Akiyoshi Hoshino

On the Cyto-Toxicity Caused by Quantum Dots

Quantum dots (QDs) such as CdSe QDs have been introduced as new fluorophores. The QDs conjugated with antibody are starting to be widely used for immunostaining. However there is still not sufficient analysis of the toxicity of QDs in the literature. Therefore we evaluated the cell damage caused by the quantum dots for biological applications. We performed cell viability assay to determine the difference in cell damage depending on the sizes and colors of mercapto‐undecanoic acid (MUA) QDs and the cell types. The results showed that the cell viability decreased with increasing concentration of MUA‐QDs. But in the case of Vero cell (African green monkeys kidney cell) with red fluorescence QD (QD640), the cell damage was less than for the others. Furthermore through the flow cytometry assay we found that this cell damage caused by MUA‐QD turned out to be cell death after 4‐6‐hr incubation. From the two assays described above, we found that there is a range of concentration of MUA‐QDs where the cell viability decreased without cell death occurring and thus we conclude that attention should be given when MUA‐QDs are applied to living organisms even in low concentrations.

Quantum Dots Targeted to the Assigned Organelle in Living Cells

Fluorescent nanocrystal quantum dots (QDs) have the potential to be applied to bioimaging since QDs emit higher and far longer fluorescence than conventional organic probes. Here we show that QDs conjugated with signal peptide obey the order to transport the assigned organelle in living cells. We designed the supermolecule of luminescent QDs conjugated with nuclear‐ and mitochondria‐targeting ligands. When QDs with nuclear‐localizing signal peptides were added to the culture media, we can visualize the movements of the QDs being delivered into the nuclear compartment of the cells with 15 min incubation. In addition, mitochondrial signal peptide can also transport QDs to the mitochondria in living cells. In conclusion, these techniques have the possibility that QDs can reveal the transduction of proteins and peptides into specific subcellular compartments as a powerful tool for studying intracellular analysis in vitro and even in vivo.

Characterization of β-Glucan Recognition Site on C-Type Lectin, Dectin 1

ABSTRACT Dectin 1 is a mammalian cell surface receptor for (1→3)-β-d-glucans. Since (1→3)-β-d-glucans are commonly present on fungal cell walls, it has been suggested that dectin 1 is important for recognizing fungal invasion. In this study we tried to deduce the amino acid residues in dectin 1 responsible for β-glucan recognition. HEK293 cells transfected with mouse dectin 1 cDNA could bind to a gel-forming (1→3)-β-d-glucan, schizophyllan (SPG). The binding of SPG to a dectin 1 transfectant was inhibited by pretreatment with other β-glucans having a (1→3)-β-d-glucosyl linkage but not by pretreatment with α-glucans. Dectin 1 has a carbohydrate recognition domain (CRD) consisting of six cysteine residues that are highly conserved in C-type lectins. We prepared 32 point mutants with mutations in the CRD and analyzed their binding to SPG. Mutations at Trp221 and His223 resulted in decreased binding to β-glucan. Monoclonal antibody 4B2, a dectin- 1 monoclonal antibody which had a blocking effect on the β-glucan interaction, completely failed to bind the dectin-1 mutant W221A. A mutant with mutations in Trp221 and His223 did not have a collaborative effect on Toll-like receptor 2-mediated cellular activation in response to zymosan. These amino acid residues are distinct from residues in other sugar-recognizing peptide sequences of typical C-type lectins. These results suggest that the amino acid sequence W221-I222-H223 is critical for formation of a β-glucan binding site in the CRD of dectin 1.

Use of fluorescent quantum dot bioconjugates for cellular imaging of immune cells, cell organelle labeling, and nanomedicine: surface modification regulates biological function, including cytotoxicity.

With the development of nanotechnology, nanoscale products that are smaller than several hundred nanometers have been applied to all areas of science and technology. Nanoscale products, including carbon nanotubes, fullerene derivatives, and nanocrystal quantum dots (QDs), are wide spread as novel tools in various fields, not only in materials engineering, electronics, plastics, and the automobile and aerospace industries, but also in molecular biology and medicine. At present, QDs have been widely used in biological and medical studies because of their superior photoemission and photostability. Although the physical and chemical properties of QDs have been circumstantially investigated, little is known about any harmful effects of QDs on human health. Here we report on the toxicity and biological behavior of QDs in vitro and in vivo. The toxicity of the core constituent chemicals such as cadmium and selenium has been identified. Recently, the surface molecules surrounding QDs have been intensively investigated. Accumulating evidence that toxic surface-covering molecules showed their cytotoxicity and biomolecules conjugated with QDs maintained their biological effects indicates that at least the biological properties of QDs are attributable to the QD-capping material rather than to the core metalloid complex itself.

Inhibition of CCL1-CCR8 Interaction Prevents Aggregation of Macrophages and Development of Peritoneal Adhesions

Peritoneal adhesions are a significant complication of surgery and visceral inflammation; however, the mechanism has not been fully elucidated. The aim of this study was to clarify the mechanism of peritoneal adhesions by focusing on the cell trafficking and immune system in the peritoneal cavity. We investigated the specific recruitment of peritoneal macrophages (PMφ) and their expression of chemokine receptors in murine models of postoperative and postinflammatory peritoneal adhesions. PMφ aggregated at the site of injured peritoneum in these murine models of peritoneal adhesions. The chemokine receptor CCR8 was up-regulated in the aggregating PMφ when compared with naive PMφ. The up-regulation of CCR8 was also observed in PMφ, but not in bone marrow-derived Mφ, treated with inflammatory stimulants including bacterial components and cytokines. Importantly, CCL1, the ligand for CCR8, a product of both PMφ and peritoneal mesothelial cells (PMCs) following inflammatory stimulation, was a potent enhancer of CCR8 expression. Cell aggregation involving PMφ and PMCs was induced in vitro in the presence of CCL1. CCL1 also up-regulated mRNA levels of plasminogen activator inhibitor-1 in both PMφ and PMCs. CCR8 gene-deficient mice or mice treated with anti-CCL1-neutralizing Ab exhibited significantly reduced postoperational peritoneal adhesion. Our study now establishes a unique autocrine activation system in PMφ and the mechanism for recruitment of PMφ together with PMCs via CCL1/CCR8, as immune responses of peritoneal cavity, which triggers peritoneal adhesions.

Quantum Dot as a Drug Tracer In Vivo

Quantum dots (QDs) have been applied to a wide range of biological studies by taking advantage of their fluorescence properties. There is almost no method to trace small molecules including medicine. Here, we used QDs for fluorescent tracers for medicine and analyzed their kinetics and dynamics. We conjugated QDs with captopril, anti-hypertensive medicine, by an exchange reaction while retaining the medicinal properties. We investigated the medicinal effect of QD-conjugated captopril (QD-cap) in vitro and in vivo. We also evaluated the concentration and the distribution of the QD-cap in the blood and the organs with their fluorescence. We demonstrate that the QD-cap inhibits the activity of ACE in vitro. The QD-cap reduced the blood pressure of hypertensive model rats. The concentration of the QD-cap in the blood was measured by using the standard curve of the fluorescence intensity. The blood concentration of the QD-cap decrease exponentially and QD-cap has approximately the same half-life as that of captopril. In addition, the fluorescence of the QDs revealed that QD-cap accumulates in the liver, lungs, and spleen. We succeeded in analyzing the dynamics and kinetics of small molecules using fluorescence of QDs

Deficiency of Chemokine Receptor CCR1 Causes Osteopenia Due to Impaired Functions of Osteoclasts and Osteoblasts

Chemokines are characterized by the homing activity of leukocytes to targeted inflammation sites. Recent research indicates that chemokines play more divergent roles in various phases of pathogenesis as well as immune reactions. The chemokine receptor, CCR1, and its ligands are thought to be involved in inflammatory bone destruction, but their physiological roles in the bone metabolism in vivo have not yet been elucidated. In the present study, we investigated the roles of CCR1 in bone metabolism using CCR1-deficient mice. Ccr1−/− mice have fewer and thinner trabecular bones and low mineral bone density in cancellous bones. The lack of CCR1 affects the differentiation and function of osteoblasts. Runx2, Atf4, Osteopontin, and Osteonectin were significantly up-regulated in Ccr1−/− mice despite sustained expression of Osterix and reduced expression of Osteocalcin, suggesting a lower potential for differentiation into mature osteoblasts. In addition, mineralized nodule formation was markedly disrupted in cultured osteoblastic cells isolated from Ccr1−/− mice. Osteoclastogenesis induced from cultured Ccr1−/− bone marrow cells yielded fewer and smaller osteoclasts due to the abrogated cell-fusion. Ccr1−/− osteoclasts exerted no osteolytic activity concomitant with reduced expressions of Rank and its downstream targets, implying that the defective osteoclastogenesis is involved in the bone phenotype in Ccr1−/− mice. The co-culture of wild-type osteoclast precursors with Ccr1−/− osteoblasts failed to facilitate osteoclastogenesis. This finding is most likely due to a reduction in Rankl expression. These observations suggest that the axis of CCR1 and its ligands are likely to be involved in cross-talk between osteoclasts and osteoblasts by modulating the RANK-RANKL-mediated interaction.

Simultaneous multicolor detection system of the single-molecular microbial antigen with total internal reflection fluorescence microscopy.

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystal QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD‐conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD‐conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high‐sensitivity detection systems.

Organ distribution of quantum dots after intraperitoneal administration, with special reference to area-specific distribution in the brain

Quantum dots (QDs) are well known for their potential application in biosensing, ex vivo live-cell imaging and in vivo animal targeting. The brain is a challenging organ for drug delivery, because the blood brain barrier (BBB) functions as a gatekeeper guarding the body from exogenous substances. Here, we evaluated the distribution of bioconjugated QDs, i.e., captopril-conjugated QDs (QDs-cap) following intraperitoneal injection into male ICR mice as a model system for determining the tissue localization of QDs, employing ICP-MS and confocal microscopy coupled with spectrometric analysis. We have demonstrated that intraperitoneally administered QDs-cap were delivered via systemic blood circulation into liver, spleen, kidney and brain at 6 h after injection. QDs-cap were located predominantly inside the blood vessels in the liver, kidney and brain, but a few were distributed in the parenchyma, especially noteworthy in the brain. Careful studies on acute as well as chronic toxicity of QDs in the brain are required prior to clinical application to humans.

Separation of murine neutrophils and macrophages by thermoresponsive magnetic nanoparticles.

Magnetic particles have been used widely in both biotechnological and medical fields, including for immunoassay, enzyme immobilization, drug transport, and immunological diagnosis. Especially particles with bioactive molecules such as antibodies and streptavidin are very useful tools for cell separation. Here we report affinity selection of neutrophils and macrophages from peritoneal inflammatory cells performed by thermoresponsive magnetic nanoparticles conjugated with macrophage‐specific anti‐F4/80 antibody. The magnetic nanoparticles, which are capped with thermoresponsive polymers, are aggregated by heating the particles over 30 °C and show their intrinsic magnetism. The neutrophils are concentrated approximately 90% by these magnetic nanoparticles without any activation, indicating that this novel cell separation method could fulfill a wide range of applications in analysis of the isolation of fragile cells such as neutrophils.