Hiroaki Kanazawa

The rational design of a synthetic polymer nanoparticle that neutralizes a toxic peptide in vivo

Synthetic polymer nanoparticles (NPs) that bind venomous molecules and neutralize their function in vivo are of significant interest as “plastic antidotes.” Recently, procedures to synthesize polymer NPs with affinity for target peptides have been reported. However, the performance of synthetic materials in vivo is a far greater challenge. Particle size, surface charge, and hydrophobicity affect not only the binding affinity and capacity to the target toxin but also the toxicity of NPs and the creation of a “corona” of proteins around NPs that can alter and or suppress the intended performance. Here, we report the design rationale of a plastic antidote for in vivo applications. Optimizing the choice and ratio of functional monomers incorporated in the NP maximized the binding affinity and capacity toward a target peptide. Biocompatibility tests of the NPs in vitro and in vivo revealed the importance of tuning surface charge and hydrophobicity to minimize NP toxicity and prevent aggregation induced by nonspecific interactions with plasma proteins. The toxin neutralization capacity of NPs in vivo showed a strong correlation with binding affinity and capacity in vitro. Furthermore, in vivo imaging experiments established the NPs accelerate clearance of the toxic peptide and eventually accumulate in macrophages in the liver. These results provide a platform to design plastic antidotes and reveal the potential and possible limitations of using synthetic polymer nanoparticles as plastic antidotes.

Metallothionein-3 is expressed in the brain and various peripheral organs of the rat

Metallothionein-3 (MT-3), also known as growth inhibitory factor (GIF), was originally identified in the brain. An essential step in elucidating the potential roles of MT-3 is to evaluate its expression levels in organs other than the brain. In this present study, we carried out RT-PCR, Western blot and immunohistochemical analyses to quantify MT-3 mRNA and its protein in the cerebrum, eye, heart, kidney, liver, prostate, testis, tongue, and muscle in male Wistar rats. MT-3 mRNA was detected in the cerebrum, the dorsolateral lobe of the prostate, testis, and tongue. Using a monoclonal anti-MT-3 antibody, we detected MT-3 in the cerebrum, the dorsolateral lobe of the prostate, testis, and tongue as a single band on an immunoblot. Immunohistochemical staining showed MT-3 in some astrocytes in the deep cortex, ependymal cells, and choroidal cells in the cerebrum. MT-3 was also detected in some cells of the glomerulus and the collective tubules in the kidney, some cells in the glandular epithelium of the dorsolateral lobe of the prostate, some Sertoli cells and Lydig cells in the testis, and taste bud cells in the tongue. Although MT-3 immunopositivity was obviously demonstrated in the kidney by the immnunohistochemical method, the expression of MT-3 was not fully detectable by RT-PCR and Western blot analyses. Interestingly, only a subset of cells showed positivity for MT-3, not all cells in all tissues. The localization of MT-3 in peripheral organs outside the brain suggests that MT-3 has roles in these tissues besides its role in growth inhibition of neurites.

PET imaging of brain cancer with positron emitter-labeled liposomes

Since nanocarriers such as liposomes are known to accumulate in tumors of tumor-bearing animals, and those that have entrapped a positron emitter can be used to image a tumor by PET, we applied (18)F-labeled 100-nm-sized liposomes for the imaging of brain tumors. Polyethylene glycol (PEG)-modified liposomes, which are known to accumulate in tumors by passive targeting and those modified with Ala-Pro-Arg-Pro-Gly, which are known to home into angiogenic sites were used. Those liposomes labeled with DiI fluorescence accumulated in a glioma implanted in a rat brain 1h after the injection, although they did not accumulate in the normal brain tissues due to the protection afforded by the blood-brain barrier. Preformed liposomes were easily labeled with 1-[(18)F]fluoro-3,6-dioxatetracosane, and enabled the imaging of gliomas by PET with higher contrast than that obtained with [(18)F]deoxyfluoroglucose. In addition, the smallest tumor among those tested, having a diameter of 1mm was successfully imaged by the liposomal (18)F. Therefore, nanocarrier-based imaging of brain tumors is promising for the diagnosis of brain cancer and possible drug delivery-based therapy.

Shiga-Like Toxin II Impairs Hepatobiliary Transport of Doxorubicin in Rats by Down-Regulation of Hepatic P Glycoprotein and Multidrug Resistance-Associated Protein Mrp2

ABSTRACT We investigated the effect of Shiga-like toxin II (SLT-II), derived from Escherichia coli O157:H7, on the hepatobiliary excretion of doxorubicin, a substrate for P glycoprotein and the multidrug resistance-associated protein Mrp2, and on the expression of P glycoprotein and Mrp2 in rats. Histopathological examination did not show any liver injury in SLT-II-treated rats. A significant delay in the disappearance of doxorubicin from plasma after its intravenous administration (5 mg/kg of body weight) was observed in rats treated 24 h earlier with SLT-II (2 μg/animal). When rats received an infusion of doxorubicin (2.6 μg/min) 24 h after intravenous injection of SLT-II, the steady-state concentration of doxorubicin in plasma increased and the bile flow decreased, whereas the concentration in liver did not alter. SLT-II significantly increased the unbound fraction of doxorubicin in plasma but did not alter the concentration in liver tissue. SLT-II significantly decreased the biliary excretion rate and biliary clearance of doxorubicin based on the total concentration and concentration of the unbound fraction in plasma and liver. Western blot analysis revealed that SLT-II down-regulated P glycoprotein and Mrp2 in the liver, which could explain the observed decrease in the biliary excretion of doxorubicin by SLT-II. A tumor necrosis factor alpha (TNF-α) production inhibitor, pentoxifylline, could not protect SLT-II-induced decreases in the biliary clearance of doxorubicin and down-regulation of both transporters. It is unlikely that TNF-α plays a major role in the SLT-II-induced decrease in the hepatobiliary transport of doxorubicin and the down-regulation of both transporters.

Effect of endotoxin on doxorubicin transport across blood-brain barrier and P-glycoprotein function in mice.

The aim of this study was to investigate whether Klebsiella pneumoniae endotoxin modifies transport of doxorubicin, a P-glycoprotein substrate, across the blood-brain barrier and P-glycoprotein function in mice. Doxorubicin (30 mg/kg) was administered into the tail vein or fluorescein isothiocyanate-labeled dextran (FD-4) was infused (20 microg/min) into the right jugular vein of mice intravenously injected with endotoxin (10 mg/kg) 6 or 24 h earlier. Blood and brain samples were collected 4 h after injection of doxorubicin or 1 h after infusion of FD-4. We examined using Western blotting the influence of endotoxin on the expression of P-glycoprotein in brains obtained 6, 12 and 24 h after injection. Endotoxin did not change the plasma and brain concentrations and brain-to-plasma concentration ratio (K(p) value) of FD-4. No histopathological changes in brain capillaries were observed. These results suggest that endotoxin does not cause damage to brain capillaries. Plasma and brain concentrations of doxorubicin in mice treated 6 h earlier with endotoxin were significantly higher than those in control and mice treated 24 h earlier. However, endotoxin did not significantly change the K(p) value of doxorubicin. The protein level of P-glycoprotein was significantly, but slightly down-regulated 6 h after endotoxin treatment. However, the levels remained almost unchanged after 12 and 24 h. The present results suggest that Klebsiella pneumoniae endotoxin has no effect on the brain capillary integrity and doxorubicin transport across the blood-brain barrier in mice. It is likely that P-glycoprotein function might be sufficient to transport doxorubicin in spite of decreased levels of P-glycoprotein in the brain.

Consecutive oral administration of Bifidobacterium longum MM-2 improves the defense system against influenza virus infection by enhancing natural killer cell activity in a murine model

Bifidobacterium, one of the major components of intestinal microflora, shows anti‐influenza virus (IFV) potential as a probiotic, partly through enhancement of innate immunity by modulation of the intestinal immune system. Bifidobacterium longum MM‐2 (MM‐2), a very safe bacterium in humans, was isolated from healthy humans and its protective effect against IFV infection in a murine model shown. In mice that were intranasally inoculated with IFV, oral administration of MM‐2 for 17 consecutive days improved clinical symptoms, reduced mortality, suppressed inflammation in the lower respiratory tract, and decreased virus titers, cell death, and pro‐inflammatory cytokines such as IL‐6 and TNF‐α in bronchoalveolar lavage fluid. The anti‐IFV mechanism of MM‐2 involves innate immunity through significant increases in NK cell activities in the lungs and spleen and a significant increase in pulmonary gene expression of NK cell activators such as IFN‐γ, IL‐2, IL‐12 and IL‐18. Even in non‐infected mice, MM‐2 administration also induced significant enhancement of both IFN‐γ production by Peyers patch cells (PPs) and splenetic NK cell activity. Oral administration of MM‐2 for 17 days activates systemic immunoreactivity in PPs, which contributes to innate immunity, including NK cell activation, resulting in an anti‐IFV effect. MM‐2 as a probiotic may function as a prophylactic agent in the management of an IFV epidemic.

Visualization of Sialidase Activity in Mammalian Tissues and Cancer Detection with a Novel Fluorescent Sialidase Substrate

Sialidase removes sialic acid from sialoglycoconjugates and plays crucial roles in many physiological and pathological processes. Various human cancers express an abnormally high level of the plasma membrane-associated sialidase isoform.Visualization of sialidase activity in living mammalian tissues would be useful not only for understanding sialidase functions but also for cancer diagnosis. However, since enzyme activity of mammalian sialidase is remarkably weak compared with that of bacterial and viral sialidases, it has been difficult to detect sialidase activity in mammalian tissues. We synthesized a novel benzothiazolylphenol-based sialic acid derivative (BTP-Neu5Ac) as a fluorescent sialidase substrate. BTP-Neu5Ac can visualize sialidase activities sensitively and selectively in acute rat brain slices. Cancer cells implanted orthotopically in mouse colons and human colon cancers (stages T3-T4) were also clearly detected with BTP-Neu5Ac. The results suggest that BTP-Neu5Ac is useful for histochemical imaging of sialidase activities.

Shiga-like toxin II modifies brain distribution of a P-glycoprotein substrate, doxorubicin, and P-glycoprotein expression in mice

The effect of Shiga-like toxin II (SLT-II), which was derived from Escherichia coli O157:H7, on doxorubicin transport across the blood-brain barrier (BBB) and P-glycoprotein function, was investigated in ddY mice. Doxorubicin (30 mg kg(-1)) was administered intravenously or fluorescein isothiocyanate labeled dextran (FD-4) was infused (20 microg min(-1)) to the mice, who had received an intravenous injection of SLT-II (0.2 microg/animal) 6 or 24 h earlier. Blood and brain were removed 4 h after injection of doxorubicin or 60 min after infusion of FD-4. SLT-II significantly elevated the brain concentration and brain-to-plasma concentration ratio (K(p)) of doxorubicin and FD-4 24 h after injection, but did not alter 6 h after. Cyclosporin A (200 mg kg(-1)) significantly increased the K(p) value of doxorubicin in the control mice, but did not alter it in mice treated 24 h earlier with SLT-II. Pentoxifylline (100 mg kg(-1)) a TNF-alpha production inhibitor, ameliorated SLT-II-induced increases in the brain concentrations of both drugs and the K(p) value of FD-4, suggesting that TNF-alpha, at least in part, causes damage to the brain capillaries. Western blot analysis revealed that SLT-II increased the protein level of P-glycoprotein in the brain of mice 6 h after injection and the increased level remained unchanged for 24 h. SLT-II did not change ATP content in the brain of mice. These results suggest that the increased P-glycoprotein level cannot explain SLT-II-induced increase in the doxorubicin accumulation in brain. The present findings indicate that SLT-II impairs the BBB function and doxorubicin transport across the BBB, while it overexpresses P-glycoprotein.

Imaging of sialidase activity in rat brain sections by a highly sensitive fluorescent histochemical method

Sialidase (EC 3.2.1.18) removes sialic acid from sialoglycoconjugates. Since sialidase extracellularly applied to the rat hippocampus influences many neural functions, including synaptic plasticity and innervations of glutamatergic neurons, endogenous sialidase activities on the extracellular membrane surface could also affect neural functions. However, the distribution of sialidase activity in the brain remains unknown. To visualize extracellular sialidase activity on the membrane surface in the rat brain, acute brain slices were incubated with 5-bromo-4-chloroindol-3-yl-α-d-N-acetylneuraminic acid (X-Neu5Ac) and Fast Red Violet LB (FRV LB) at pH 7.3. After 1h, myelin-abundant regions showed intense fluorescence in the rat brain. Although the hippocampus showed weak fluorescence in the brain, mossy fiber terminals in the hippocampus showed relatively intense fluorescence. These fluorescence intensities were attenuated with a sialidase-specific inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA, 1mM). Additionally, the fluorescence intensities caused by X-Neu5Ac and FRV LB were correlated with the sialidase activity measured with 4-methylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac), a classical substrate for quantitative measurement of sialidase activity, in each brain region. Therefore, staining with X-Neu5Ac and FRV LB is specific for sialidase and useful for quantitative analysis of sialidase activities. The results suggest that white matter of the rat brain has intense sialidase activity.

Topographical Relation between Serotonin-Containing Paraneurons and Peptidergic Neurons in the Intestine and Urethra

Luminal stimulation of enterochromaffin (EC) cells is known to cause their release of serotonin which in turn may induce a variety of reflexes via mucosal intrinsic neurons. To morphologically support this idea, the present study demonstrates a close topographical relationship between EC cells and nerves, using whole-mount preparations. Beaded nerve fibers containing vasoactive intestinal polypeptide (VIP) were observed in close proximity to serotonin-immunoreactive EC cells in the small and large intestines of the rat and guinea pig; in whole-mount preparations, the VIP nerve fibers appeared to underlie EC cells. This finding correlates with the physiological datum that the intra-arterial infusion of serotonin causes VIP release from the intestine. Canine urethral serotonin cells, a counterpart of the intestinal EC cells, were shown to contact intraepithelial nerves immunoreactive for calcitonin gene-related peptide. The neuroparaneuronal connection in the urethra may play an important role in the serotonin-evoked urethrogenital reflex. Intestinal and urethral serotonin-containing paraneurons, which are sensory in nature, may release serotonin in response to luminal stimuli, and directly activate adjacent peptidergic neurons to initiate the reflex arcs.