Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

Inhibition of in-stent restenosis by oral copper chelation in porcine coronary arteries

Stress-induced release of IL-1alpha and fibroblast growth factor-1 is dependent on intracellular copper and is a major driver of neointimal hyperplasia. Therefore, we assessed the effect of tetrathiomolybdate (TTM), a clinically proven copper chelator, on in-stent restenosis. Nine pigs were treated with TTM (5 mg/kg po) twice daily for 2 wk before stent implantation and for 4 wk thereafter, and nine pigs served as controls. In-stent restenosis was assessed by quantitative coronary angiography (QCA), intravascular ultrasound (IVUS), and histomorphometry. Serum ceruloplasmin activity was used as a surrogate marker of copper bioavailability. In TTM-treated animals, ceruloplasmin dropped 70 +/- 10% below baseline levels. Baseline characteristics were comparable in TTM-treated and control animals. At 4-wk follow-up, all parameters relevant to in-stent restenosis were significantly reduced in TTM-treated animals: minimal lumen diameter by QCA was 2.03 +/- 0.57 and 1.47 +/- 0.45 mm in TTM-treated and control animals, respectively (P < 0.05), percent stenosis diameter was 39% less in TTM-treated animals (27.1 +/- 16.6% vs. 44.5 +/- 16.1%, P < 0.05), minimal lumen area by IVUS was 60% larger in TTM-treated animals (4.27 +/- 1.56 vs. 2.67 +/- 1.19 mm(2), P < 0.05), and neointimal volume by histomorphometry was 37% less in TTM-treated animals (34.9 +/- 11.5 vs. 55.2 +/- 19.6 mm(3), P < 0.05). We conclude that systemic copper chelation with a clinically approved chelator significantly inhibits in-stent restenosis.     

Selective regulation of arterial branching morphogenesis by synectin

Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.     

Overcoming immunity to a viral vaccine by DNA priming before vector boosting

Replication-defective adenovirus (ADV) and poxvirus vectors have shown potential as vaccines for pathogens such as Ebola or human immunodeficiency virus in nonhuman primates, but prior immunity to the viral vector in humans may limit their clinical efficacy. To overcome this limitation, the effect of prior viral exposure on immune responses to Ebola virus glycoprotein (GP), shown previously to protect against lethal hemorrhagic fever in animals, was studied. Prior exposure to ADV substantially reduced the cellular and humoral immune responses to GP expressed by ADV, while exposure to vaccinia inhibited vaccine-induced cellular but not humoral responses to GP expressed by vaccinia. This inhibition was largely overcome by priming with a DNA expression vector before boosting with the viral vector. Though heterologous viral vectors for priming and boosting can also overcome this effect, the paucity of such clinical viral vectors may limit their use. In summary, it is possible to counteract prior viral immunity by priming with a nonviral, DNA vaccine.     

Reward ignorant modeling of dynamic treatment regimes

Personalized medicine optimizes patient outcome by tailoring treatments to patient‐level characteristics. This approach is formalized by dynamic treatment regimes (DTRs): decision rules that take patient information as input and output recommended treatment decisions. The DTR literature has seen the development of increasingly sophisticated causal inference techniques that attempt to address the limitations of our typically observational datasets. Often overlooked, however, is that in practice most patients may be expected to receive optimal or near‐optimal treatment, and so the outcome used as part of a typical DTR analysis may provide limited information. In light of this, we propose considering a more standard analysis: ignore the outcome and elicit an optimal DTR by modeling the observed treatment as a function of relevant covariates. This offers a far simpler analysis and, in some settings, improved optimal treatment identification. To distinguish this approach from more traditional DTR analyses, we term it reward ignorant modeling, and also introduce the concept of multimethod analysis, whereby different analysis methods are used in settings with multiple treatment decisions. We demonstrate this concept through a variety of simulation studies, and through analysis of data from the International Warfarin Pharmacogenetics Consortium, which also serve as motivation for this work.     

Response definition criteria for ELISPOT assays revisited

No consensus has been reached on how to determine if an immune response has been detected based on raw data from an ELISPOT assay. The goal of this paper is to enable investigators to understand and readily implement currently available methods for response determination. We describe empirical and statistical approaches, identifying the strengths and limitations of each approach to allow readers to rationally select and apply a scientifically sound method appropriate to their specific laboratory setting. Five representative approaches were applied to data sets from the CIMT Immunoguiding Program and the response detection and false positive rates were compared. Simulation studies were also performed to compare empirical and statistical approaches. Based on these, we recommend the use of a non-parametric statistical test. Further, we recommend that six medium control wells or four wells each for both medium control and experimental conditions be performed to increase the sensitivity in detecting a response, that replicates with large variation in spot counts be filtered out, and that positive responses arising from experimental spot counts below the estimated limit of detection be interpreted with caution. Moreover, a web-based user interface was developed to allow easy access to the recommended statistical methods. This interface allows the user to upload data from an ELISPOT assay and obtain an output file of the binary responses.     

Effect of rAd5-Vector HIV-1 Preventive Vaccines on HIV-1 Acquisition: A Participant-Level Meta-Analysis of Randomized Trials

Background

Three phase 2b, double-blind, placebo-controlled, randomized efficacy trials have tested recombinant Adenovirus serotype-5 (rAd5)-vector preventive HIV-1 vaccines: MRKAd5 HIV-1 gag/pol/nef in Step and Phambili, and DNA/rAd5 HIV-1 env/gag/pol in HVTN505. Due to efficacy futility observed at the first interim analysis in Step and HVTN505, participants of all three studies were unblinded to their vaccination assignments during the study but continued follow–up. Rigorous meta-analysis can provide crucial information to advise the future utility of rAd5-vector vaccines.

Methods

We included participant-level data from all three efficacy trials, and three Phase 1–2 trials evaluating the HVTN505 vaccine regimen. We predefined two co-primary analysis cohorts for assessing the vaccine effect on HIV-1 acquisition. The modified-intention-to-treat (MITT) cohort included all randomly assigned participants HIV-1 uninfected at study entry, who received at least the first vaccine/placebo, and the Ad5 cohort included MITT participants who received at least one dose of rAd5-HIV vaccine or rAd5-placebo. Multivariable Cox regression models were used to estimate hazard ratios (HRs) of HIV-1 infection (vaccine vs. placebo) and evaluate HR variation across vaccine regimens, time since vaccination, and subgroups using interaction tests.

Findings

Results are similar for the MITT and Ad5 cohorts; we summarize MITT cohort results. Pooled across the efficacy trials, over all follow-up time 403 (n = 224 vaccine; n = 179 placebo) of 6266 MITT participants acquired HIV-1, with a non-significantly higher incidence in vaccine recipients (HR 1.21, 95% CI 0.99–1.48, P = 0.06). The HRs significantly differed by vaccine regimen (interaction P = 0.03; MRKAd5 HR 1.41, 95% CI 1.11–1.78, P = 0.005 vs. DNA/rAd5 HR 0.88, 95% CI 0.61–1.26, P = 0.48). Results were similar when including the Phase 1–2 trials. Exploratory analyses based on the efficacy trials supported that the MRKAd5 vaccine-increased risk was concentrated in Ad5-positive or uncircumcised men early in follow-up, and in Ad5-negative or circumcised men later. Overall, MRKAd5 vaccine-increased risk was evident across subgroups except in circumcised Ad5-negative men (HR 0.97, 95% CI 0.58−1.63, P = 0.91); there was little evidence that the DNA/rAd5 vaccine, that was tested in this subgroup, increased risk (HR 0.88, 95% CI 0.61–1.26, P = 0.48). When restricting the analysis of Step and Phambili to follow-up time before unblinding, 114 (n = 65 vaccine; n = 49 placebo) of 3770 MITT participants acquired HIV-1, with a non-significantly higher incidence in MRKAd5 vaccine recipients (HR 1.30, 95% CI 0.89–1.14, P = 0.18).

Interpretation and Significance

The data support increased risk of HIV-1 infection by MRKAd5 over all follow-up time, but do not support increased risk of HIV-1 infection by DNA/rAd5. This study provides a rationale for including monitoring plans enabling detection of increased susceptibility to infection in HIV-1 at-risk populations.