-induced T-cell responses, the intervention did not confer any additional benefit on any measures of the HIV reservoir compared with antiretroviral therapy alone. Implications of all the available evidence. RIVER was the first RCT in treated recent HIV infection, and was not able to show any impact of 'kick and kill' on the primary outcome measure, or any marker of the HIV reservoir size. This is consistent with other studies which had tested HDACi alone. We did not, however, stop antiretroviral therapy in the RIVER trial participants, and future studies may include a treatment interruption as a further measure of impact. Whilst the RIVER trial suggests that this specific 'kick and kill' approach may not be an effective approach towards achieving HIV cure, the overall principle can not yet be dismissed, as more potent future interventions may have a greater impact.

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␤-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A␤42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A␤ lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A␤ in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC 50 ϳ 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A␤ levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A␤ levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP␤, A␤40, A␤42, and plasma A␤40 levels. CSF A␤42 lowering showed an EC 50 of ϳ20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A␤ lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.