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Kirwin S.J.,DuPont Company | Kirwin S.J.,Eye-Q | Kanaly S.T.,Allergan, Inc. | Hansen C.R.,DuPont Company | And 4 more authors.
Investigative Ophthalmology and Visual Science | Year: 2011

Purpose. Patients with diabetic retinopathy may experience severe vision loss due to macular edema and neovascularization secondary to vascular abnormalities. However, before these abnormalities become apparent, there are functional deficits in contrast sensitivity, color perception, and dark adaptation. The goals of this study are to evaluate early changes (up to 3 months) in retinal gene expression, selected visual cycle proteins, and optokinetic tracking (OKT) in streptozotocin (STZ)-induced diabetic rats. Methods. Retinal gene expression in diabetic Long Evans rats was measured by whole genome microarray 7 days, 4 weeks, and 3 months after the onset of hyperglycemia. Select gene and protein changes were probed by polymerase chain reaction (PCR) and immunohistochemistry, respectively, and OKT thresholds were measured using a virtual optokinetics system. Results. Microarray analysis showed that the most consistently affected molecular and cellular functions were cell-to-cell signaling and interaction, cell death, cellular growth and proliferation, molecular transport, and cellular movement. Further analysis revealed reduced expression of several genes encoding visual cycle proteins including lecithin/retinol acyltransferase (LRAT), retinal pigment epithelium (RPE)-specific protein 65 kDa (RPE65), and RPE retinal G protein-coupled receptor (RGR). These molecular changes occurred simultaneously with a decrease in OKT thresholds by 4 weeks of diabetes. Immunohistochemistry revealed a decrease in RPE65 in the RPE layer of diabetic rats after 3 months of hyperglycemia. Conclusions. The data presented here are further evidence that inner retinal cells are affected by hyperglycemia simultaneously with blood retinal barrier breakdown, suggesting that glial and neuronal dysfunction may underlie some of the early visual deficits in persons with diabetes. © 2011 The Association for Research in Vision and Ophthalmology, Inc.

Drake A.W.,Takeda San Francisco Inc. | Klakamp S.L.,Takeda San Francisco Inc.
Journal of Immunological Methods | Year: 2011

Determining the optimal conditions for surface regeneration is fundamental for performance of efficient and robust protein-protein interaction kinetic studies. We devised a systematic methodology comprised of an automated seven-cycle analyte and buffer injection Biacore scheme and data interpretation algorithm. The efficiency and utility is illustrated using an antigen/monoclonal antibody interaction that required ultimately six pulses of acid for regeneration. This technique has broad applicability to any biosensor assay that requires regeneration of a surface. © 2011 Elsevier B.V.

Sato S.,Takeda San Francisco Inc. | Sato S.,Takeda Pharmaceutical | Kamada H.,Takeda Pharmaceutical | Watanabe T.,Takeda Pharmaceutical | And 2 more authors.
PLoS ONE | Year: 2013

Purpose: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The membrane-bound proHB-EGF is known to be a precursor of the soluble form of HB-EGF (sHB-EGF), which promotes cell proliferation and survival. While the functions of sHB-EGF have been extensively studied, it is not yet fully understood if proHB-EGF is also involved in cellular signaling events. In this study, we utilized the anti-HB-EGF monoclonal antibodies Y-142 and Y-073, which have differential specificities toward proHB-EGF, in order to elucidate proHB-EGF functions in cancer cells. Experimental Design: The biological activities of proHB-EGF were assessed in cell proliferation, caspase activation, and juxtacrine activity assays by using a 3D spheroid culture of NUGC-3 cells. Results: Y-142 and Y-073 exhibited similar binding and neutralizing activities for sHB-EGF. However, only Y-142 bound to proHB-EGF. We could detect the function of endogenously expressed proHB-EGF in a 3D spheroid culture. Blocking proHB-EGF with Y-142 reduced spheroid formation, suppressed cell proliferation, and increased caspase activation in the 3D spheroid culture of NUGC-3 cells. Conclusions: Our results show that proHB-EGF acts as a cell proliferation and cell survival factor in cancer cells. The results suggest that proHB-EGF may play an important role in tumor progression. © 2013 Sato et al.

Sato S.,Takeda San Francisco Inc. | Sato S.,Takeda Pharmaceutical | Drake A.W.,Takeda San Francisco Inc. | Tsuji I.,Takeda Pharmaceutical | Fan J.,Takeda San Francisco Inc.
PLoS ONE | Year: 2012

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathological functions. Modulation of HB-EGF activity might have a therapeutic potential in the oncology area. We explored the therapeutic possibilities by characterizing the in vitro biological activity of anti-HB-EGF monoclonal antibody Y-142. EGF receptor (EGFR) ligand and species specificities of Y-142 were tested. Neutralizing activities of Y-142 against HB-EGF were evaluated in EGFR and ERBB4 signaling. Biological activities of Y-142 were assessed in cancer cell proliferation and angiogenesis assays and compared with the anti-EGFR antibody cetuximab, the HB-EGF inhibitor CRM197, and the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab. The binding epitope was determined with alanine scanning. Y-142 recognized HB-EGF as well as the EGFR ligand amphiregulin, and bound specifically to human HB-EGF, but not to rodent HB-EGF. In addition, Y-142 neutralized HB-EGF-induced phosphorylation of EGFR and ERBB4, and blocked their downstream ERK1/2 and AKT signaling. We also found that Y-142 inhibited HB-EGF-induced cancer cell proliferation, endothelial cell proliferation, tube formation, and VEGF production more effectively than cetuximab and CRM197 and that Y-142 was superior to bevacizumab in the inhibition of HB-EGF-induced tube formation. Six amino acids in the EGF-like domain were identified as the Y-142 binding epitope. Among the six amino acids, the combination of F115 and Y123 determined the amphiregulin cross-reactivity and that F115 accounted for the species selectivity. Furthermore, it was suggested that the potent neutralizing activity of Y-142 was derived from its recognition of R142 and Y123 and its high affinity to HB-EGF. Y-142 has a potent HB-EGF neutralizing activity that modulates multiple biological activities of HB-EGF including cancer cell proliferation and angiogenic activities. Y-142 may have a potential to be developed into a therapeutic agent for the treatment of HB-EGF-dependent cancers. © 2012 Sato et al.

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