Yoshichika Yoshioka

Chronic polyarthritis caused by mammalian DNA that escapes from degradation in macrophages.

A large amount of chromosomal DNA is degraded during programmed cell death and definitive erythropoiesis. DNase II is an enzyme that digests the chromosomal DNA of apoptotic cells and nuclei expelled from erythroid precursor cells after macrophages have engulfed them. Here we show that DNase II-/-IFN-IR-/- mice and mice with an induced deletion of the DNase II gene develop a chronic polyarthritis resembling human rheumatoid arthritis. A set of cytokine genes was strongly activated in the affected joints of these mice, and their serum contained high levels of anti-cyclic citrullinated peptide antibody, rheumatoid factor and matrix metalloproteinase-3. Early in the pathogenesis, expression of the gene encoding tumour necrosis factor (TNF)-α was upregulated in the bone marrow, and administration of anti-TNF-α antibody prevented the development of arthritis. These results indicate that if macrophages cannot degrade mammalian DNA from erythroid precursors and apoptotic cells, they produce TNF-α, which activates synovial cells to produce various cytokines, leading to the development of chronic polyarthritis.

Critical role of Trib1 in differentiation of tissue-resident M2-like macrophages

Macrophages consist of at least two subgroups, M1 and M2 (refs 1, 2, 3). Whereas M1 macrophages are proinflammatory and have a central role in host defence against bacterial and viral infections, M2 macrophages are associated with responses to anti-inflammatory reactions, helminth infection, tissue remodelling, fibrosis and tumour progression. Trib1 is an adaptor protein involved in protein degradation by interacting with COP1 ubiquitin ligase. Genome-wide association studies in humans have implicated TRIB1 in lipid metabolism. Here we show that Trib1 is critical for the differentiation of F4/80+MR+ tissue-resident macrophages—that share characteristics with M2 macrophages (which we term M2-like macrophages)—and eosinophils but not for the differentiation of M1 myeloid cells. Trib1 deficiency results in a severe reduction of M2-like macrophages in various organs, including bone marrow, spleen, lung and adipose tissues. Aberrant expression of C/EBPα in Trib1-deficient bone marrow cells is responsible for the defects in macrophage differentiation. Unexpectedly, mice lacking Trib1 in haematopoietic cells show diminished adipose tissue mass accompanied by evidence of increased lipolysis, even when fed a normal diet. Supplementation of M2-like macrophages rescues the pathophysiology, indicating that a lack of these macrophages is the cause of lipolysis. In response to a high-fat diet, mice lacking Trib1 in haematopoietic cells develop hypertriglyceridaemia and insulin resistance, together with increased proinflammatory cytokine gene induction. Collectively, these results demonstrate that Trib1 is critical for adipose tissue maintenance and suppression of metabolic disorders by controlling the differentiation of tissue-resident M2-like macrophages.

Preparation and characterization of highly fluorescent, glutathione-coated near infrared quantum dots for in vivo fluorescence imaging.

Fluorescent probes that emit in the near-infrared (NIR, 700–1,300 nm) region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs) have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH)-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs) were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell) QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4). The GSH-QDs (800 nm emission) were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer), and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM), the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIR-fluorescence imaging of a lymph node in a mouse is presented.

Multilayered, core/shell nanoprobes based on magnetic ferric oxide particles and quantum dots for multimodality imaging of breast cancer tumors

Multilayered, core/shell nanoprobes (MQQ-probe) based on magnetic nanoparticles (MNPs) and quantum dots (QDs) have been successfully developed for multimodality tumor imaging. This MQQ-probe contains Fe(3)O(4) MNPs, visible-fluorescent QDs (600 nm emission) and near infrared-fluorescent QDs (780 nm emission) in multiple silica layers. The fabrication of the MQQ-probe involves the synthesis of a primer Fe(3)O(4) MNPs/SiO(2) core by a reverse microemulsion method. The MQQ-probe can be used both as a fluorescent probe and a contrast reagent of magnetic resonance imaging. For breast cancer tumor imaging, anti-HER2 (human epidermal growth factor receptor 2) antibody was conjugated to the surface of the MQQ-probe. The specific binding of the antibody conjugated MQQ-probe to the surface of human breast cancer cells (KPL-4) was confirmed by fluorescence microscopy and fluorescence-activated cell sorting analysis in vitro. Due to the high tissue permeability of near-infrared (NIR) light, NIR fluorescence imaging of the tumor mice (KPL-4 cells transplanted) was conducted by using the anti-HER2 antibody conjugated MQQ-probe. In vivo multimodality images of breast tumors were successfully taken by NIR fluorescence and T(2)-weighted magnetic resonance. Antibody conjugated MQQ-probes have great potential to use for multimodality imaging of cancer tumors in vitro and in vivo.

Carnosic acid prevents obesity and hepatic steatosis in ob/ob mice

Aim:  Carnosic acid (CA) inhibits adipogenesis in vitro. The present study evaluated the therapeutic effects of CA in ob/ob mice.

Identification of an atypical monocyte and committed progenitor involved in fibrosis

Monocytes and macrophages comprise a variety of subsets with diverse functions. It is thought that these cells play a crucial role in homeostasis of peripheral organs, key immunological processes and development of various diseases. Among these diseases, fibrosis is a life-threatening disease of unknown aetiology. Its pathogenesis is poorly understood, and there are few effective therapies. The development of fibrosis is associated with activation of monocytes and macrophages. However, the specific subtypes of monocytes and macrophages that are involved in fibrosis have not yet been identified. Here we show that Ceacam1+Msr1+Ly6C−F4/80−Mac1+ monocytes, which we term segregated-nucleus-containing atypical monocytes (SatM), share granulocyte characteristics, are regulated by CCAAT/enhancer binding protein β (C/EBPβ), and are critical for fibrosis. Cebpb deficiency results in a complete lack of SatM. Furthermore, the development of bleomycin-induced fibrosis, but not inflammation, was prevented in chimaeric mice with Cebpb−/− haematopoietic cells. Adoptive transfer of SatM into Cebpb−/− mice resulted in fibrosis. Notably, SatM are derived from Ly6C−FcεRI+ granulocyte/macrophage progenitors, and a newly identified SatM progenitor downstream of Ly6C−FcεRI+ granulocyte/macrophage progenitors, but not from macrophage/dendritic-cell progenitors. Our results show that SatM are critical for fibrosis and that C/EBPβ licenses differentiation of SatM from their committed progenitor.

Quantitation by 1H‐NMR of dolichol, cholesterol and choline‐containing lipids in extracts of normal and phathological thyroid tissue

Proton magnetic resonance spectroscopy at 1.9 T was used to quantify dolichols, cholesterols, choline‐containing phospholipids and double bonds in unsaturated acyl chains in lipid extracts of four types of thyroid tissue [normal (n = 27), papillary cancer (n = 15), adenoma (n = 13) and Basedow disease (n = 6)]. In normal thyroid the mean concentrations of dolichol, cholesterol and phospholipids were 1.2, 3.6 and 2.1 µmol/g wet weight, respectively. The concentrations of these lipids exhibited positive mutual correlations and positive correlations with patient age. The increase in dolichol in elderly human thyroid may be due to the accumulation of lysosomes and may help to compensate for the decrease in the activity of lysosomal enzymes and in thyroid hormone production and release. Dolichol concentrations were significantly lower in papillary cancer (0.4 µmol/g) and Basedow disease (0.3 µmol/g) compared to normal thyroid (p < 0.01 and p < 0.05, respectively), while cholesterol was enhanced only in cancer tissue (10.7 µmol/g). Benign adenoma exhibited normal levels of both dolichol and cholesterol. These results suggest that the synthesis and accumulation of isoprenoids are normal in adenoma but not in cancer. Copyright

Contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state.

The reduced form of glutathione (GSH; gamma-glutamyl cysteinyl glycine) is supposedly the most powerful reducing battery in the central nervous system against oxidative stress. We evaluated the contribution of GSH and GSSG to MEGA-PRESS (a frequency-selective refocusing technique) signals assessed by magnetic resonance spectroscopy (MRS). GSH gave a single positive signal (2.95 ppm) by the MEGA-PRESS. In contrast, GSSG gave a multiplet of reversed signals (3.03, 3.23, and 3.34 ppm). A phantom solution mimicking the normal in vivo condition (GSH:GSSG=100:1) gave a single positive peak. Even when the ratio was changed to 10:1, corresponding to toxic oxidative stress, GSH was prominent and GSSG signals were minimal. Thus, GSSG signals could be negligible. In the phantom solution (creatine:GSH:aspartate:gamma-aminobutyric acid=7:3:1:1), the creatine signal overshadowed the other signals. Through the MEGA-PRESS, a single peak of GSH stood out over other signals. In vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from GSH. The estimated concentration of GSH in the human brain was 1.9+/-0.37 mM (mean+/-S.D., n=4). In conclusion, MEGA-PRESS allowed us to assess GSH levels in vivo non-invasively.

Multifunctional Core-Shell Silica Nanoparticles for Highly Sensitive19F Magnetic Resonance Imaging

19F magnetic resonance imaging (19F MRI) is useful for monitoring particular signals from biological samples, cells, and target tissues, because background signals are missing in animal bodies. Therefore, highly sensitive 19F MRI contrast agents are in great demand for their practical applications. However, we have faced the following challenges: 1) increasing the number of fluorine atoms decreases the solubility of the molecular probes, and 2) the restriction of the molecular mobility attenuates the 19F MRI signals. Herein, we developed novel multifunctional core–shell nanoparticles to solve these issues. They are composed of a core micelle filled with liquid perfluorocarbon and a robust silica shell. These core–shell nanoparticles have superior properties such as high sensitivity, modifiability of the surface, biocompatibility, and sufficient in vivo stability. By the adequate surface modifications, gene expression in living cells and tumor tissue in living mice were successfully detected by 19F MRI.

Impact of surface coating and particle size on the uptake of small and ultrasmall superparamagnetic iron oxide nanoparticles by macrophages.

Purpose Magnetic resonance imaging (MRI) using contrast agents like superparamagnetic iron oxide (SPIO) is an extremely versatile technique to diagnose diseases and to monitor treatment. This study tested the relative importance of particle size and surface coating for the optimization of MRI contrast and labeling efficiency of macrophages migrating to remote inflammation sites. Materials and methods We tested four SPIO and ultrasmall superparamagnetic iron oxide (USPIO), alkali-treated dextran magnetite (ATDM) with particle sizes of 28 and 74 nm, and carboxymethyl dextran magnetite (CMDM) with particle sizes of 28 and 72 nm. Mouse macrophage RAW264 cells were incubated with SPIOs and USPIOs, and the labeling efficiency of the cells was determined by the percentage of Berlin blue-stained cells and by measuring T2 relaxation times with 11.7-T MRI. We used trypan blue staining to measure cell viability. Results Analysis of the properties of the nanoparticles revealed that ATDM-coated 74 nm particles have a lower T2 relaxation time than the others, translating into a higher ability of MRI negative contrast agent. Among the other three candidates, CMDM-coated particles showed the highest T2 relaxation time once internalized by macrophages. Regarding labeling efficiency, ATDM coating resulted in a cellular uptake higher than CMDM coating, independent of nanoparticle size. None of these particle formulations affected macrophage viability. Conclusion This study suggests that coating is more critical than size to optimize the SPIO labeling of macrophages. Among the formulations tested in this study, the best MRI contrast and labeling efficiency are expected with ATDM-coated 74 nm nanoparticles.