Cerebral amyloid angiopathy (CAA), characterized by the deposition of amyloid aggregates in the walls of cerebral vasculature, is a major factor in intracerebral hemorrhage and vascular cognitive impairment and is also associated closely with Alzheimer's disease (AD). We previously reported (99m)Tc-hydroxamamide ((99m)Tc-Ham) complexes with a bivalent amyloid ligand showing high binding affinity for β-amyloid peptide (Aβ(1-42)) aggregates present frequently in the form in AD. In this article, we applied them to CAA-specific imaging probes, and evaluated their utility for CAA-specific imaging. In vitro inhibition assay using Aβ(1-40) aggregates deposited mainly in CAA and a brain uptake study were performed for (99m)Tc-Ham complexes, and all (99m)Tc-Ham complexes with an amyloid ligand showed binding affinity for Aβ(1-40) aggregates and very low brain uptake. In vitro autoradiography of human CAA brain sections and ex vivo autoradiography of Tg2576 mice were carried out for bivalent (99m)Tc-Ham complexes ([(99m)Tc]SB2A and [(99m)Tc]BT2B), and they displayed excellent labeling of Aβ depositions in human CAA brain sections and high affinity and selectivity to CAA in transgenic mice. These results may offer new possibilities for the development of clinically useful CAA-specific imaging probes based on the (99m)Tc-Ham complex.

We examined whether the amino acid PET tracers, trans-1-amino-3-(18)F-fluorocyclobutanecarboxylic acid (anti-(18)F-FACBC) and (11)C-methyl-l-methionine ((11)C-Met), are suitable for detecting early responses to combination therapies including temozolomide (TMZ), interferon-β (IFN), and bevacizumab (Bev) in glioblastoma.

Human T-cell leukemia virus type 1 (HTLV-1) is the causal agent of a neoplastic disease of CD4+ T cells, adult T-cell leukemia (ATL), and inflammatory diseases including HTLV-1 associated myelopathy/tropical spastic paraparesis, dermatitis, and inflammatory lung diseases. ATL cells, which constitutively express CD25, resemble CD25+CD4+ regulatory T cells (T(reg)). Approximately 60% of ATL cases indeed harbor leukemic cells that express FoxP3, a key transcription factor for T(reg) cells. HTLV-1 encodes an antisense transcript, HTLV-1 bZIP factor (HBZ), which is expressed in all ATL cases. In this study, we show that transgenic expression of HBZ in CD4+ T cells induced T-cell lymphomas and systemic inflammation in mice, resembling diseases observed in HTLV-1 infected individuals. In HBZ-transgenic mice, CD4+Foxp3+ T(reg) cells and effector/memory CD4+ T cells increased in vivo. As a mechanism of increased T(reg) cells, HBZ expression directly induced Foxp3 gene transcription in T cells. The increased CD4+Foxp3+ T(reg) cells in HBZ transgenic mice were functionally impaired while their proliferation was enhanced. HBZ could physically interact with Foxp3 and NFAT, thereby impairing the suppressive function of T(reg) cells. Thus, the expression of HBZ in CD4+ T cells is a key mechanism of HTLV-1-induced neoplastic and inflammatory diseases.

The interferon-inducible transmembrane (Ifitm/Fragilis) genes encode homologous proteins that are induced by IFNs. Here, we show that IFITM proteins regulate murine CD4+ Th cell differentiation. Ifitm2 and Ifitm3 are expressed in wild-type (WT) CD4+ T cells. On activation, Ifitm3 was downregulated and Ifitm2 was upregulated. Resting Ifitm-family-deficient CD4+ T cells had higher expression of Th1-associated genes than WT and purified naive Ifitm-family-deficient CD4+ T cells differentiated more efficiently to Th1, whereas Th2 differentiation was inhibited. Ifitm-family-deficient mice, but not Ifitm3-deficient mice, were less susceptible than WT to induction of allergic airways disease, with a weaker Th2 response and less severe disease and lower Il4 but higher Ifng expression and IL-27 secretion. Thus, the Ifitm family is important in adaptive immunity, influencing Th1/Th2 polarization, and Th2 immunopathology.

T cells are known to be plastic and to change their phenotype according to the cellular and biochemical milieu they are embedded in. In this study, we transposed this concept at a macroscopic level assessing whether changes in the environmental housing conditions of C57/BL6 mice would influence the phenotype and function of T cells. Our study shows that exposure to 2 weeks in an enriched environment (EE) does not impact the T cell repertoire in vivo and causes no changes in the early TCR-driven activation events of these cells. Surprisingly, however, T cells from enriched mice showed a unique T helper effector cell phenotype upon differentiation in vitro. This was featured by a significant reduction in their ability to produce IFN-γ and by an increased release of IL-10 and IL-17. Microarray analysis of these cells also revealed a unique gene fingerprint with key signaling pathways involved in autoimmunity being modulated. Together, our results provide first evidence for a specific effect of EE on T cell differentiation and its associated changes in gene expression profile. In addition, our study sheds new light on the possible mechanisms by which changes in environmental factors can significantly influence the immune response of the host and favor the resolution of the inflammatory response.

Photoacoustic imaging, which enables high-resolution imaging in deep tissues, has lately attracted considerable attention. For tumor imaging, photoacoustic probes have been proposed to enhance the photoacoustic effect to improve detection sensitivity. Here, we evaluated the feasibility of using a biocompatible hydrophilic polymer, polyoxazoline, conjugated with indocyanine green (ICG) as a tumor-targeted photoacoustic probe via enhanced permeability and retention effect. ICG molecules were multivalently conjugated to partially hydrolyzed polyoxazoline, thereby serving as highly sensitive photoacoustic probes. Interestingly, loading multiple ICG molecules to polyoxazoline significantly enhanced photoacoustic signal intensity under the same ICG concentration. In vivo biodistribution studies using tumor bearing mice demonstrated that 5% hydrolyzed polyoxazoline (50 kDa) conjugated with ICG (ICG/polyoxazoline = 7.8), P14-ICG7.8, showed relatively high tumor accumulation (9.4%ID/g), resulting in delivery of the highest dose of ICG among the probes tested. P14-ICG7.8 enabled clear visualization of the tumor regions by photoacoustic imaging 24 h after administration; the photoacoustic signal increased in proportion with the injected dose. In addition, the signal intensity in blood vessels in the photoacoustic images did not show much change, which was attributed to the high tumor-to-blood ratios of P14-ICG7.8. These results suggest that polyoxazoline-ICG would serve as a robust probe for sensitive photoacoustic tumor imaging.

Zinc-finger protein 143 (ZNF143) is a transcription factor that is involved in anticancer drug resistance and cancer cell survival. In the present study, we identified a novel small molecule N-(5-bromo-2-methoxyphenyl)-3-(pyridine-3-yl) propiolamide (YPC-21661) that inhibited ZNF143 promoter activity and down-regulated the expression of the ZNF143-regulated genes, RAD51, PLK1, and Survivin, by inhibiting the binding of ZNF143 to DNA. In addition, YPC-21661 was cytotoxic and induced apoptosis in the human colon cancer cell line, HCT116 and human prostate cancer cell line, PC-3. 2-(pyridine-3-ylethynyl)-5-(2-(trifluoromethoxy)phenyl)-1,3,4-oxadiazole (YPC-22026), a metabolically stable derivative of YPC-21661, induced tumor regression accompanied by the suppression of ZNF143-regulated genes in a mouse xenograft model. The present study revealed that the inhibition of ZNF143 activity by small molecules induced tumor regression in vitro and in vivo; therefore, ZNF143 is a promising target of cancer therapeutics.

The understanding of pathological processes is based on the comparison between physiological and pathological conditions, and transcriptomic analysis has been extensively applied to various diseases for this purpose. However, the way in which the transcriptomic data of pathological cells relate to the transcriptomes of normal cellular counterparts has not been fully explored, and may provide new and unbiased insights into the mechanisms of these diseases. To achieve this, it is necessary to develop a method to simultaneously analyse components across different levels, namely genes, normal cells, and diseases. Here we propose a multidimensional method that visualises the cross-level relationships between these components at three different levels based on transcriptomic data of physiological and pathological processes, by adapting Canonical Correspondence Analysis, which was developed in ecology and sociology, to microarray data (CCA on Microarray data, CCAM). Using CCAM, we have analysed transcriptomes of haematological disorders and those of normal haematopoietic cell differentiation. First, by analysing leukaemia data, CCAM successfully visualised known relationships between leukaemia subtypes and cellular differentiation, and their characteristic genes, which confirmed the relevance of CCAM. Next, by analysing transcriptomes of myelodysplastic syndromes (MDS), we have shown that CCAM was effective in both generating and testing hypotheses. CCAM showed that among MDS patients, high-risk patients had transcriptomes that were more similar to those of both haematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors (MEP) than low-risk patients, and provided a prognostic model. Collectively, CCAM reveals hidden relationships between pathological and physiological processes and gene expression, providing meaningful clinical insights into haematological diseases, and these could not be revealed by other univariate and multivariate methods. Furthermore, CCAM was effective in identifying candidate genes that are correlated with cellular phenotypes of interest. We expect that CCAM will benefit a wide range of medical fields.

Recently, we developed a benzofuran derivative for the imaging of β-amyloid plaques, 5-(5-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)benzofuran-2-yl)-N-methylpyridin-2-amine (18F-FPYBF-2) (Ono et al., J Med Chem 54:2971-9, 2011). The aim of this study was to assess the feasibility of 18F-FPYBF-2 as an amyloid imaging PET tracer in a first clinical study with healthy volunteers and patients with various dementia and in comparative dual tracer study using 11C-Pittsburgh Compound B (11C-PiB).

Amyloid-β (Aβ) accumulation, accelerated by traumatic brain injury (TBI), may play a crucial role in neurodegeneration in chronic-stage TBI. The injury type could influence Aβ dynamics because of TBI's complex, heterogeneous nature. We, therefore, investigated spatial patterns of amyloid deposition according to injury type after TBI using 5-(5-(2-(2-(2-[F]-fluoroethoxy)ethoxy)ethoxy)benzofuran-2-yl)-N-methylpyridin-2-amine (18F-FPYBF-2) positron emission tomography (PET).

To understand the pathways involved in drug clearance from the body, quantitative evaluations of the hepatobiliary transport of drugs are important. The organic anion transporting polypeptide (OATP) family transporter, particularly OATP1B1 and 1B3, are considered to play an important role in hepatic uptake of organic anion compounds. Pitavastatin is a substrate of OATP, and it includes a fluorine group. Therefore, it represents an acceptable positron-emission tomography (PET) tracer using fluorine-18 to image in vivo hepatic transporter functions.

Exogenously hypercholesterolemic (ExHC) rats develop diet-induced hypercholesterolemia (DIHC) when fed with dietary cholesterol. Previously, we reported that, under the high-sucrose-diet-feeding condition, a loss-of-function mutation in Smek2 results in low activity of fatty acid synthase (FAS) followed by the shortage of hepatic triacylglycerol content in ExHC rats and the onset of DIHC. However, the relationship between the Smek2 mutation and FAS dysfunction is still unclear. Here, we focused on carbohydrate metabolism, which provides substrates for FAS, and analyzed carbohydrate and lipid metabolisms in ExHC rats to clarify how the deficit of Smek2 causes DIHC. Male ExHC and SD rats were fed high-sucrose or high-starch diets containing 1% cholesterol for 2 weeks. Serum cholesterol levels of the ExHC rats were higher, regardless of the dietary carbohydrate. Hepatic triacylglycerol levels were higher in only the SD rats fed the high-sucrose diet. Moreover, the ExHC rats exhibited a diabetes-like status and accumulation of hepatic glycogen and low hepatic mRNA levels of liver-type phosphofructokinase (Pfkl), which encodes a rate-limiting enzyme for glycolysis. These results suggest that the glucose utilization, particularly glycolysis, is impaired in the liver of ExHC rats. To evaluate how the diet with extremely low glucose affect to DIHC, ExHC.BN-Dihc2BN, a congenic strain that does not develop DIHC, and ExHC rats were fed a high-fructose diet containing 1% cholesterol for 2 weeks. The serum cholesterol and hepatic triacylglycerol levels were similar in the strains. Results of water-soluble metabolite analysis with primary hepatocytes, an increase in fructose-6-phosphate and decreases in succinate, malate and aspartate in ExHC rats, support impaired glycolysis in the ExHC rats. Thus, the Smek2 mutation causes abnormal hepatic glucose utilization via downregulation of Pfkl expression. This abnormal glucose metabolism disrupts hepatic fatty acid synthesis and causes DIHC in the ExHC rats.

In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation.

Hyperphosphorylated tau proteins are one of the neuropathological hallmarks in the Alzheimer's disease (AD) brain. The in vivo imaging of tau aggregates with nuclear medical imaging probes is helpful for the further comprehension of and medical intervention in the AD pathology. For tau-selective PET imaging, we newly designed and synthesized 18F-labeled benzimidazopyridine (BIP) derivatives with fluoroalkylamino groups, [18F]IBIPF1 and [18F]IBIPF2, and evaluated their utilities as tau imaging probes. They both bound selectively to tau against amyloid β (Aβ) aggregates in AD brain sections in vitro, and showed good pharmacokinetics in mouse brains in vivo. Notably, [18F]IBIPF1 exhibited high tau-selectivity (Tau/Aβ ratio = 34.8), high brain uptake (6.22% ID/g at 2 min postinjection), and subsequent washout (2.77% ID/g at 30 min postinjection). In vivo analysis of radiometabolites indicated that [18F]IBIPF1 was stable against metabolism in the mouse brain. These encouraging preclinical results suggest that further structural optimization based on the BIP scaffold may lead to the development of more useful tau imaging probes.

Living in isolation is considered an emerging societal problem that negatively affects the physical wellbeing of its sufferers in ways that we are just starting to appreciate. This study investigates the immunomodulatory effects of social isolation in mice, utilising a two-week program of sole cage occupancy followed by the testing of immune-inflammatory resilience to bacterial sepsis. Our results revealed that mice housed in social isolation showed an increased ability to clear bacterial infection compared to control socially housed animals. These effects were associated with specific changes in whole blood gene expression profile and an increased production of classical pro-inflammatory cytokines. Interestingly, equipping socially isolated mice with artificial nests as a substitute for their natural huddling behaviour reversed the increased resistance to bacterial sepsis. Together these results suggest that the control of body temperature through social housing and huddling behaviour are important factors in the regulation of the host immune response to infection in mice and might provide another example of the many ways by which living conditions influence immunity.

TJs maintain the epithelial barrier by regulating paracellular permeability. Since TJs are under dynamically fluctuating intercellular tension, cells must continuously survey and repair any damage. However, the underlying mechanisms allowing cells to sense TJ damage and repair the barrier are not yet fully understood. Here, we showed that proteinases play an important role in the maintenance of the epithelial barrier. At TJ break sites, EpCAM-claudin-7 complexes on the basolateral membrane become accessible to apical membrane-anchored serine proteinases (MASPs) and the MASPs cleave EpCAM. Biochemical data and imaging analysis suggest that claudin-7 released from EpCAM contributes to the rapid repair of damaged TJs. Knockout (KO) of MASPs drastically reduced barrier function and live-imaging of TJ permeability showed that MASPs-KO cells exhibited increased size, duration, and frequency of leaks. Together, our results reveal a novel mechanism of TJ maintenance through the localized proteolysis of EpCAM at TJ leaks, and provide a better understanding of the dynamic regulation of epithelial permeability.

Patients suffering from autoimmune diseases are more susceptible to mental disorders yet, the existence of specific cellular and molecular mechanisms behind the co-morbidity of these pathologies is far from being fully elucidated. By generating transgenic mice overexpressing Annexin-A1 exclusively in T cells to study its impact in models of autoimmune diseases, we made the unpredicted observation of an increased level of anxiety. Gene microarray of Annexin-A1 CD4+ T cells identified a novel anxiogenic factor, a small protein of approximately 21 kDa encoded by the gene 2610019F03Rik which we named Immuno-moodulin. Neutralizing antibodies against Immuno-moodulin reverted the behavioral phenotype of Annexin-A1 transgenic mice and lowered the basal levels of anxiety in wild type mice; moreover, we also found that patients suffering from obsessive compulsive disorders show high levels of Imood in their peripheral mononuclear cells. We thus identify this protein as a novel peripheral determinant that modulates anxiety behavior. Therapies targeting Immuno-moodulin may lead to a new type of treatment for mental disorders through regulation of the functions of the immune system, rather than directly acting on the nervous system.

Chronic inflammation is involved in the formation and enlargement of cerebral aneurysms (CAs), with macrophages playing a key role in the process. The present study evaluated visualization of macrophages present in CAs using an activatable fluorescent probe (IONP-ICG) comprising an iron oxide nanoparticles (IONPs) conjugated with indocyanine green (ICG).

Innate-like T cell populations expressing conserved TCRs play critical roles in immunity through diverse developmentally acquired effector functions. Focusing on the prototypical lineage of invariant natural killer T (iNKT) cells, we sought to dissect the mechanisms and timing of fate decisions and functional effector differentiation. Utilizing induced expression of the semi-invariant NKT cell TCR on double positive thymocytes, an initially highly synchronous wave of iNKT cell development was triggered by brief homogeneous TCR signaling. After reaching a uniform progenitor state characterized by IL-4 production potential and proliferation, effector subsets emerged simultaneously, but then diverged toward different fates. While NKT17 specification was quickly completed, NKT1 cells slowly differentiated and expanded. NKT2 cells resembled maturing progenitors, which gradually diminished in numbers. Thus, iNKT subset diversification occurs in dividing progenitor cells without acute TCR input but utilizes multiple active cytokine signaling pathways. These data imply a two-step model of iNKT effector differentiation.

(99m)Tc-HYNIC-annexin A5 can be considered as a benchmark in the field of apoptosis imaging. However, (99m)Tc-HYNIC-annexin A5 has characteristics of high uptake and long retention in non-target tissues such as kidney and liver. To minimize this problem, we developed a novel (99m)Tc-labeled annexin A5 using a bis(hydroxamamide) derivative [C3(BHam)2] as a bifunctional chelating agent, and evaluated its usefulness as an imaging agent for detecting apoptosis. The amino group of C3(BHam)2 was converted to a maleimide group, and was coupled to thiol groups of annexin A5 pretreated with 2-iminothiolane. (99m)Tc labeling was performed by a ligand exchange reaction with (99m)Tc-glucoheptonate. Biodistribution experiments for both (99m)Tc-C3(BHam)2-annexin A5 and (99m)Tc-HYNIC-annexin A5 were performed in normal mice. In addition, in tumor-bearing mice, the relationship between the therapeutic effects of chemotherapy (5-FU) and the tumor accumulation of (99m)Tc-C3(BHam)2-annexin A5 just after the first treatment of 5-FU was evaluated. (99m)Tc-C3(BHam)2-annexin A5 was prepared with a radiochemical purity of over 95%. In biodistribution experiments, (99m)Tc-C3(BHam)2-annexin A5 had a much lower kidney accumulation of radioactivity than (99m)Tc-HYNIC-annexin A5. In the organs for metabolism, such as liver and kidney, radioactivity after the injection of (99m)Tc-HYNIC-annexin A5 was residual for a long time. On the other hand, radioactivity after the injection of (99m)Tc-C3(BHam)2-annexin A5 gradually decreased. In therapeutic experiments, tumor growth in the mice treated with 5-FU was significantly inhibited. Accumulation of (99m)Tc-C3(BHam)2-annexin A5 in tumors significantly increased after 5-FU treatment. The accumulation of radioactivity in tumor correlated positively with the counts of TUNEL-positive cells. These findings suggest that (99m)Tc-C3(BHam)2-annexin A5 may contribute to the efficient detection of apoptotic tumor response after chemotherapy.