Incidence of and risk factors for glaucoma in lost-to-follow-up normal-tension glaucoma suspect patients.

BACKGROUND: To investigate the incidence and risk factors of glaucoma in normal-tension glaucoma (NTG) suspect patients who had been lost-to-follow-up for at least 24 months. METHODS: Seventy-two eyes of 72 NTG suspect patients who returned to the hospital after at least 24 months of follow-up loss were enrolled in this study between January 2009 and June 2013. The data were collected retrospectively. The incidence of glaucoma was investigated using a comprehensive glaucoma evaluation in lost-to-follow-up NTG suspect patients. The patients were classified into the glaucoma group, who developed glaucoma during the study period, and the glaucoma suspect group, who did not, to analyse the risk factors for glaucoma. RESULTS: The number of patients who developed glaucoma was 7 (9.7 %) out of the 72 NTG suspect patients who had been mean lost-to-follow-up for 44 months. The rate of progression from suspected to glaucoma was 2.6 %/year. In the glaucoma group, the baseline intraocular pressure (IOP) was 18.43 ± 2.44 mmHg, and the average retinal nerve fiber layer (RNFL) thickness was 78.14 ± 7.60 µm; in the glaucoma suspect group, the baseline IOP was 14.95 ± 2.47 mmHg, and the average RNFL thickness was 92.55 ± 7.65 µm. The study results showed that the glaucoma group had higher baseline IOP and a thinner average RNFL (p = 0.003; p < 0.001). The results of the multivariable logistic regression analysis showed that the risk factors for glaucoma were high baseline IOP (OR = 1.63; p = 0.037) and a thin average RNFL (OR = 0.841; p = 0.004). CONCLUSIONS: The incidence of glaucoma in the lost-to-follow-up NTG suspect patients was 9.7 % for approximately 44 months, at a rate of 2.6 %/year. The risk factors for glaucoma in these patients were high baseline IOP and a thin average RNFL.

Inhibition of hepatitis C virus (HCV) replication by specific RNA aptamers against HCV NS5B RNA replicase.

This study identified specific and avid RNA aptamers consisting of 2'-hydroxyl- or 2'-fluoropyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication. These aptamers acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity. Cytoplasmic expression of the 2'-hydroxyl aptamer efficiently inhibited HCV replicon replication in human liver cells through specific interaction with, and sequestration of, the target protein without either off-target effects or escape mutant generation. A selected 2'-fluoro aptamer could be truncated to a chemically manufacturable length of 29 nucleotides (nt), with increase in the affinity to HCV NS5B. Noticeably, transfection of the truncated aptamer efficiently suppressed HCV replication in cells without escape mutant appearance. The aptamer was further modified through conjugation of a cholesterol or galactose-polyethylene glycol ligand for in vivo availability and liver-specific delivery. The conjugated aptamer efficiently entered cells and inhibited genotype 1b subgenomic and genotype 2a full-length HCV JFH-1 RNA replication without toxicity and innate immunity induction. Importantly, a therapeutically feasible amount of the conjugated aptamer was delivered in vivo to liver tissue in mice. Therefore, cytoplasmic expression of 2'-hydroxyl aptamer or direct administration of chemically synthesized and ligand-conjugated 2'-fluoro aptamer against HCV NS5B could be a potent anti-HCV approach.

hnRNP L is required for the translation mediated by HCV IRES.

Translation of hepatitis C virus (HCV) RNA is initiated by internal loading of the ribosome into the HCV internal ribosome entry site (IRES). Previously, heterogeneous ribonucleoprotein L (hnRNP L) was shown to bind specifically to the 3' border region of the HCV IRES and enhance HCV mRNA translation. Here, we provide evidence for the functional requirement of hnRNP L for the HCV IRES-mediated translation initiation using specific RNA aptamers. In vitro selection techniques were employed to isolate RNA aptamers against hnRNP L, which were shown to contain consensus sequences with repetitive ACAC/U. The hnRNP L-specific RNA aptamers efficiently inhibited the in vitro translation reactions mediated by the HCV IRES in rabbit reticulocyte lysates. RNA ligands with only (ACAU)5 or (AC)10 nucleotide sequences could also specifically bind to hnRNP L, and specifically and effectively impeded in vitro translation reactions controlled by the HCV IRES. Importantly, the hnRNP L-specific RNA aptamers inhibited the HCV IRES function in cells in a dose-dependent manner, and the aptamer-mediated inhibition of the HCV IRES was considerably relieved by the addition of hnRNP L-expressing vector. These results strongly demonstrate the functional requirement of cellular hnRNP L for the HCV IRES activity.

PET imaging of HER2 expression with an 18F-fluoride labeled aptamer.

BACKGROUND/PURPOSE: Aptamers are oligonucleotide or peptide molecules that bind to a target molecule with high affinity and specificity. The present study aimed to evaluate the target specificity and applicability for in vivo molecular imaging of an aptamer labeled with a radioisotope. METHODS: The human epidermal growth factor receptor 2 (HER2/ErbB2) aptamer was radiolabeled with 18F-fluoride. HER2-positive tumor cell uptake of the aptamer was evaluated in comparison to negative controls by flow cytometry and confocal microscopy. Using 18F-labeled HER2-specific aptamer positron emission tomography (PET), in vivo molecular images of BT474 tumor-bearing mice were taken at 60, 90 and 120 minutes after injection. RESULTS: In flow cytometric analysis, HER2 aptamer showed strong binding to HER2-positive BT474 cells, while binding to HER2-negative MDA-MB231 cells was quite low. Likewise, in confocal microscopic images, the aptamer was bound to HER2-positive breast cancer cells, with minimal binding to HER2-negative cells. In vivo PET molecular imaging of BT474 tumor-bearing mice revealed significant higher uptake of the 18F-labeled HER2 specific aptamer into the tumor compared to the that of HER2-negative cell tumor(p = 0.033). HER2 aptamer was able to preferentially bind to HER2-positive breast cancer cells both in vitro and in vivo, by recognizing HER2 structure on the surface of these cells. CONCLUSION: The 18F-labeled aptamer enabled appropriate visualization of HER2 expression by human breast cancer cells. The results suggest that a radiolabeled HER2 aptamer could potentially be applied in the development of treatment strategies or in targeted therapy against HER2-positive breast cancer cells.

Hybridization-based aptamer labeling using complementary oligonucleotide platform for PET and optical imaging.

Aptamers are promising next-generation ligands used in molecular imaging and theragnosis. Aptamers are synthetic nucleic acids that can be held together with complementary sequences by base-pair hybridization. In this study, the complementary oligonucleotide (cODN) hybridization-based aptamer conjugation platform was developed to use aptamers as the molecular imaging agent. The cODN was pre-labeled with fluorescent dye or radioisotope and hybridized with a matched sequence containing aptamers in aqueous conditions. The cODN platform-hybridized aptamers exhibited good serum stability and specific binding affinity towards target cancer cells both in vitro and in vivo. These results suggest that the newly designed aptamer conjugation platform offers great potential for the versatile application of aptamers as molecular imaging agents.

PI3K-C2α Knockdown Results in Rerouting of Insulin Signaling and Pancreatic Beta Cell Proliferation.

Insulin resistance is a syndrome that affects multiple insulin target tissues, each having different biological functions regulated by insulin. A remaining question is to mechanistically explain how an insulin target cell/tissue can be insulin resistant in one biological function and insulin sensitive in another at the same time. Here, we provide evidence that in pancreatic ß cells, knockdown of PI3K-C2α expression results in rerouting of the insulin signal from insulin receptor (IR)-B/PI3K-C2α/PKB-mediated metabolic signaling to IR-B/Shc/ERK-mediated mitogenic signaling, which allows the ß cell to switch from a highly glucose-responsive, differentiated state to a proliferative state. Our data suggest the existence of IR-cascade-selective insulin resistance, which allows rerouting of the insulin signal within the same target cell. Hence, factors involved in the rerouting of the insulin signal represent tentative therapeutic targets in the treatment of insulin resistance.

Cilostazol protects endothelial cells against lipopolysaccharide-induced apoptosis through ERK1/2- and P38 MAPK-dependent pathways.

BACKGROUND/AIMS: We examined the effects of cilostazol on mitogen-activated protein kinase (MAPK) activity and its relationship with cilostazol-mediated protection against apoptosis in lipopolysaccharide (LPS)-treated endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to LPS and cilostazol with and without specific inhibitors of MAPKs; changes in MAPK activity in association with cell viability and apoptotic signaling were investigated. RESULTS: Cilostazol protected HUVECs against LPS-induced apoptosis by suppressing the mitochondrial permeability transition, cytosolic release of cytochrome c, and subsequent activation of caspases, stimulating extracellullar signal-regulated kinase (ERK1/2) and p38 MAPK signaling, and increasing phosphorylated cAMP-responsive element-binding protein (CREB) and Bcl-2 expression, while suppressing Bax expression. These cilostazol-mediated cellular events were effectively blocked by MAPK/ERK kinase (MEK1/2) and p38 MAPK inhibitors. CONCLUSIONS: Cilostazol protects HUVECs against LPS-induced apoptosis by suppressing mitochondria-dependent apoptotic signaling. Activation of ERK1/2 and p38 MAPKs, and subsequent stimulation of CREB phosphorylation and Bcl-2 expression, may be responsible for the cellular signaling mechanism of cilostazol-mediated protection.