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#28899088   2017/09/13 Save this To Up

Identification of Siglec ligands using a proximity labeling method.

Siglecs are a family of receptor-type glycan recognition proteins (lectins) involved in self-nonself discrimination by the immune system. Identification of Siglec ligands is necessary to understand how Siglec-ligand interaction translates into biological outcomes. However, this is challenging because the interaction is weak. To facilitate identification of Siglec ligands, we adopted a proximity labeling method based on the tyramide radicalization principle. Cells that express Siglec ligands were labeled with Siglec-peroxidase complexes and incubated with biotin tyramide and hydrogen peroxide, to generate short-lived tyramide radicals that covalently label the proteins near the Siglec-peroxidase complex. A proof-of-principle experiment using CD22 (Siglec-2) probe identified its known ligands on B cells, including CD22 itself, CD45, and IgM among others, demonstrating the validity of this method. The specificity of labeling was confirmed by sialidase treatment of target cells and using glycan recognition-deficient mutant CD22 probes. Moreover, possible interactions between biotin-labeled proteins were revealed by literature-based protein-protein interaction network analysis, implying the presence of a molecular cluster comprising CD22 ligands. Further application of this method identified CD44 as a hitherto unknown Siglec-15 ligand on RAW264.7-derived osteoclasts. These results demonstrated the utility of proximity labeling for the identification of Siglec ligands, which may extend to other lectins.

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#28515058   2017/05/18 Save this To Up

Tissue-engineered cartilage constructed by a biotin-conjugated anti-CD44 avidin binding technique for the repairing of cartilage defects in the weight-bearing area of knee joints in pigs.

The lack of effective treatment for cartilage defects has prompted investigations using tissue engineering techniques for their regeneration and repair. The success of tissue-engineered repair of cartilage may depend on the rapid and efficient adhesion of transplanted cells to a scaffold. Our aim in this study was to repair full-thickness defects in articular cartilage in the weight-bearing area of a porcine model, and to investigate whether the CD44 monoclonal antibody biotin-avidin (CBA) binding technique could provide satisfactory tissue-engineered cartilage.

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#28273973   2017/03/09 Save this To Up

Evaluation of the Role of ALDH1 as Cancer Stem Cell Marker in Colorectal Carcinoma: An Immunohistochemical Study.

Colorectal Carcinoma (CRC) is the third most commonly diagnosed cancer in males. Stem Cells (SC) may be involved in tumour growth, including colon cancer. Aldehyde Dehydrogenase 1 (ALDH1) is a detoxifying enzyme that might modulate SC proliferation.

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#28230204   2017/02/23 Save this To Up

High-resolution imaging of living mammalian cells bound by nanobeads-connected antibodies in a medium using scanning electron-assisted dielectric microscopy.

Nanometre-scale-resolution imaging technologies for liquid-phase specimens are indispensable tools in various scientific fields. In biology, observing untreated living cells in a medium is essential for analysing cellular functions. However, nanoparticles that bind living cells in a medium are hard to detect directly using traditional optical or electron microscopy. Therefore, we previously developed a novel scanning electron-assisted dielectric microscope (SE-ADM) capable of nanoscale observations. This method enables observation of intact cells in aqueous conditions. Here, we use this SE-ADM system to clearly observe antibody-binding nanobeads in liquid-phase. We also report the successful direct detection of streptavidin-conjugated nanobeads binding to untreated cells in a medium via a biotin-conjugated anti-CD44 antibody. Our system is capable of obtaining clear images of cellular organelles and beads on the cells at the same time. The direct observation of living cells with nanoparticles in a medium allowed by our system may contribute the development of carriers for drug delivery systems (DDS).

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#27742685   2016/10/15 Save this To Up

A Hyaluronidase-Responsive Nanoparticle-Based Drug Delivery System for Targeting Colon Cancer Cells.

The ability of nanoparticles to target tumors and to enable site-specific drug release provides a unique system for the delivery of effective therapy with reduced toxic side effects. In this study, we used mesoporous silica nanoparticles (MSN) to fabricate a targeted drug delivery system that is responsive to hyaluronidase (HAase). Following engraftment of desthiobiotin onto the surface of MSN, a streptavidin complex was generated, which was functionalized with biotin-modified hyaluronic acid (HA) to enable controlled drug release at cancer cells expressing HAase. Various technologies were used to confirm the successful fabrication of this MSN-based nanocarrier system for targeted drug delivery. In vitro analyses showed that the release of doxorubicin hydrochloride (Dox) was accelerated significantly in the presence of biotin or HAase and accelerated further in the presence of biotin and HAase. Uptake by cancer cells was mediated efficiently by CD44 receptor-mediated endocytosis and the MSN exhibited good biocompatibility in vitro and in vivo MSN-HA/Dox nanoparticles induced apoptosis in cancer cells more efficiently than free doxorubicin and inhibited tumor growth with minimal systemic toxicity in vivo Collectively, our findings offered a preclinical proof of concept for a novel targeted drug delivery carrier system for cancer therapy. Cancer Res; 76(24); 7208-18. ©2016 AACR.

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#26897523   2016/02/22 Save this To Up

CD44-mediated monocyte transmigration across Cryptococcus neoformans-infected brain microvascular endothelial cells is enhanced by HIV-1 gp41-I90 ectodomain.

Cryptococcus neoformans (Cn) is an important opportunistic pathogen in the immunocompromised people, including AIDS patients, which leads to fatal cryptococcal meningitis with high mortality rate. Previous researches have shown that HIV-1 gp41-I90 ectodomain can enhance Cn adhesion to and invasion of brain microvascular endothelial cell (BMEC), which constitutes the blood brain barrier (BBB). However, little is known about the role of HIV-1 gp41-I90 in the monocyte transmigration across Cn-infected BBB. In the present study, we provide evidence that HIV-1 gp41-I90 and Cn synergistically enhance monocytes transmigration across the BBB in vitro and in vivo. The underlying mechanisms for this phenomenon require further study.

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#25993512   2015/05/21 Save this To Up

Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs) conjugated with antibodies (i.e., targeted biotin-MBs). Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs), which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+)) and MDA-MB-453 cells (CD44-), which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+) is a commonly used cancer-stem-cell biomarker, our targeted biotin-MBs could be a potent tool to sort cancer stem cells from dissected tumor tissue for use in preclinical experiments and clinical trials.

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#25352745   2014/10/29 Save this To Up

Hepatitis C virus core and NS3 antigens induced conjunctival inflammation via toll-like receptor-mediated signaling.

Dry eye condition is an extrahepatic manifestation associated with chronic hepatitis C virus (HCV) infection. Since conjunctival inflammation can contribute to the dry eye condition, in the present study we analyzed the conjunctival inflammatory response to HCV core and NS3 proteins.

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#24960585   2014/08/04 Save this To Up

Light-triggered, efficient cytosolic release of IM7-saporin targeting the putative cancer stem cell marker CD44 by photochemical internalization.

We have used the site specific and light-depended drug delivery method photochemical internalization (PCI) to release an immunotoxin (IT), targeting the CD44 receptor, into the cytosol of target cells. The IT consisted of a pan CD44 mAb (clone IM7) bound to the ribosome inactivating protein (RIP) saporin by a biotin-streptavidin linker named IM7-saporin. PCI is based upon photosensitizing compounds localized in the membrane of endosomes and lysosomes causing membrane rupture upon illumination followed by release of the IT into the cytosol. In this in vitro study, we have used 7 different human cancer cell lines of various origins to investigate the cytotoxic effect of PCI-based targeting of the cancer stem cell (CSC) marker CD44. Epi-fluorescence microscopy shows both specific binding and uptake of the IM7-Alexa488, after 30 min and 18 h of incubation, and colocalization with the PCI-photosensitizer TPCS2a prior to light-triggered cytosolic release of the CD44-targeting IT. PCI of IM7-saporin resulted in efficient and specific cytotoxicity in CD44-expressing but not in CD44-negative cancer cells. A higher level of reactive oxygen species (ROS) was found in untreated and photodynamic therapy (PDT)-treated LNCaP (CD44(neg)) compared to that of DU145 (CD44(pos)) prostate cancer (PC) cells. This may explain the PDT-resistance observed in the DU145 cells. PCI-based targeting of CD44-expressing cancer cells gives very potent and specific cytotoxic effects and may represent a rational strategy for achieving site-selective elimination of CSCs in aggressive androgen-independent and treatment-resistant PC cells preventing cytotoxic effects on distant normal stem cells.

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#24891594   2014/08/26 Save this To Up

Characterization of a novel recombinant hyaluronan binding protein for tissue hyaluronan detection.

Tumor necrosis factor-Stimulated Gene 6 protein (TSG-6) is a hyaluronan (HA)-binding glycoprotein containing an HA-binding Link module. Because of its well-defined structure, HA binding properties and small size, TSG-6 is an excellent candidate as an alternative to animal-derived HA-binding protein (HABP) for the detection of HA. The present work describes the generation and characterization of a novel recombinant HA-binding probe obtained by fusion of a modified TSG-6 Link module with mutationally inactivated heparin-binding sequence and the Fc portion of human IgG1 (TSG-6-ΔHep-Fc) for tissue HA detection in histological samples. Direct binding assays indicated strong binding of TSG-6-ΔHep-Fc to HA, with little residual binding to heparin. Histolocalization of HA in formalin-fixed, paraffin-embedded tissue sections using biotin-TSG-6-ΔHep-Fc resulted in hyaluronidase-sensitive staining patterns similar to those obtained with biotin-HABP, but with improved sensitivity. HA was detected in many human tissues, and was most abundant in soft connective tissues such as the skin dermis and the stroma of various glands. Digital image analysis revealed a linear correlation between biotin-HABP and biotin-TSG-6-ΔHep-Fc staining intensity in a subset of normal and malignant human tissues. These results demonstrate that TSG-6-ΔHep-Fc is a sensitive and specific probe for the detection of HA by histological methods.

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