Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer

Development of Methods for Cross-Sectional HIV Incidence Estimation in a Large, Community Randomized Trial

Background Accurate methods of HIV incidence determination are critically needed to monitor the epidemic and determine the population level impact of prevention trials. One such trial, Project Accept, a Phase III, community-randomized trial, evaluated the impact of enhanced, community-based voluntary counseling and testing on population-level HIV incidence. The primary endpoint of the trial was based on a single, cross-sectional, post-intervention HIV incidence assessment. Methods and Findings Test performance of HIV incidence determination was evaluated for 403 multi-assay algorithms [MAAs] that included the BED capture immunoassay [BED-CEIA] alone, an avidity assay alone, and combinations of these assays at different cutoff values with and without CD4 and viral load testing on samples from seven African cohorts (5,325 samples from 3,436 individuals with known duration of HIV infection [1 month to >10 years]). The mean window period (average time individuals appear positive for a given algorithm) and performance in estimating an incidence estimate (in terms of bias and variance) of these MAAs were evaluated in three simulated epidemic scenarios (stable, emerging and waning). The power of different test methods to detect a 35% reduction in incidence in the matched communities of Project Accept was also assessed. A MAA was identified that included BED-CEIA, the avidity assay, CD4 cell count, and viral load that had a window period of 259 days, accurately estimated HIV incidence in all three epidemic settings and provided sufficient power to detect an intervention effect in Project Accept. Conclusions In a Southern African setting, HIV incidence estimates and intervention effects can be accurately estimated from cross-sectional surveys using a MAA. The improved accuracy in cross-sectional incidence testing that a MAA provides is a powerful tool for HIV surveillance and program evaluation

Efforts to Improve the Seasonal Influenza Vaccine

Seasonal influenza is an acute syndrome, principally involving the respiratory tract caused by influenza viruses that are globally present [...

Gut Microbiota as a Driver of Inflammation in Nonalcoholic Fatty Liver Disease

The prevalence of nonalcoholic fatty liver disease and the consequent burden of metabolic syndrome have increased in recent years. Although the pathogenesis of nonalcoholic fatty liver disease is not completely understood, it is thought to be the hepatic manifestation of the dysregulation of insulin-dependent pathways leading to insulin resistance and adipose tissue accumulation in the liver. Recently, the gut-liver axis has been proposed as a key player in the pathogenesis of NAFLD, as the passage of bacteria-derived products into the portal circulation could lead to a trigger of innate immunity, which in turn leads to liver inflammation. Additionally, higher prevalence of intestinal dysbiosis, larger production of endogenous ethanol, and higher prevalence of increased intestinal permeability and bacterial translocation were found in patients with liver injury. In this review, we describe the role of intestinal dysbiosis in the activation of the inflammatory cascade in NAFLD

Assembly of Biologically Functional Structures by Nucleic Acid Templating: Implementation of a Strategy to Overcome Inhibition by Template Excess

Delivery of therapeutic molecules to pathogenic cells is often hampered by unintended toxicity to normal cells. In principle, this problem can be circumvented if the therapeutic effector molecule is split into two inactive components, and only assembled on or within the target cell itself. Such an in situ process can be realized by exploiting target-specific molecules as templates to direct proximity-enhanced assembly. Modified nucleic acids carrying inert precursor fragments can be designed to co-hybridize on a target-specific template nucleic acid, such that the enforced proximity accelerates assembly of a functional molecule for antibody recognition. We demonstrate the in vitro feasibility of this adaptation of nucleic acid-templated synthesis (NATS) using oligonucleotides bearing modified peptides (“haplomers”), for templated assembly of a mimotope recognized by the therapeutic antibody trastuzumab. Enforced proximity promotes mimotope assembly via traceless native chemical ligation. Nevertheless, titration of participating haplomers through template excess is a potential limitation of trimolecular NATS. In order to overcome this problem, we devised a strategy where haplomer hybridization can only occur in the presence of target, without being subject to titration effects. This generalizable NATS modification may find future applications in enabling directed targeting of pathological cells

Kinetics of Melan-A/MART-1 increase.

<p>The flow cytometry data show effect of four Hsp90 inhibitors on the MU89 MART::EGFP cell line at the indicated doses as assessed over time. The same number of cells per well were plated in each well of a 24 well plate and drug was added on day zero. Each day cells were collected and assayed for that time point. Control untreated cells are shown for comparison. The data are from one representative experiment.</p

Effect of Hsp90 Inhbitors on differentiation antigens and MHC Class I.

a<p>Cells were untreated (control) or treated with 5000 Units/ml of IFN-beta, or with the Hsp90 inhibitors as indicated for 3 days.</p>b<p>Dose indicated is optimal dose for antigen increase</p>c<p>Number represents geometric mean of intracellular staining with an antibody to Melan-A/MART-1, gp100 or TRP-2 of live gated cells. Number is parenthesis is fold increase relative to untreated control.</p>d<p>Number represents geometric mean of surface staining with the MHC Class I antibody W6/32 (or H2kb for B16) of live gated cells.</p><p>Antigen status of human melanoma cell lines: (+)  =  Melan-A/MART-1and gp100 high (-) = Melan-A/MART-1and gp100 low.</p><p>n.a.  =  not applicable, glioma do not express Melan-A/MART-1, and human Melan-A/MART-1 antibody did not cross react with murine Melan-A/MART-1</p><p>n.d.  =  not determined; these cells were not stained with the TRP-2 antibody.</p><p>Effect of Hsp90 Inhbitors on differentiation antigens and MHC Class I.</p

Effect of Hsp90 inhibition on MU89 growth.

<p>A WST assay was used to assess cell numbers in control and Hsp90-inhibitor treated tumors. WST levels were assayed at time zero and after 3 days. Cells were treated with the indicated Hsp90 inhibitors at the doses indicated. Percent growth was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114506#s2" target="_blank">Methods</a> and is plotted on the left y-axis. Data represent the average and standard deviation of triplicate wells. The level of Melan-A/MART-1 (geometric mean), as assayed by intracellular staining and flow cytometry, is shown for comparison on the right y-axis. The data for Melan-A/MART-1 staining are from one representative experiment.</p

Effect of Hsp90 Inhibitors on Melan-A/MART-1 promoter.

<p>Data shown are flow cytometry-generated histograms of EGFP production in reporter cell lines with EGFP linked to the Melan-A/MART-1 promoter. In each histogram, the thin line curve represents the untreated control, and bold line is Hsp90 inhibitor treated cells. In each case, the reporter cells were treated for three days prior to assessing EGFP-related fluorescence. Data are from one representative experiment. The first and third columns are low antigen A375 cells and the second and fourth columns are high antigen-expressing MM96L+ cells. Doses of Hsp90 inhibitor used are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114506#pone-0114506-t001" target="_blank">Table 1</a>.</p