Qiong Dong

Specific siRNA Targeting Receptor for Advanced Glycation End Products (RAGE) Decreases Proliferation in Human Breast Cancer Cell Lines

Receptor for Advanced Glycation End Products (RAGE) is an oncogenic trans-membranous receptor overexpressed in various human cancers. However, the role of RAGE in breast cancer development and proliferation is still unclear. In this study, we demonstrated that RAGE expression levels are correlated to the degree of severity of breast cancer. Furthermore, there is a decrease in the proliferation of all sub-types of breast cancer, MCF-7, SK-Br-3 and MDA-MB-231, as a result of the effect of RAGE siRNA. RAGE siRNA arrested cells in the G1 phase and inhibited DNA synthesis (p < 0.05). Moreover, qRT-PCR and Western Blot results demonstrated that RAGE siRNA decreases the expression of transcriptional factor NF-κB p65 as well as the expression of cell proliferation markers PCNA and cyclinD1. RAGE and RAGE ligands can thus be considered as possible targets for breast cancer management and therapy.

A highly selective turn-off fluorescent probe for Cu(II) based on a dansyl derivative and its application in living cell imaging

A novel dansyl derivative (1) was designed and synthesized with high yield. It is highly selective for Cu2+ over other competing metal ions such as Ca2+, Co2+, Cr3+, Cu+, Fe2+, Ga3+, Hg2+, Mg2+, Na+, Ni2+ and Pb2+. Zn2+ and Fe3+ only slightly changed the fluorescence of probe 1. The linear relationship between fluorescence intensity and Cu2+ concentration indicates that 1 can be used for quantification. The binding ratio of probe 1 to Cu2+ was found to be 1 : 1 according to Jobs plot experiments. Probe 1 can be used in a broad pH value window ranging from 5 to 11. The limit of detection (LOD) based on 3 × δblank/k was calculated with a value as low as 1.6 × 10−6 M for Cu2+. Additionally, the association constant of probe 1 − Cu2+ complexes was found to be 5.08 × 104 M−1. Moreover, fluorescence microscopy experiments showed that 1 can be used as a fluorescent probe for evaluating the presence of exogenous Cu2+ in living cells.

A Wash-Free Homogeneous Colorimetric Immunoassay Method.

Rapid and convenient biosensing platforms could be beneficial to timely diagnosis and treatment of diseases in virtually any care settings. Sandwich immunoassays, the most commonly used methods for protein detection, often rely on expensive tags such as enzyme and tedious wash and incubation procedures operated by skilled labor. In this report, we revolutionized traditional sandwich immunoassays by providing a wash-free homogeneous colorimetric immunoassay method without requirement of any separation steps. The proposed strategy was realized by controlling the growth of gold nanoparticles (AuNPs) to mediate the interparticle spacing in the protein-AuNP oligomers. We have demonstrated the successful in vitro detection of cancer biomarker in serum samples from patients with high clinical sensitivity and specificity.

Resveratrol inhibits Hexokinases II mediated glycolysis in non-small cell lung cancer via targeting Akt signaling pathway

Deregulation of glycolysis was often observed in human cancer cells. In the present study, we reported resveratrol, a small polyphenol, which has been intensively studied in various tumor models, has a profound anti-tumor effect on human non-small cell lung cancer (NSCLC) via regulation of glycolysis. Resveratrol impaired hexokinase II (HK2)-mediated glycolysis, and markedly inhibited anchorage-dependent and -independent growth of NSCLC cells. Exposure to resveratrol decreased EGFR and downstream kinases Akt and ERK1/2 activation. Moreover, we revealed that resveratrol impaired glucose metabolism by mainly inhibiting expression of HK2 mediated by the Akt signaling pathway, and exogenous overexpression of constitutively activated Akt1 in NSCLC cells substantially rescued resveratrol-induced glycolysis suppression. The in vivo data indicated that resveratrol obviously suppressed tumor growth in a xenograft mouse model. Our results suggest targeting HK2 or metabolic enzymes appears to be a new approach for clinical NSCLC prevention or treatment.

Investigation of Porcine Endogenous Retrovirus in the Conservation Population of Ningxiang Pig

Porcine endogenous retrovirus (PERV) varies between pig breeds. Screening and analysis of PERV in putative pig breeds may provide basic parameters to evaluate the biological safety of xenotransplantation from pigs to humans. In this study, PERV was investigated among the conservation population of the Ningxiang pig. The result revealed that the genotype of PERV distribution was subtype A, 100%; subtype B, 100%; and subtype C, 100%. The env sequences of PERV-A and -B showed 11 clones detected by KpnI and MboI digestion, indicating that there existed multiple variants of PERV-A and -B in the Ningxiang pig. Reverse transcriptase polymerase chain reaction results showed that PERV had transcriptional activity in these individuals. In addition, PERV A/C recombinant was detected in most individuals of Ningxiang pig. Because PERV A/C recombinants increase the potential infectious risk, the breed may not be a proper donor for xenotransplantation.

Characterization of PERV in a new conserved pig herd as potential donor animals for xenotransplantation in China.

BackgroundXenotransplantation has drawn increased attention in recent years as a potential solution to the scarcity of human source donor organs. Researchers have highlighted the need to characterize the influence of porcine endogenous retroviruses (PERV) in xenotransplantation. Screening and analyzing the presence and subtype of PERV in donor source animal breeds could provide basic parameters to evaluate the biological safety of xenotransplantation from pigs to humans. We bred a new miniature porcine herd (XENO-1) after decades of investigation, the herd was purpose bred to produce a potential donor animal source for xenotransplantation. To this end we studied the animals’ PERV expression characteristics.MethodsWe randomly selected 37 animals of the herd, PCR and RT-PCR based on specific primers were utilized to determine their PERV viral subtype. High fidelity PCR and restriction enzyme digestion were employed for variants detection. To thoroughly understand the PERV expression pattern, quantitative PCR was applied to measure mRNA expression levels in different tissues, At last, transfection capacity was assessed using a in vitro co-culture system.ResultsOur results revealed that the XENO-1 herd was free of PERV-C and exhibited low levels of PERVs in different tissues compared to commercial pig (landrace). The XENO-1 herd showed unique variants of A/B recombination. In addition, even though there were A/B variants in the XENO-1 herd, co-culturing revealed no evidence of PERV transmission from XENO-1 tissue to human cells.ConclusionOverall, Our results displayed an unique PERV expression pattern in a new pig herd and demonstrated its non-transfection capacity in vitro. Data in the research indicate that XENO-1 animals can serve as a better potential donor source for xenotransplantation.

Coatting NICCs With MSCs and EPCs to Alleviate Instant Blood Mediated Inflammatory Reaction

A42 Immunosuppressive Capacities of Human Renal Tubular Epithelial Cells; a Role for Indoleamine 2,3-Dioxygenase? M. Demmers, C. Baan, M. Roemeling-van Rhijn, T. van den Bosch, M. Hoogduijn, M. Betjes, W. Weimar, A. Rowshani. Internal Medicine, Section Nephrology and Transplantation, Erasmus MC University Medical Center, Rotterdam, Netherlands. Introduction Renal tubular epithelial cells (TECs) are one of the main targets of T cell attack during acute cellular rejection. We hypothesize that TECs modulate the outcome of allo-immunity in a bi-directional way executing immunosuppressive effects and dampening the local infl ammation. Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme inhibiting T-cell proliferation. TECs express cytoplasmic IDO during acute rejection. We studied whether TECs possess immunosuppressive capacities and if IDO might play a role suppressing T-cell alloactivity. Materials and Methods Anti CD3/CD28 activated peripheral blood mononuclear cells were cocultured with IFN-γ/TNF-α activated TECs for 3 days. We analysed CD4+ T-cell and CD8+ T-cell proliferation response in the absence or presence of IDO inhibitor 1-L-MT. Next we analysed early and late apoptosis as increased IDO acitivity is associated with increased apoptosis. Further we examined whether inhibition of T cell proliferation was cell-cell contact dependent using transwell membrane experiments. Results We found that TECs dose-dependently inhibited CD4+ T-cell and CD8+ T-cell proliferation. TEC mRNA analysis and supernatant L-kynurenine showed that activated TECs express IDO mRNA expression and signifi cantly upregulated L-kynureninen, which was signifi cantly downregulated using 1-L-MT. Transwell experiments showed that TEC-mediated immunosuppression is cell-cell contact dependent. Downregulated CD4+ T-cell proliferation was partly recovered after addition of 1-L-MT, while CD8+ T-cell proliferation was not affected by 1-L-MT. Activated TECs increased early and late apoptosis of proliferating CD4+ T-cells, 1-L-MT abrogated both early and late CD4+ T-cell apoptosis. Discussion Our data show that TECs possess immunosuppressive capacities and inhibit the allo-reactive T cell proliferation that can partly be explained by indoleamine 2,3-dioxygenase immune regulation. Abstract# A43 Introduction of a New Cell Model of Biopsy-Derived Human Proximal Tubule Cells to Study the Role of Pharmacogenetics in CNIAssociated Nephrotoxicity. N. Knops,1,2 D. Kuypers,3 R. Masereeuw,4 E. Levtchenko,1,2 L. Van den Heuvel.2 1Pediatric Nephrology and Solid Organ Transplantation, University Hospital Leuven, Leuven, Belgium; 2Labarotory for Pediatrics, Dept of Development & Regeneration, KU Leuven, Leuven, Belgium; 3Nephrology, University Hospital Leuven, Leuven, Belgium; 4Pharmacology and Toxicology, Radboud University, Nijmegen, Netherlands. Background: Calcineurin inhibitors (CNI) constitute the basis of immunosuppressive regimes in transplantation, but are associated with the development of histological lesions leading to kidney failure. CNI’s are metabolized by CYP3A and excreted by Pgp (ABCB1) in the gut and liver but also in proximal tubular cells (PTC). Clinical studies demonstrated a relation between common variants of CYP3A5/ ABCB1 genes and CNI-associated nephrotoxicity (CNIT). The mechanism is unknown. We established a model of human PTC that can be used to study the pathogenesis of CNIT. Methods: A technique was developed to culture cells from a protocol biopsy in renal allograft recipients. Primary cells were transfected with SV40T and hTERT virus for conditional immortalization and differentiation. Subclones were selected based upon specifi c PTC markers (AQP1 and CD13) using Western Blot (WB) and FACS. Light and scanning electron microscopy were performed to detect PTC morphology. PCR and sequencing was used to assess genotype. Quantative RT-PCR, WB and immunohistochemistry was performed for CYP3A5 an ABCB1 expression. CYP3A5 activity was assessed by differential midazolam(MDZ) hydroxylation using LC-MS and Pgp activity by calcein effl ux. Results: From 27 out of 38 biopsies cell lines were generated. Based upon genotype 11 subclones with PTC biomarkers were selected. In vitro PTC morphology with brush border microvilli was observed. We confi rmed CYP3A5 and Pgp mRNA and protein expression. CYP3A5*1 carriers had increased 1OH/4OH MDZ formation vs.*3/*3 (1,44 vs 0,7; p<0,05). Pgp activity was confirmed by 39% calcein accumulation(95% CI:33-44), but not related ABCB1 3435CT genotype. Tacrolimus disappearance was 49 times higher in CYP3A5*1 vs.*3/*3 carriers, but again not related to ABCB1 3435CT genotype. Conclusion: PTC cell lines can be generated from a kidney biopsy and demonstrate functional expression of genes involved in CNI metabolism after immortalization. Differences in protein function were detected for CYP3A5 genotype. This in vitro model can be used to study the role of pharmacogenetic variation in CNIT. DISCLOSURES: Knops, N.: Grant/Research Support, Astellas. Kuypers, D.: Grant/ Research Support, Astellas. Levtchenko, E.: Grant/Research Support, Astellas. A43 Introduction of a New Cell Model of Biopsy-Derived Human Proximal Tubule Cells to Study the Role of Pharmacogenetics in CNIAssociated Nephrotoxicity. N. Knops,1,2 D. Kuypers,3 R. Masereeuw,4 E. Levtchenko,1,2 L. Van den Heuvel.2 1Pediatric Nephrology and Solid Organ Transplantation, University Hospital Leuven, Leuven, Belgium; 2Labarotory for Pediatrics, Dept of Development & Regeneration, KU Leuven, Leuven, Belgium; 3Nephrology, University Hospital Leuven, Leuven, Belgium; 4Pharmacology and Toxicology, Radboud University, Nijmegen, Netherlands. Background: Calcineurin inhibitors (CNI) constitute the basis of immunosuppressive regimes in transplantation, but are associated with the development of histological lesions leading to kidney failure. CNI’s are metabolized by CYP3A and excreted by Pgp (ABCB1) in the gut and liver but also in proximal tubular cells (PTC). Clinical studies demonstrated a relation between common variants of CYP3A5/ ABCB1 genes and CNI-associated nephrotoxicity (CNIT). The mechanism is unknown. We established a model of human PTC that can be used to study the pathogenesis of CNIT. Methods: A technique was developed to culture cells from a protocol biopsy in renal allograft recipients. Primary cells were transfected with SV40T and hTERT virus for conditional immortalization and differentiation. Subclones were selected based upon specifi c PTC markers (AQP1 and CD13) using Western Blot (WB) and FACS. Light and scanning electron microscopy were performed to detect PTC morphology. PCR and sequencing was used to assess genotype. Quantative RT-PCR, WB and immunohistochemistry was performed for CYP3A5 an ABCB1 expression. CYP3A5 activity was assessed by differential midazolam(MDZ) hydroxylation using LC-MS and Pgp activity by calcein effl ux. Results: From 27 out of 38 biopsies cell lines were generated. Based upon genotype 11 subclones with PTC biomarkers were selected. In vitro PTC morphology with brush border microvilli was observed. We confi rmed CYP3A5 and Pgp mRNA and protein expression. CYP3A5*1 carriers had increased 1OH/4OH MDZ formation vs.*3/*3 (1,44 vs 0,7; p<0,05). Pgp activity was confirmed by 39% calcein accumulation(95% CI:33-44), but not related ABCB1 3435CT genotype. Tacrolimus disappearance was 49 times higher in CYP3A5*1 vs.*3/*3 carriers, but again not related to ABCB1 3435CT genotype. Conclusion: PTC cell lines can be generated from a kidney biopsy and demonstrate functional expression of genes involved in CNI metabolism after immortalization. Differences in protein function were detected for CYP3A5 genotype. This in vitro model can be used to study the role of pharmacogenetic variation in CNIT. DISCLOSURES: Knops, N.: Grant/Research Support, Astellas. Kuypers, D.: Grant/ Research Support, Astellas. Levtchenko, E.: Grant/Research Support, Astellas. Abstract# A44 Angiogenin Promotes Cell Survival During Cyclosporine-Induced Endoplasmic Reticulum Stress. I. Mami,1 N. Bovier,1 S. Pezet,1 P. Beaune,1,2 N. Pallet,1,2 E. Thervet.1,3 1INSERM U-775, INSERM, Paris, France; 2Service de Biochimie, Hopital Europeen Georges Pompidou, Paris, France; 3Service de Nephrologie, Hopital Europeen Georges Pompidou, Paris, France. Background Calcineurin inhibitors nephrotoxicity promotes chronic kidney injury, and contributes to chronic allograft nephropathy. We have demonstrated previously that cyclosporine is an ER stress inducer, ER stress mediates its nephrotoxicity. ER stress contributes to kidney disease, and constitutes a progression factor. Recent studies suggest that Angiogenin (ANG), a stress-activated and secreted ribonuclease, cleaves tRNA to generate fragments called tiRNA. These tiRNA contribute to stress-induced translational repression, indicating that ANG and tiRNA help to reprogram protein translation during stress, and are previously unappreciated components of the stress response. The implication of tiRNA in the ER stress-induced translational repression is unknown. Objectives Our hypothesis is that cyclosporine regulates the production and activation of ANG during the Unfolded Protein Response (UPR), the adaptive program activated in response to ER stress, in the kidney epithelium. That ANG promotes cellular adaptation during stress, mediated by tiRNA integrated in the UPR-induced translational repression. The purpose of this study is to characterize the mechanisms of ANG synthesis, cellular localization and biological functions, during ER stress activated by cyclosporine. Results In a model of human epithelial cells, we have demonstrated that ANG expression is induced during ER stress, that ANG production depends on IRE1a, and that ANG expression is regulated by the transcription factor sXBP1 and NF-kB. ER stress promotes a nucleo-cytoplasmic transfert of ANG which localizes in part in stress granules. ANG inhibits ER stress-indu