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BACKGROUND - Proteomic approaches allow measurement of thousands of proteins in a single specimen, which can accelerate biomarker discovery. However, applying these technologies to massive biobanks is not currently feasible because of the practical barriers and costs of implementing such assays at scale. To overcome these challenges, we used a "virtual proteomic" approach, linking genetically predicted protein levels to clinical diagnoses in >40 000 individuals.
METHODS - We used genome-wide association data from the Framingham Heart Study (n=759) to construct genetic predictors for 1129 plasma protein levels. We validated the genetic predictors for 268 proteins and used them to compute predicted protein levels in 41 288 genotyped individuals in the Electronic Medical Records and Genomics (eMERGE) cohort. We tested associations for each predicted protein with 1128 clinical phenotypes. Lead associations were validated with directly measured protein levels and either low-density lipoprotein cholesterol or subclinical atherosclerosis in the MDCS (Malmö Diet and Cancer Study; n=651).
RESULTS - In the virtual proteomic analysis in eMERGE, 55 proteins were associated with 89 distinct diagnoses at a false discovery rate q<0.1. Among these, 13 associations involved lipid (n=7) or atherosclerosis (n=6) phenotypes. We tested each association for validation in MDCS using directly measured protein levels. At Bonferroni-adjusted significance thresholds, levels of apolipoprotein E isoforms were associated with hyperlipidemia, and circulating C-type lectin domain family 1 member B and platelet-derived growth factor receptor-β predicted subclinical atherosclerosis. Odds ratios for carotid atherosclerosis were 1.31 (95% CI, 1.08-1.58; P=0.006) per 1-SD increment in C-type lectin domain family 1 member B and 0.79 (0.66-0.94; P=0.008) per 1-SD increment in platelet-derived growth factor receptor-β.
CONCLUSIONS - We demonstrate a biomarker discovery paradigm to identify candidate biomarkers of cardiovascular and other diseases.
Parietal cell atrophy is considered to cause metaplasia in the stomach. We developed mice that express the diphtheria toxin receptor specifically in parietal cells to induce their death, and found this to increase proliferation in the normal stem cell zone and neck but not to cause metaplastic reprogramming of chief cells. Furthermore, the metaplasia-inducing agents tamoxifen or DMP-777 still induced metaplasia even after previous destruction of parietal cells by diphtheria toxin. Atrophy of parietal cells alone therefore is not sufficient to induce metaplasia: completion of metaplastic reprogramming of chief cells requires mechanisms beyond parietal cell injury or death.
Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.
The glandular stomach has two major zones: the acid secreting corpus and the gastrin cell-containing antrum. Nevertheless, a single gland lies at the transition between the forestomach and corpus in the mouse stomach. We have sought to define the lineages that make up this gland unit at the squamocolumnar junction. The first gland in mice showed a notable absence of characteristic corpus lineages, including parietal cells and chief cells. In contrast, the gland showed strong staining of Griffonia simplicifolia-II (GSII)-lectin-positive mucous cells at the bases of glands, which were also positive for CD44 variant 9 and Clusterin. Prominent numbers of doublecortin-like kinase 1 (DCLK1) positive tuft cells were present in the first gland. The first gland contained Lgr5-expressing putative progenitor cells, and a large proportion of the cells were positive for Sox2. The cells of the first gland stained strongly for MUC4 and EpCAM, but both were absent in the normal corpus mucosa. The present studies indicate that the first gland in the corpus represents a unique anatomic entity. The presence of a concentration of progenitor cells and sensory tuft cells in this gland suggests that it may represent a source of reserve reparative cells for adapting to severe mucosal damage.
The two-pore-domain potassium (K2P) channel TREK-2 serves to modulate plasma membrane potential in dorsal root ganglia c-fiber nociceptors, which tunes electrical excitability and nociception. Thus, TREK-2 channels are considered a potential therapeutic target for treating pain; however, there are currently no selective pharmacological tools for TREK-2 channels. Here we report the identification of the first TREK-2 selective activators using a high-throughput fluorescence-based thallium (Tl) flux screen (HTS). An initial pilot screen with a bioactive lipid library identified 11-deoxy prostaglandin F2α as a potent activator of TREK-2 channels (EC ≈ 0.294 μM), which was utilized to optimize the TREK-2 Tl flux assay (Z' = 0.752). A HTS was then performed with 76 575 structurally diverse small molecules. Many small molecules that selectively activate TREK-2 were discovered. As these molecules were able to activate single TREK-2 channels in excised membrane patches, they are likely direct TREK-2 activators. Furthermore, TREK-2 activators reduced primary dorsal root ganglion (DRG) c-fiber Ca influx. Interestingly, some of the selective TREK-2 activators such as 11-deoxy prostaglandin F2α were found to inhibit the K2P channel TREK-1. Utilizing chimeric channels containing portions of TREK-1 and TREK-2, the region of the TREK channels that allows for either small molecule activation or inhibition was identified. This region lies within the second pore domain containing extracellular loop and is predicted to play an important role in modulating TREK channel activity. Moreover, the selective TREK-2 activators identified in this HTS provide important tools for assessing human TREK-2 channel function and investigating their therapeutic potential for treating chronic pain.
Serine-rich repeat glycoproteins are adhesins expressed by commensal and pathogenic Gram-positive bacteria. A subset of these adhesins, expressed by oral streptococci, binds sialylated glycans decorating human salivary mucin MG2/MUC7, and platelet glycoprotein GPIb. Specific sialoglycan targets were previously identified for the ligand-binding regions (BRs) of GspB and Hsa, two serine-rich repeat glycoproteins expressed by Streptococcus gordonii While GspB selectively binds sialyl-T antigen, Hsa displays broader specificity. Here we examine the binding properties of four additional BRs from Streptococcus sanguinis or Streptococcus mitis and characterize the molecular determinants of ligand selectivity and affinity. Each BR has two domains that are essential for sialoglycan binding by GspB. One domain is structurally similar to the glycan-binding module of mammalian Siglecs (sialic acid-binding immunoglobulin-like lectins), including an arginine residue that is critical for glycan recognition, and that resides within a novel, conserved YTRY motif. Despite low sequence similarity to GspB, one of the BRs selectively binds sialyl-T antigen. Although the other three BRs are highly similar to Hsa, each displayed a unique ligand repertoire, including differential recognition of sialyl Lewis antigens and sulfated glycans. These differences in glycan selectivity were closely associated with differential binding to salivary and platelet glycoproteins. Specificity of sialoglycan adherence is likely an evolving trait that may influence the propensity of streptococci expressing Siglec-like adhesins to cause infective endocarditis.
Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.
IL-15 is currently undergoing clinical trials to assess its efficacy for treatment of advanced cancers. The combination of IL-15 with soluble IL-15Rα generates a complex termed IL-15 superagonist (IL-15 SA) that possesses greater biological activity than IL-15 alone. IL-15 SA is considered an attractive antitumor and antiviral agent because of its ability to selectively expand NK and memory CD8(+) T (mCD8(+) T) lymphocytes. However, the adverse consequences of IL-15 SA treatment have not been defined. In this study, the effect of IL-15 SA on physiologic and immunologic functions of mice was evaluated. IL-15 SA caused dose- and time-dependent hypothermia, weight loss, liver injury, and mortality. NK (especially the proinflammatory NK subset), NKT, and mCD8(+) T cells were preferentially expanded in spleen and liver upon IL-15 SA treatment. IL-15 SA caused NK cell activation as indicated by increased CD69 expression and IFN-γ, perforin, and granzyme B production, whereas NKT and mCD8(+) T cells showed minimal, if any, activation. Cell depletion and adoptive transfer studies showed that the systemic toxicity of IL-15 SA was mediated by hyperproliferation of activated NK cells. Production of the proinflammatory cytokine IFN-γ, but not TNF-α or perforin, was essential to IL-15 SA-induced immunotoxicity. The toxicity and immunological alterations shown in this study are comparable to those reported in recent clinical trials of IL-15 in patients with refractory cancers and advance current knowledge by providing mechanistic insights into IL-15 SA-mediated immunotoxicity.
Copyright © 2015 by The American Association of Immunologists, Inc.
The histologic differential diagnosis between intramuscular myxoma and low-grade myxofibrosarcoma can be quite difficult in some cases. To identify a diagnostic immunohistochemical marker, we compared the staining profiles of 19 different antigens, including cell cycle proteins, apoptosis proteins, and proliferative markers, and selected other signaling and structural proteins in these two tumors. Ten cases each of intramuscular myxoma and low-grade myxofibrosarcoma were stained with antibodies directed against apoptosis regulatory proteins (Bcl2, activated caspase-3, phospho-H2A.X, and cleaved PARP), cell cycle regulatory proteins (Rb1, Cyclin-A, CDKN1B, and Cdt1), proliferative markers (KI67, MCM2, phospho-histone H3, and geminin), cell signalling molecules (c-Myc, EGF, EGFR, PLA2G4A, and HSP90), a dendritic cell marker (CD209), and the extracellular matrix proteoglycan decorin. Staining patterns of myxoma and myxofibrosarcoma were compared using Fisher's exact test and the Mann-Whitney test. For each potential diagnostic marker studied, the proportions of cases scored as positive on both dichotomous or ordinal scales were not significantly different between myxoma and myxofibrosarcoma. Myxoma and myxofibrosarcoma share a common immunophenotype for each of the markers studied. Distinction between these tumors is still predominantly based on morphologic criteria.
Current histopathologic classification schemes for gastric adenocarcinoma have limited clinical utility and are difficult to apply due to tumor heterogeneity. Elucidation of molecular subtypes of gastric cancer may contribute to our understanding of gastric cancer biology and to the development of new molecular markers that may lead to improved diagnosis, therapy, or prognosis. We previously demonstrated that Epstein-Barr virus (EBV)-infected gastric cancers have a distinct human gene expression profile compared with uninfected cancers. We now examine the histopathologic features characterizing infected (n=14) and uninfected (n=89) cancers; the latter of which are now further divided into 2 major molecular subtypes based on expression patterns of 93 RNAs. One uninfected gastric cancer subtype was distinguished by upregulation of 3 genes with neuroendocrine (NE) function (CHGA, GAST, and REG4 encoding chromogranin, gastrin, and the secreted peptide REG4 involved in epithelial cell regeneration), implicating hormonal factors in the pathogenesis of a major class of gastric adenocarcinomas. Evidence of NE differentiation (molecular, immunohistochemical, or morphologic) was mutually exclusive of EBV infection. EBV-infected tumors tended to have solid-type morphology with lymphoid stroma. This study reveals novel molecular subtypes of gastric cancer and their associated morphologies that demonstrate divergent NE features.
We have been investigating an imaging agent that enables real-time and accurate diagnosis of early colorectal cancer at the intestinal mucosa by colonoscopy. The imaging agent is peanut agglutinin-immobilized polystyrene nanospheres with surface poly(N-vinylacetamide) chains encapsulating coumarin 6. Intracolonically-administered lectin-immobilized fluorescent nanospheres detect tumor-derived changes through molecular recognition of lectin for the terminal sugar of cancer-specific antigens on the mucosal surface. The focus of the present study was to evaluate imaging abilities of the nanospheres in animal models that reflect clinical environments. We previously developed an orthotopic mouse model with human colorectal tumors growing on the mucosa of the descending colon to better resemble the clinical disease. The entire colon of the mice in the exposed abdomen was monitored in real time with an in vivo imaging apparatus. Fluorescence from the nanospheres was observed along the entire descending colon after intracolonical administration from the anus. When the luminal side of the colon was washed with phosphate-buffered saline, most of the nanospheres were flushed. However, fluorescence persisted in areas where cancer cells were implanted. Histological evaluation demonstrated that tumors were present in the mucosal epithelia where the nanospheres fluoresced. In contrast, no fluorescence was observed when control mice, without tumors were tested. The lectin-immobilized fluorescent nanospheres were tumor-specific and remained bound to tumors even after vigorous washing. The nanospheres nonspecifically bound to normal mucosa were easily removed through mild washing. These results indicate that the nanospheres combined with colonoscopy, will be a clinically-valuable diagnostic tool for early-stage primary colon carcinoma.
Copyright © 2014 John Wiley & Sons, Ltd.
Realizing personalized medicine, which promises to enable early disease detection, efficient diagnostic staging, and therapeutic efficacy monitoring, hinges on biomarker quantification in patient samples. Yet, the lack of a sensitive technology and assay methodology to rapidly validate biomarker candidates continues to be a bottleneck for clinical translation. In our first direct and quantitative comparison of backscattering interferometry (BSI) to fluorescence sensing by ELISA, we show that BSI could aid in overcoming this limitation. The analytical validation study was performed against ELISA for two biomarkers for lung cancer detection: Cyfra 21-1 and Galectin-7. Spiked serum was used for calibration and comparison of analytical figures of merit, followed by analysis of blinded patient samples. Using the ELISA antibody as the probe chemistry in a mix-and-read assay, BSI provided significantly lower detection limits for spiked serum samples with each of the biomarkers. The limit of quantification (LOQ) for Cyrfa-21-1 was measured to be 230 pg/mL for BSI versus 4000 pg/mL for ELISA, and for Galectin-7, it was 13 pg/mL versus 500 pg/mL. The coefficient of variation for 5 day, triplicate determinations was <15% for BSI and <10% for ELISA. The two techniques correlated well, ranging from 3-29% difference for Cyfra 21-1 in a blinded patient sample analysis. The label-free and free-solution operation of BSI allowed for a significant improvement in analysis speed, with greater ease, improved LOQ values, and excellent day-to-day reproducibility. In this unoptimized format, BSI required 5.5-fold less sample quantity needed for ELISA (a 10 point calibration curve measured in triplicate required 36 μL of serum for BSI vs 200 μL for ELISA). The results indicate that the BSI platform can enable rapid, sensitive analytical validation of serum biomarkers and should significantly impact the validation bottleneck of biomarkers.